VIRULENCE 2021, VOL. 12, NO. 1, 444–469 https://doi.org/10.1080/21505594.2021.1871823 REVIEW ARTICLE Host Defence RNases as Antiviral Agents against Enveloped Single Stranded RNA Viruses Jiarui Li and Ester Boix Dpt. Of Biochemistry and Molecular Biology, Faculty of Biosciences, Universitat Autònoma De Barcelona, Spain ABSTRACT ARTICLE HISTORY Owing to the recent outbreak of Coronavirus Disease of 2019 (COVID-19), it is urgent to develop Received 21 September 2020 effective and safe drugs to treat the present pandemic and prevent other viral infections that Revised 26 December 2020 might come in the future. Proteins from our own innate immune system can serve as ideal sources Accepted 30 December 2020 of novel drug candidates thanks to their safety and immune regulation versatility. Some host KEYWORDS defense RNases equipped with antiviral activity have been reported over time. Here, we try to Enveloped single-stranded summarize the currently available information on human RNases that can target viral pathogens, RNA Viruses; virus-host with special focus on enveloped single-stranded RNA (ssRNA) viruses. Overall, host RNases can interplay; RNases; innate fight viruses by a combined multifaceted strategy, including the enzymatic target of the viral Immunity; antiviral drugs genome, recognition of virus unique patterns, immune modulation, control of stress granule formation, and induction of autophagy/apoptosis pathways. The review also includes a detailed description of representative enveloped ssRNA viruses and their strategies to interact with the host and evade immune recognition. For comparative purposes, we also provide an exhaustive revision of the currently approved or experimental antiviral drugs. Finally, we sum up the current perspectives of drug development to achieve successful eradication of viral infections. Introduction 40 years ago[7], most of them are still not effective for the treatment of viral infections caused by corona­ It is almost one century since the initial isolation of viruses (CoVs) or Respiratory Syncytial Virus (RSV). individual viruses and their assignment to specific Therefore, novel strategies are urgently needed to solve diseases[1]. During these 100 years, diseases caused by this issue. viruses have posed a huge threat to human health. It has been proposed that host defense Ribonucleases Particular attention is drawn by viruses that contain (RNases) can act as drug candidates, which offer an RNA in their genome, which are estimated to represent alternative way of fighting against viral infections[8]. about 75% of the virus-related human diseases[2]. RNases, targeting a diversity of cellular RNAs, play an While we are still struggling to find effective drugs or important role in many biological processes such as vaccines to fight against human immunodeficiency immune modulation, angiogenesis, neurogenesis, and virus (HIV) or Hepatitis C virus (HCV), both of which will lead to chronic diseases, more enveloped host defense [8–16]. Already in 1968, researchers ssRNA viruses that can cause fatal respiratory symp­ observed that patients with tick-born encephalitis had toms have gradually (or suddenly) emerged. According higher RNA-catalytic activity in their blood and cere­ to World Health Organization (WHO), the spread of brospinal fluids[17]. Since then, the number of RNases severe acute respiratory syndrome (SARS) in 2003 has with reported antiviral properties has increased slowly led to over 700 deaths[3] and the mortality rate of the but significantly. For example, eosinophil-derived neu­ Middle East respiratory syndrome (MERS), first identi­ rotoxin (EDN/hRNase 2), one member of RNase fied in Saudi Arabia in 2012, has already reached to A superfamily, displays a high activity against ssRNA 35%[4]. Most recently, the dreaded COVID-19 has viruses like HIV and RSV [18,19]. Also, the antiviral spread in most countries of the world and caused mechanisms of RNase L are nowadays studied in deep: more than 600,000 people's death at the end of the protein is not only involved in degrading viral RNA July 2020[5]. The coronavirus pandemic has become but is also associated with the activation of interferon a global public health emergency[6]. Although there (IFN) production[20]. Although there are still limited are more antiviral drugs currently available than references about RNases against human CoV CONTACT Ester Boix Ester.Boix@uab.es © 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. VIRULENCE 445 pathogens, it has already been demonstrated that RNase protein (S), a glycoprotein trimer that belongs to class L can exert a significant effect on a murine coronavirus I fusion protein at the virion surface[36], binds to the mouse hepatitis beta CoV strain (MHV-JHM)[21]. angiotensin-converting enzyme 2 (ACE2) for SARS and Taken together these positive results about the anti­ SARS-CoV-2 and to the dipeptidyl peptidase 4 (DPP4) viral properties of human RNases, this review aims to for MERS-CoV infection [37–40]. This recognition step summarize the main characteristics of enveloped is crucial to induce membrane fusion through the con­ ssRNA viruses that nowadays represent a threat to formational transition of spike [41–43]. Also, there are human health, together with the main mechanisms many reports that describe HCV entrance into cells, that RNases can exert against viral infections, with the mainly mediated by endocytosis and subsequent fusion aim to provide the basis for the development of novel into endosome by envelope glycoproteins, E1 and E2 and safe antiviral drugs. [44–46]. Following entrance, the (+) ssRNA of the viruses can serve as both genome and mRNA and is directly trans­ Mechanism of enveloped single-stranded RNA lated into protein by host ribosomes[47]. The replica­ viral infection tion of the positive strand will produce a double- stranded RNA (dsRNA), which is then transcribed The entire infection viral particle, virion, consists of into a positive single-stranded genome/mRNA. a nucleic acid molecule surrounded by a protein shell Notably, the replication and transcription of (+) named capsid which, together with the genome, forms ssRNA viruses can take place in membrane invagina­ the nucleocapsid [22,23]. In particular, for enveloped tions in order to increase the efficiency of replication viruses, there is also a lipid bilayer derived from the and evade host defense[48]. The taken membrane may host cell membranes outside its nucleocapsid. Both be derived from various organelles, including endoplas­ nucleocapsid and envelope of virion contribute to the mic reticulum (ER), late endosome/lysosome, or mito­ viral infection[24]. Viruses must enter into the host chondrial outer membrane[49]. For example, cells to deliver their genomic information. Fusion and accumulation of dsRNA has been found to locate into endocytosis are two general ways for enveloped viruses the double-membrane vesicles (DMVs) induced by entry (Figure 1) [25–27]. The viral membrane fusion SARS-CoV infection, suggesting the probable site of protein can help the viruses to directly fuse its mem­ viral RNA synthesis[50]. brane with the cytoplasmic membrane [28] and fusion The CoV genome is the largest one among positive- may also occur at the endosome after endocytosis[29]. strand RNA viruses. It expresses two co-terminal poly­ For most enveloped viruses, the entry is mediated by proteins, pp1a and pp1ab, which are processed into a series of envelope glycoproteins such as the spike in coronavirus non-structural proteins with specific biolo­ CoVs and gp120 in HIV-1[25]. After the viral genome gical properties, named nsp1-16 [31,51]. The endonu­ penetrates the host cytoplasm, the viruses will exert clease nsp15, targeting viral polyuridine sequences, their function by inhibiting the normal metabolism of participates in the evasion of dsRNA sensors. the host cell, activating replication of its genome, tran­ Accordingly, the loss of nsp15 activity leads to strong scription and translation of its own RNA, and final attenuation of the disease in mice by stimulating the assembly and release of virions[30]. Here, we will detail immune response through activation of interferon the structural characteristics and intracellular behaviors (IFN), protein kinase R(PKR), and the 2′-5′ oligoade­ of some representative enveloped ssRNA viruses that nylate synthetase (OAS)/RNase L system [52,53]. recently pose a great threat to human health (Figure 1). Likewise, the exoribonuclease, nsp14 is crucial for repli­ cation proofreading and also works as a (guanine-N7) methyltransferase (N7-MTase) for mRNA capping Coronaviruses (CoVs) and Hepatitis C virus (HCV) [54,55]. The nsp14 protein function is also key to the CoVs and HCV are enveloped positive ssRNA [(+) development of viral resistance[56]. In addition, nsp6 ssRNA] viruses with a diameter of about 125 nm and in the avian CoV infectious bronchitis virus (IBV) is 40–100 nm, respectively [31,32]. The notorious viruses, capable of inducing autophagy, a strategy that might SARS-CoV, MERS-CoV, and SARS-CoV-2 belong to aim to alter the adaptive immune response and pro­ beta-CoVs and we find eight genotypes for HCV mote degradation of host immunomodulatory proteins [33,34]. [57]. On the other hand, HCV only encodes a single The CoVs can enter into the cell either by fusion or polyprotein, which is processed by the host and viral by endocytosis[35]. The entrance into human cells proteases to create 10 viral proteins, including Core, E1, relies on a receptor recognition mechanism. The spike E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B. 446 J. LI AND E. BOIX Figure 1. The Viral Life Cycle. The life cycle of positive single-strand RNA [(+)ssRNA], negative single-strand RNA viruses [(-)ssRNA] and also HIV are indicated. Normally, viruses firstly enter into the host cell by either fusion or endocytosis, then the viral genome is replicated and the viral polyproteins are translated within the cytoplasm. Many (+)ssRNA viruses can replicate and be transcribed in special DMV. Finally, virion assembly and release takes place. The life cycle for (+)ssRNA viruses are shown in red color and for (-) ssRNA in blue. For HIV, the genome will be reverse transcribed into dsDNA and then integrated into the host genome, as shown in light blue color. Among these, the non-structural proteins, protease respiratory tract infections among children younger NS3/NS4A, phosphoprotein NS5A, and polymerase than 5 years [62,63]. NS5B, are the major players in viral replication and Direct fusion with the cytoplasmic membrane by also the main targets for the design of direct-acting a pH-independent mode has generally been antiviral agents [58,59]. considered as the main method of viral entry, although endocytosis followed by acid-independent membrane fusion is also observed for RSV [61,64–66]. Respiratory Syncytial Virus (RSV) and Parainfluenza There are two main envelope glycoproteins on the viral Virus (PIV) surface, the attachment protein and the fusion protein, RSV and PIV (PIV 1–4) belong to the Paramyxoviridae which facilitate the attachment and cell membrane family, which are enveloped, spherical but negative fusion for viral entry, respectively. In the case of PIV, ssRNA [(-)ssRNA] viruses with a diameter of about the attachment protein is a hemagglutinin- 150 nm for RSV and 150–300 nm for PIV [60,61]. neuraminidase [61,67]. Interestingly, in comparison They are both the leading cause of acute lower with other paramyxoviruses, the RSV fusion protein VIRULENCE 447 alone is enough to mediate membrane fusion as well as protective immunity against HIV [81,86]. Also, lots of viral infection [68] and the glycoprotein is also an strategies are developed by HIV to regulate the cell unusual attachment protein that can bind to the cell autophagy and establish a chronic infection[87]. surface glycosaminoglycans containing iduronic acid, like heparan sulfate and chondroitin sulfate B[69]. The replication and transcription of this kind of Host-virus interplay virus take place in the cell cytoplasm. Unlike many According to the characteristics of viruses introduced positive-sense RNA viruses that are translated immedi­ above, we find that interactions between the host and ately by the host cell, the genome of a negative-sense the virus can occur either to protect the host or to RNA virus alone is not infectious, so the viral RNA- promote the viral infection. Generally, when the virion dependent RNA polymerase (RdRp) is needed to enters into the cell, it exerts multiple strategies to evade synthesize the positive-sense RNA [60,70]. Notably, the host immune functions, including avoidance of replication and transcription are controlled by the identification by Pattern-Recognition Receptors same polymerase[71]. Two proteins, large polymerase (PRRs), inhibition of IFN signaling, and interference subunit L and phosphoprotein (P), contribute to the with host protein expression[88]. In its turn, the host core polymerase complex. The L protein exerts its responds to the virus by activation of the adaptive enzyme activity to participate in RNA synthesis, cap­ immunity, formation of Stress Granules (SGs), autop­ ping, and cap methylation while the P protein is an hagy, or even apoptosis[89]. essential cofactor[71]. Paramyxoviruses also can pro­ Many reviews have exhaustively discussed the overall duce IFN-antagonists to evade the host IFN-mediated host-virus interplay [88,90,91]. Here, we will focus on innate immune response[72]. the interaction between the host and the virus with enveloped ssRNA, with a special emphasis on the type Human Immunodeficiency Virus (HIV) of viruses mentioned above. There are two types of HIV, HIV-1 and HIV-2, both of which are spherical, enveloped (+)ssRNA viruses. The Pathogen-associated molecular patterns (PAMPs) diameter of the virion is about 120 nm for HIV-1 and 110 nm for HIV-2 [73,74]. HIV-2, which is mainly It is widely known that dsRNA and RNA with a 5′- present in West Africa, has lower mortality than HIV- triphosphate (5′-pppRNA) are commonly produced by 1[75]. RNA viruses during replication [92] and the host Normally, both fusions with the cell membrane and immunity will detect these “foreigners” by PRRs, such endocytic pathways have been indicated for HIV entry as Toll-like receptors (TLRs), RIG-I-like receptors [76–79]. The envelope glycoproteins are gp120/gp41 for (RLRs), and Nod-like receptors (NLRs) [93–95]. In HIV-1 and gp125/gp36 for HIV-2, among which the TLRs, ssRNA can be identified by TLR7 and TLR8 former ones (gp120 and gp125) recognize host CD4, while dsRNA by TLR3 and TLR9. Also, TLR2 and CCR5, CXCR4 receptors, and the latter ones (gp41 and TLR4 may contribute to the recognition of viral glyco­ gp36) act as fusion or transmembrane proteins [74,80]. proteins[89]. In addition, TLR2 heterodimers with Therefore, all CD4-positive cells such as dendritic cells, either TLR1 or TLR6 can provide additional recogni­ T helper cells, macrophages, and astrocytes are suscep­ tion specificity for some viral proteins[96]. In their tible to HIV[81]. turn, RLRs are conformed by Retinoic Acid-Inducible The cytoplasm is the location where HIV transcrip­ gene I (RIG-I), also named DExD/H-box helicase 58, tion is activated. However, in contrast with CoVs that and Melanoma Differentiation-Associated protein 5 replicate in the cytoplasm[82], the HIV genome repli­ (MDA5)[97], among which RIG-I is responsible for cates in the nucleus. The RNA of HIV is reverse tran­ the short RNA ligands with 5′-triphosphate caps and scribed and copied into a linear dsDNA molecule that MDA5 for long dsRNA[92]. The recognition process of then enters into the nucleus and is integrated into the PAMPs (Figure 2a) activates IFNs signaling, as well as cell genome, where the virus remains in latency unde­ the expression of inflammatory chemokines and cyto­ tected by the immune recognition system[83]. The kines [90,98,99]. It should be noted that many key integration step is key for viral replication as well as molecules such as 2′,5′-oligoadenylate synthetase for transcription and Tat (Trans-activator of transcrip­ (OAS), together with RNase L to form OAS/RNase tion) is the viral-encoded transcription factor needed L pathway (Figure 2b), and PKR (Figure 2c), are not for the expression of viral genes [84,85]. Unfortunately, only induced by IFN but also by dsRNA-specific PRRs HIV can destroy the immune system and there is no that trigger the antiviral response [100,101]. 448 J. LI AND E. BOIX Figure 2. Host-virus interplay. (a). Pathogen-associated molecular patterns (PAMPs). The three most common PAMPs in case of viral intrusion are shown. From left to right, they are RLRs, TLRs and NLRs respectively. Different receptors can recognize various RNAs produced by viruses. Normally, after the recognition, IFNs and cytokines are induced through a series of signal cascades. (b). OAS/RNase L pathway. The related processes in response to dsRNA are shown. An IFN-induced 2–5A synthetase (OAS) is expressed to synthesize 2′5′ oligoadenylates (2–5A), which activate RNase L, and then small RNAs cleaved by active RNase L can exert multiple functions to fight viruses. The related processes are shown. (c). PKR pathway. The important processes are indicated. PKR is also an IFN-induced, dsRNA-activated protein kinase. Once active, PKR can phosphorylate the eukaryotic translation initiation factor (eIF2α), which later suppresses viral translation and induces stress granules (SGs) formation. Specifically, long dsRNA produced during the repli­ IFN (IFN-λ). A recent review has detailed the interac­ cation of a (+)ssRNA virus, such as the murine CoV or tion between the host and HCV[105]. MHV, can be recognized by MDA5 and then activate Likewise, (-)ssRNA viruses, such as RSV, also produce the type I IFN response[102]. TLR3 is also a dsRNA dsRNA intermediates during replication. Therefore, in sensor, pre-stimulation of which can block MHV infec­ the RSV-infected epithelial cells, TLR3 pathways can tion through induction of IFN-β in macrophages[103]. mediate the expression of chemokines CXCL8 and Besides the recognition of dsRNA, it is demonstrated CCL5[106]. Interestingly, RIG-I but not MDA5 has that the membrane protein from SARS-CoV can been identified as key for immune defense against RSV directly promote the production of IFN-β through [107]. Similarly, the fusion protein of RSV contributes to a TLR-related signaling pathway[104]. In addition, the induction of interleukin 6 (IL-6), which depends on HCV RNA can also be recognized by TLR3, MDA5, the presence of CD14 and TLR4[108]. In addition, activa­ and RIG-I in infected hepatocytes and TLR7 on plas­ tion of interferon-regulatory factor 3 (IRF3) and produc­ macytoid dendritic cells (pDC), thereby inducing the tion of IFN-β was reported to be triggered by nucleotide- secretion of type I IFN (INF-α and IFN-β) and type III binding oligomerization domain 2 (Nod2), a protein that VIRULENCE 449 belongs to NLRs, in RSV-infected human embryonic viral mRNA function is the acetylation of cytidine kidney (HEK-293) cells[109]. residues. Incorporation by HIV-1 of acetylation at N4 On the other hand, the particular case of (+)ssRNA position (ac4C) is associated with an increase of gene viruses, HIV, which produces dsDNA in a host cell, can expression and enhance the mRNA stability[129]. also be detected by diverse PRRs. The ssRNA of HIV-1 Secondly, noncoding RNAs (ncRNAs), such as may firstly interact with pDC, thereby stimulating INF- transfer RNA (tRNA), ribosomal RNA (rRNA), α production by TLR7, which later suppresses the microRNA (miRNA), small nuclear RNA (snRNA), or expression of CXCR4 and CCR5 [110–112]. long non-coding RNA (lncRNA), can be derived from Concurrently, TLR8 is related to the production of IL- both the virus and the host[130]. They play important 1β and release of Tumor Necrosis Factor α (TNF-α) by roles in host-virus interplay [131,132] and can be recognizing the ssRNA [113,114], whereas microRNAs manipulated by the invading viruses to establish produced by HIV-1 also serve as ligands for TLR8 a favorable host environment for its replication cycle. signaling[115]. In addition, HIV-1 can be perceived Virus-host RNA interactome has been explored and by DNA sensors like Cyclic GMP–AMP synthase a database resource has been created (ViRBase) to (cGAS) and interferon gamma inducible protein 16 integrate experimental and predicted ncRNA- (IFI16)[116]. More details about the sensing of HIV associated host–virus interactions [131,133]. Viral PAMPs are discussed elsewhere[117]. ncRNAs have a variety of biological effects that regulate Unfortunately, viruses have developed many strate­ the distinct steps of the viral life cycle [132,134,135]. gies to evade the host recognition. Firstly, viruses can Many examples are related to host recognition evasion: escape from the host RNA sensors by specially modify­ the miRNA from RSV, miR-26b, has been found to ing their RNA[118]. 2′-O-methylation refers to the suppress TLR4 and inhibition of miR-26 can increase methylation of RNA ribose at the 2′-OH position, the expression of CCL5 and IFN-β[136]. One lncRNA, which is common to all life kingdoms. In mammals, the eosinophil granule ontogeny transcript (EGOT), 2′-O-methylation of the 5′- guanosine cap by methyl­ induced by HCV is also involved in RIG-I and PKR transferases (MTases) is a molecular signature to dis­ pathway, and inhibition of EGOT can increase the tinguish “self” from “non-self” mRNA, a process where expression of IFN-stimulated genes (ISGs)[137]. On RIG-I and MDA5 can play a role [119–121]. Thus, 2′- the other hand, viral mRNAs frequently incorporate O-methylation is one of the commonest modifications Internal Ribosome Entry Sites (IRESs) sequences, adopted by viruses to mimic eukaryotic RNA by utiliz­ which can adopt specific folds to promote their efficient ing 2′-O-MTases either from themselves or from the translation. In addition, RNA viruses can also take host. For example, the SARS-CoV can encode its own profit from some host miRNAs, such as the miR-122, 2′-O-MTases(nsp16) and viral defective mutants are to facilitate their structuration and stabilize their gen­ more sensitive to IFN[122]. In its turn, HIV-1 can ome or enhance the RNA translation rate. Interestingly, take advantage of a cellular 2′-O-MTase, FTSJ3, to viruses can incorporate specific mutations in their gen­ achieve its 5′-cap methylation[123]. In addition, N6- ome to increase their stability, avoid recognition and methyladenosine (m6A) modification is another favor­ cleavage by host proteins, or facilitate their proper ite modification of viral RNA[124]. Indeed, m6A is the folding in the absence of miRNAs helpers[138]. most abundant modification in mammals and regulates Besides, viruses can adopt tRNA-like structures that the mRNA stability, transport, metabolism, and effi­ can intervene in their replication or translation steps cient translation. RNA viruses are observed to incorpo­ [139–141]. Together with tRNA mimicry, viruses can rate this modification early in their infection cycle, in initiate infection by taking profit from the host tRNAs. order to be properly recognized by their host machin­ The use of the host tRNA-Lys3 by HIV-1 for priming ery and facilitate translation. However, m6A mapping reverse transcription was identified a long time ago, methodologies have only recently been implemented although the particular details of the tRNA priming and epitranscriptomes are slowly been analyzed[125]. binding site (PBS) and viral genome interactions were Therefore, the importance of this modification in viral not elucidated till recently[142]. infectivity and immune response need further explor­ Besides, the viruses have also adapted strategies to ing. Among others, this modification has been charac­ evade directly the host immunity. They can produce terized during HIV-1 infection[126]. Likewise, m6A molecules to act as antagonists against IFN induction modification helps human metapneumovirus to evade [72,143]. We observe that most of the antagonists are from RIG-I [127] and may represent a key regulator of non-structural or accessory viral proteins. For example, the immune system[128]. Another modification that is the MHV lacking nsp15 is easily detected by dsDNA now recognized as a key regulator of both host and sensors (such as MDA5, PKR, and OAS/RNase L), 450 J. LI AND E. BOIX thereby stimulating more type I IFN induction. This antiviral proteins and non-self RNA ligand, activating result suggests that nsp15 may be an IFN antagonist of innate antiviral responses related to RLRs, PKR, or CoVs [52,144]. The non-structural proteins nsp1, nsp7, OAS/RNase L pathways, and IFN production. PLP, and the accessory proteins ORF3b, ORF6 of Alternatively, avSGs can inhibit the host cell translation SARS-CoV also have been reported as potential IFN machinery and prevent viral replication [163–166]. In antagonists[145]. Another antagonist molecule is the their turn, some viruses manage to spread their infec­ RSV non-structural protein 1 (NS1), which can bind tivity by preventing the formation of SGs, suggesting to mitochondrial antiviral signaling protein (MAVS) again immune protection of SGs [164,166]. and suppress the association of MAVS to RIG-I and Noteworthy, we find cases where the viruses can induce thereby undermine the IFN production[146]. SGs to serve themselves and even some possess both Regarding HIV-1, either interaction with cellular mole­ functions: SGs induction at early time points and SGs cules or dependence of accessory proteins, such as Vpu blockage at later stages[167]. For example, MHV- and Nef, can impair key signaling of PRRs including induced SGs lead to the shutdown of host translation RIG-I, cGAS, and IFI16[147]. It is interesting that the without affecting the production of viral proteins [156] core protein of HCV also can show IFN-antagonistic while the accessory protein 4a produced by MERS-CoV properties[148]. prevents the SGs formation and thereby promotes the Last but not the least, positive RNA viruses can viral replication[168]. It is also reported that HIV-1 hijack intracellular membranes to form unique DMVs Gag undermines both types of SGs assembly by inter­ in order to hide their RNA and avoid the innate anti­ acting with eEF2, eIF4E translation factors, and recruit­ viral responses[149]. The formation of DMV owes to ment of GTPase activating protein – SH3 domain the nsp3-4 polyproteins for MERS-CoV and another binding protein 1 (G3BP1) [169,170]. On the other nsp6 for SARS-CoV [150,151]. For HCV, it is indicated hand, the nucleocapsid of HIV-1 can induce SGs that several non-structural proteins as well as host assembly, a process that can be inhibited by Staufen1, factors are involved in the formation of DMVs[152]. a host protein related to mRNA transport and transla­ Interestingly, in the cells infected by the negative-sense tion [171,172]. Similarly, the role of SGs in RSV infec­ RNA virus RSV, we can find large cytoplasmic inclu­ tion is still controversial[173]. The RSV-induced SGs sion bodies that contain multifunctional proteins and can enhance RSV replication by mediating PKR [157] even viral RNA. It is suggested that the cytoplasmic while RSV suppresses the SGs assembly by sequestering bodies may also have a protection mechanism for the the phosphorylated p38 (p38-P) and O-linked N-acetyl replication and innate immunity evasion of negative- glucosamine (OGN) transferase (OGT) into viral inclu­ sense RNA viruses [153,154]. sion bodies[174]. In addition, RSV can also induce the specific production of tRNA-derived stress-induced RNAs (tiRNAs) and it has been found that the pro­ duced 5′-tiRNAGlu inhibits the expression of antiviral Formation of stress granules, autophagy and protein APOER2 [175,176]. Interestingly, the authors apoptosis observed that the tRNA cleavage products were Stress granules (SGs) are cytoplasmic aggregates of mediated by the endonuclease activity of angiogenin protein and RNA, which appear when the cell is (ANG; also named RNase 5). At the same time, it is under stress, including viral infections such as MHV noteworthy that ANG-induced tiRNAs are important and RSV [155–157]. There are two types of formation components for host stress response and SGs assembly mode for SGs according to whether it depends or not [177,178]. on the initiation factor eIF2α, classified as type I and II Autophagy is another cellular stress response path­ [158]. Here, we briefly introduce the best-studied pro­ way that can participate in the host-virus interplay. It is cess, which requires the participation of eIF2α (Figure a process of cell recycling, by which cells can eliminate 2C). Under cell stress by a viral infection, the PKR can damaged or diseased components and favor healthier trigger the phosphorylation of eIF2α subunit and sub­ cells. Host cells can identify and degrade virus intruders sequent increase of the affinity of eIF2B for eIF2:GDP, by a process called virophagy. Without surprise, autop­ thereby leading to the prevention of the triple complex hagy also promotes the clearance of SGs[179]. (eIF2:GTP:tRNAMet) formation and shutdown of trans­ Interestingly, the autophagy mechanism has dual func­ lation[159]. The SGs have either antiviral or proviral tions as observed for SGs. On one hand, the autopha­ effects depending on the different studied viruses gosomes formed can transfer viral cargos to lysosomes [160–162]. Antiviral SGs (avSGs) exert their specific for degradation and activate the host's innate immune. effects by providing a platform for interaction between On the other hand, the viruses also take advantage of VIRULENCE 451 autophagy to evade the immune system, support repli­ multiple biological functions of RNases, the antiviral cation, and exit the cell[180]. Many ssRNA viruses can activity has been reported [8,14,197]. Overall, the stra­ induce, suppress, or even take advantage of autophagy tegies exerted by RNases against viruses include: 1) by multiple mechanisms. It is reported that Nsp6 of inhibition of viral replication by its enzymatic activ­ CoVs restricts autophagosome expansion, but there are ity; 2) regulation of host immune recognition and no general rules, and CoVs can also manipulate the response; 3) regulation of SGs formation; 4) induction autophagy machinery for their benefit [181–183]. For of autophagy and 5) triggering of apoptosis. Here we instance, autophagy may be necessary for the formation describe the human RNases that show significant anti­ of DMVs, which will promote the efficiency of MHV viral activity together with other RNases of potential replication[184]. RSV-induced or HCV-induced autop­ therapeutic interest. In particular, we focus on the hagy also contributes to the viral replication and RNases that are active against the enveloped ssRNA thereby promotes infection [185,186]. Nevertheless, viruses. HIV requires autophagy for its early replication but has also developed many strategies to inhibit autophagy RNase A superfamily to avoid its clearance. Interestingly, pro- and anti-viral roles of autophagy are undergone associated with each The vertebrate-specific RNase A superfamily includes of the cell types in the study[87]. in human 13 members, named RNases 1–13, which are Last, when the cell injury caused during stress secreted proteins and have diverse roles, such as anti­ exceeds the capacity of the repair mechanisms, apop­ microbial and immunomodulatory [15,18,198–201]. tosis will occur to avoid excessive tissue damage. Expressed by innate immune cells and targeted to There are many reviews focused on viral infection either the extracellular or endolysosomal space, RNase and apoptosis[187]. For example, SARS-CoV induces A family members are well fit to provide a safeguard apoptosis either by exposure of its membrane pro­ action against intruding pathogens [14,202]. Within the teins or by its unique 7a protein [188,189] and family, we find several members with reported antiviral MERS-CoV can activate both the extrinsic and activity exerted by different mechanisms, depending on intrinsic apoptosis pathways in T cells[190]. the type of viruses (see Table 1) [8,14,203]. Moreover, RSV not only triggers apoptosis but can The Eosinophil-Derived Neurotoxin (EDN/RNase also escape or delay apoptosis by interfering in the 2) is one of the best studied, which can fight RSV, expression of several proteins[191]. In its turn, the PIV, and HIV [18,19]. Research about human recom­ gp120/gp41 of HIV-1 is a key component for induc­ binant EDN against RSV and PIV indicated that the tion of apoptosis in CD4 T-cell lymphocytes. In con­ ribonuclease activity of EDN is essential for the protein trast to other types of viruses, no evidence have been antiviral activity but is not unique. Indeed, bovine found about any mechanism of HIV to prevent apop­ pancreatic RNase A, the family reference, shows tosis[192]. Anyhow, apoptosis is a double-edged a much higher catalytic activity but is devoid of anti­ sword. Apoptosis caused by HCV infection may ser­ viral activity[19]. Antiviral RNases from human chor­ iously damage the liver while inhibition of apoptosis ionic gonadotropin (hCG) preparations that contain may lead to the persistence of HCV and subsequent EDN show anti-HIV activity, which may be related to development of hepatocellular carcinoma[193]. its inhibition of HIV replication in chronically infected Overall, it is important to have a good understand­ ACH-2 lymphocytes and U1 monocytes[204]. The pro­ ing of the interactions that take place between viruses tein contribution in anti-HIV activity was proven by and their host, in order to spot the most vulnerable selective blockage with anti-EDN antibodies[205]. points during viral infection and exploit adaptive Besides, the addition of recombinant EDN reduces methodologies to target each type of viruses infectivity in vitro in RSV-infected epithelial cells appropriately. [19,206]. Interestingly, EDN levels in serum are increased following RSV bronchiolitis and are used as a predictive marker of recurrent wheezing[207]. Mechanism of action of host defense RNases Another eosinophilic ribonuclease, the Eosinophil against viral infection Cationic Protein (ECP/RNase 3) has also anti-RSV Within RNases, we find endo- and exonucleases that activity, although to a much lower extent, and no catalyze the cleavage of either ssRNA or dsRNA and synergistic action with EDN was evidenced[208]. Very release selective cleavage products [194,195]. RNases recently, we have demonstrated the antiviral activity are found in all life kingdoms; they can work either against RSV of RNase 3 expressed in macrophages. within the cell cytosol or be secreted[196]. Among the Besides, a comparative transcriptome analysis indicates 452 Table 1. Anti-ssRNA viral mechanisms targeted by RNases. ssRNA Viruses RNases Proposed Mechanism (tested in vitro or in vivo)a Nameb ENVc Sense Refs [223] RNase A • Inhibits replication (in PHA-stimulated T cell blasts) HIV-1 Y + J. LI AND E. BOIX [204,223] RNase 2 /EDN • Inhibits replication (in PHA-stimulated T cell blasts) HIV-1 Y + • Degrades viral RNA (in total RNA from infected ACH2 cells) [19] • Ribonuclease-dependent activity (in RhMk cells) PIV Y - [19,212] • Ribonuclease-dependent activity (in HEp-2 cells) RSV-B Y - • A unique loop helps its interaction with viral capsid and penetration into the virion (in HEp-2 cells) [208,209] RNase 3 /ECP • Ribonuclease-dependent activity (in HEp-2 and THP-1 cells) RSV-B Y - [220,223] RNase 5 /ANG • Inhibits replication (in PHA-stimulated T cell blasts or PBMCs) HIV-1 Y + [224] BS-RNase • Inhibits replication (in H9 leukaemia cells) HIV-1 Y + [224,230,231] Onconase • Inhibits replication (in H9 and U937 leukaemia cells) HIV-1 Y + • Inhibits replication by removal of the transcription primer (tRNA synthesized in vitro) [268] RNase L • OAS/RNase L pathway is necessary for IFN-α but not for IFN- γ to inhibit viral replication (pancreatic islet from mouse) CV-B4 N + [20,247,269,270] • Inhibits viral RNA synthesis (in LS1 cells) EMCV N + • Increases the antiviral activity of IFN when overexpressed (in LS1 cells) • RNase L helps to amplify IFN-β signalling (in mouse) • Autophagy (in MEFs) • Apoptosis [244,271] • UA and UU dinucleotides in viral RNA is the main cleavage site of RNase L (HCV mRNA synthesized in vitro) HCV Y + • A small RNA cleaved by RNase L activates RIG-I and thereby promotes IFN-β response (in mouse) [245] • Inhibits HIV-1 replication by 8 times (in Jurkat cells) HIV-1 Y + [272] • RNase L cleaves viral RNAs in absence of viral resistance protein NS1 (in A549 cells) IAV Y - [246] • IFN-γ-mediated inhibition in human epithelial cells involves the OAS/RNase L pathway (in HEp-2 cells) RSV Y - [20,242,273] • RNase L helps to amplify IFN-β signalling (in mouse) SeV Y - • SGs formation (in HT1080 cells) • Autophagy (in MEFs) [274,275] • Contributes to IFN-α/β–mediated protection (in MEFs) SINV Y + • Interferes with the synthesis of minus strands of heat-resistant strain (in MEFs) [247] • Autophagy (in MEFs) VSV Y - [276,277] • Cleaves viral genomic RNA (in vitro) WNV Y + • Contributes to IFN-mediated protection (in CD11b+ cells) [278] Regnase/MCPIP1 • Inhibits replication (in hepatoma cells) HCV Y + • Degrades viral RNA (in vitro) [263] • Degrades viral RNA (in vitro) DENV Y + JEV Y + [264] RNase P • Cleaves HCV RNA transcripts (in vitro) HCV Y + [267] Binase • Inhibits replication (in MRC5 cells) MERS-CoV Y + [265,266] • Direct action on the viral mRNA (in A549 and MDCK-II cells) IAV Y - a A549: human lung adenocarcinoma epithelial cells; HEp-2: human laryngeal carcinoma/human type 2 epithelial cells; MDCK-II: Madin-Darby canine kidney epithelial cells; MEF: mouse embryonic fibroblasts; MRC5: human fetal lung fibroblasts; PBMC: peripheral blood mononuclear cells; PHA: phytohemagglutinin; RhMk: rhesus monkey kidney; THP-1: a human monocytic cell line. b CV-B4: Coxsackie virus B4; DENV: Dengue virus; EMCV: Encephalomyocarditis virus; HCV: Hepatitis C virus; HIV: Human Immunodeficiency virus; IAV: Influenza A virus; JEV: Japanese Encephalitis virus; MERS-CoV: Middle East Respiratory Syndrome Coronavirus; PIV: Human Parainfluenza virus; RSV: Respiratory Syncytial virus; SeV: Sendai virus; SINV: Sindbis virus; VSV: Vesicular Stomatitis virus; WNV: West Nile virus. c ENV: Enveloped; Y: Yes; N: No. VIRULENCE 453 that the protein antiviral properties are associated with with an elevated antiviral activity together with its catalytic activity[209]. On the other hand, increasing other appealing potential therapeutic applications levels of ECP are found during HIV infection in both [224]. The particular attraction has been drawn by adults and children [210,211]. The higher antiviral an RNase from oocytes of the leopard frog Rana activity of EDN/RNase2 with respect to ECP/RNase3 pipiens, discovered for its antitumor properties and has been attributed to the presence of a specific region named Onconase thereafter [227,228]. Onconase has in the former at the C-terminal loop. By site-directed also an unusual activity on dsRNA [229] and can mutagenesis, the authors have identified a region essen­ significantly inhibit HIV-1 replication in H9 leuke­ tial in EDN for the RSV capsid interaction and virion mia cells at nontoxicity concentration[224]. Further entry to the cell[212]. work confirmed the selective degradation of the There is no evidence that EDN/ECP has a direct virion RNA by Onconase in treated H9 and U937 effect on CoVs, but EDN is upregulated during SARS- leukemia cells with no alteration of ribosomal or CoV infection [213] and most recently, it has been assayed mRNA[230]. In addition, Onconase specific reported that eosinopenia is associated with SARS- activity on tRNAs has been associated with its abil­ CoV-2 infection [214,215]. In the same line, increasing ity to inhibit HIV replication by removal of levels of eosinophils are associated with a better prog­ tRNALys, which is known to serve as a reverse tran­ nosis of recovery from COVID-19 and EDN has been scription primer by the viruses[231]. Recently, proposed as a novel clinical biomarker of the disease Vilanova and coworkers reported that Onconase [216–218]. Moreover, it is observed that the antiviral antiviral action can also be mediated by the upre­ activity of eosinophils can be reversed by the addition gulation of the activation transcription factor 3 of the proteinaceous RNase inhibitor (RI) [19,208]. (ATF3) which promotes apoptosis and can inhibit Apart from the eosinophil-derived proteins, angiogenin the viral replication[232]. Besides, ATF3 is also (ANG/RNase 5) may play a role during viral infection reported to induce the latent state in herpes simplex [219]. ANG can suppress the replication of HIV-1 in virus (HSV)[233]. The authors also suggest that a dose-dependent manner[220]. Furthermore, when Onconase inhibition of HIV replication might be infected by RSV, the cell production of tRNA halves mediated by the induction of IL10[232]. The advan­ induced by ANG is significantly enhanced. It is now tages of the clinical development of Onconase are known that during cell stress conditions tRNA halves that the protein not only avoids the inhibition by RI promote the assembly of SGs; however, the potential con­ but is also resistant to proteolysis due to its unusual tribution of ANG in the control of infection is still unclear conformational stability[228]. Work is currently in [175]. Very recent transcriptome analysis associates epige­ progress to exploit Onconase alone or as an adju­ netic traits with the stress-induced tRNA fragment popula­ vant to fight viral infections including MERS-CoV tion and cell potential biological activities[221]. and RSV (http://tamirbio.com/) [232]. Interestingly, a posttranscriptional modification m5C, con­ Overall, the induction of the apoptosis or autop­ sidered a protection cleavage mark in higher eukaryotes, hagy pathways by several family members could be inhibits ANG action on tRNAs[222]. Another research has regarded as a strategy to facilitate the removal of also tested the anti-HIV activity of EDN, ANG, and even pathogen-infected cells [15,234–237]. A curious and RNase A, all of which inhibit the HIV-1 replication during original zymogen has been engineered by Raines primary HIV-1-infected PHA-stimulated PBMCs[223]. and coworkers, where the use of the HIV protease Another peculiar member of the RNase serves as an activation system to release the RNaseA A superfamily is the bovine seminal RNase (BS- catalytic activity that will degrade the viral gen­ RNase). It is the unique natural dimeric protein of ome[238]. the family and shows a specific enhanced activity against dsRNA. Interestingly, BS-RNase is reported to have anti-HIV activity on H9 leukemia RNase L cells[224]. In addition, dsRNA cleavage by BS- RNase L is a ubiquitous intracellular endonuclease acti­ RNase is induced by IFN-γ[225]. Moreover, direct vated by a 2′5′-oligoadenylates (2–5A), which is speci­ binding of the C-terminus of IFN with RNA is fically synthesized by the OAS. The OAS/RNase involved in the activation of the RNase antiviral L pathway is activated during viral infections and is activity[226]. regulated by IFN[239]. Briefly, when exposed to the Last, within the lower-order vertebrates of the dsRNA produced by viruses, the host cells will induce RNaseA superfamily, we find amphibian RNases IFN and then secrete 2–5A to induce the activation of 454 J. LI AND E. BOIX RNase L. Activated RNase L cleaves viral ssRNA, (L*) produced by Theiler’s murine encephalomyelitis thereby suppressing viral replication, protein synthesis, virus can bind to RNase L and prevent its activation and spread. In addition, RNase L can further amplify by 2–5A by an antagonism stratagem similar to the one IFN signaling and promote the formation of SGs, which described for MHV[255]. A wealth of literature has include many antiviral proteins [240–242]. Thereby, emerged during the last months on host-virus interplay RNase L provides a natural cell protection against mechanisms due to COVID-19 pandemic. Latest popu­ viral infection (Figure 2b). lation studies on human genetic variants related to Many kinds of viruses such as enveloped ssRNA severe COVID-19 illness have identified the deficiency viruses, the object of this study, can be inhibited in the OAS/RNase L antiviral system among the top directly or indirectly by RNase L (Table 1) [239,243]. critical phenotype markers [256,257]. Activation of RNase L then generates both host and viral RNA cleavage products, which on their turn can RNase T2 activate RIG-I and stimulate expression of IFN-β dur­ ing HCV infection [20,244]. Interestingly, HIV-1 repli­ RNase T2 family is present not only in eukaryotes but cation can be directly suppressed by overexpression of also in bacteria and viruses, with a variety of func­ RNase L, even without IFN treatment[245]. IFN-γ has tions, such as degradation of RNAs, regulation of the been found to inhibit RSV infection in human epithe­ immune response, or even control of tumor progres­ lial cells, but cells that overexpress the RNase sion [258,259]. RNase T2 activity has also been linked L inhibitor (RLI) attenuate this antiviral effect, suggest­ to the host antiviral response. One study demonstrated ing that activated RNase L is essential for IFN-γ- that RNase T2-deficiency resembles congenital cyto­ mediated anti-RSV activity[246]. RNase L can also pro­ megalovirus (CMV) infection. Strikingly, CMV infec­ tect the brain from sustained MHV infection and tion can block as an evasion strategy the antiviral thereby prevent demyelination and neurodegeneration RNase L response, which results in increased ssRNA [21]. SGs and autophagy can also be induced by RNase levels, associated with exacerbated innate immunity L in both (+)ssRNA and (-)ssRNA viral infections activation and eventually similar neuropathological [242,247]. Alternatively, RNase L activation in virus- consequences[260]. This suggests that RNase T2 infected cells can lead to cleavage of both host and viral might play a similar role as RNase L in cellular RNA and facilitate the activation of apoptosis and sub­ immune response[261]. Recent research also shows sequent removal of infected cells[248]. that RNase T2, together with EDN, can release uridine Unfortunately, viruses have developed on their turn nucleotides from pathogen RNAs, which in their turn many mechanisms to evade the OAS/RNase L system. are activators of TLR8 [202,262]. Interestingly, the Specific 2′,5′-phosphodiesterases that cleave 2–5A are enrichment in uridine products is achieved by the released by CoVs to prevent RNase L activation: ns4b complementary cleavage preferences of the two endor­ from MERS-CoV and ns2 from MHV[249]. Deletion of ibonucleases: RNase T2 at XU and EDN/RNase2 at the ns4b gene can activate RNase L during infection of UX, where X is a purine. To note, both secretory Calu-3 lung cells and also the ns2-deletion mutant virus RNases work at endolysosomal compartments, with cannot replicate in wild-type mice but is highly patho­ an optimum activity at a pH between 4.5 and 5.5, genic in RNase L deficient mice [250,251]. Likewise, the and might provide an indirect mechanism to activate Tat protein of HIV-1 binds to the 2–5A synthetase, the immune system in the presence of pathogenic undermines the TAR-mediated activation of 2–5A RNAs. In particular, the authors have analyzed the synthesis, and thus blocks OAS signaling[252]. In addi­ RNases cleavage pattern on the genome of selected tion, RLI can be induced during HIV-1 infection to RNA viruses, some of which show an abundance of inhibit the OAS/RNase L pathway[253]. A better uridine-rich sequences. understanding of the strategies of viruses to modulate the OAS/RNase L immune response pathways should Regnase1 and RNase P provide guidance for the development of novel drugs [239,241]. In fact, avoiding the recognition of the OAS/ MCPIP1 (also designated as Regnase1), is a human RNase L system is not only the privilege of the popular protein with both antiviral and endoribonuclease activ­ ssRNA viruses introduced above, some other ssRNA ities. It is a zinc finger protein involved in the cell viruses have also been found to engineer similar stra­ inflammatory response that regulates the half-life of tegies. For example, a unique RNA structure carried by mRNA and miRNA. The nuclease domain cleaves poliovirus, a (+)ssRNA virus, helps to inhibit the endo­ ssRNA and shows a high affinity for viral RNA [263] nuclease activity of RNase L[254]. A specific protein and a broad-spectrum antiviral action. Likewise, VIRULENCE 455 another a human endonuclease, human RNase P, is RNA target molecules, RNA binding proteins, and found to cleave HCV RNA transcripts[264]. RNA cleavage enzymes. In summary, we can conclude that host-derived RNases can fight multiple viruses through direct or Other RNases indirect actions, which not only remove viral RNAs Microbial RNases have also been proposed as thera­ but also regulate self-immunity. In addition, host cells peutic agents against ssRNA viruses (Table 1). Ilinskaya have evolved inhibitors to specifically protect subcellu­ and coworkers have extensively explored the potential lar compartments and prevent the potential toxic action of the RNase from Bacillus pumilus, also named Binase, of their own RNases against cellular RNAs. Another on influenza A virus. The authors observed a direct advantage of RNases is that once expressed they can be action on the viral mRNA in both in vitro and in vivo secreted, thereby exerting their antiviral activity either models [265,266]. Binase was also effectively tested at intracellular or extracellular level. This dual function against CoVs (MERS-CoV and the human low patho­ is of great benefit to improve antiviral effects at the genic CoV-229E strain)[267]. Other microbial RNases local site of infection and to reduce the potential side are extensively reviewed by Ilinskaya and Mahmud[8]. effects on the remaining healthy tissues. From Last, a very promising emerging discipline in the a perspective focused on applied therapies, some nano­ field is based on the design of artificial RNases delivery systems have been engineered to vehicle the (aRNases) for targeted RNA cleavage. Chemical con­ RNases within host cells [284–286]. We are confident jugates were first synthesized containing RNA binding that the design of novel vehicle tools should greatly and catalytic domains, although their main drawback expand the applicability of RNases with high catalytic was their poor catalytic efficiency[8]. Small molecule efficiency on RNA viral genomes but limited cleavage derived RNases can also be engineered by connecting specificity on target sequences. short mono to tripeptides[279]. The aRNases active site includes imidazole groups and perform an equivalent Current antiviral drugs design and acid-base catalysis to native enzymes. Although their development efficiency is significantly lower than natural RNases, they are active in physiological conditions and were It has been almost 60 years since the approval of the demonstrated effective against influenza A and H1N1 first antiviral drug, idoxuridine. During this period, viruses. Last but not least, we can engineer antiviral thousands of antiviral compounds were proposed, but ribozymes, RNA based-drugs with specific RNA target­ only about 100 antiviral drugs were approved for the ing and endowed with intrinsic endonucleolytic action, treatment of major human infectious diseases[287]. The some of which are in clinical trials[280]. action of antiviral drugs may include inhibition of viral Notwithstanding, to design effective antiviral drugs attachment, penetration, and uncoating, prevention of we should also consider other contributing factors, viral replication or protein synthesis, blockage of viral such as the three-dimensional structuration of RNA post-assembly and activation of innate immunity molecules, the presence of specific posttranscriptional [288,289]. Among the many antiviral drugs, some can modifications, or the involvement of RNA binding be used or even exclusively dedicated to ssRNA viral proteins that might protect the viral genome from infection. Therefore, in this part, we will briefly intro­ RNases cleavage. Fortunately, novel methodologies, duce the main mechanisms for drugs that target envel­ such as eCLIP are now providing tools to easily identify oped ssRNA viral infections (summarized in Table 2). protein-RNA binding regions[281]. Some viruses can We will also take the opportunity to prospect the stra­ stabilize and protect their genomic RNA by interaction tegies that can be applied to develop novel drugs with the host-specific miRNAs. For example, miR122 against ssRNA viruses. can modify the viral RNA base pairing and increase its half-life, promoting translation and protection of HCV Available anti-viral agents RNA [138,282]. The emergence of resistant virus strains to miR-122 inhibitors with enhanced genome By referring to Virus Pathogen Database and Analysis stability and protection against host RNases has Resource (ViPR) (https://www.viprbrc.org/brc/), it is recently been reported[283]. Therefore, the ability of shown that more than half of approved antiviral drugs host RNases to target viral RNA might be hindered by are against HIV or HCV infection, suggesting that the presence of a diversity of ncRNAs. To obtain antiviral drugs developed for human chronic viral- a more realistic scenario of the host-virus interplay we related diseases have received more attention and posi­ should bring together all the intervening agents at once, tive results. Higher percentages of approval against 456 J. LI AND E. BOIX Table 2. Main available drugs or candidates against enveloped ssRNA viruses. Virus Antiviral Mechanisms Status Representative Drugsd (+) ssRNA SARS -CoV- • Entry inhibitor Investigational • Leronlimab virus 2 • RNA polymerase inhibitors Investigational /conditionally • Favipiravir[323] • Remdesivir approved • GS-441524 • Inosine-5-monophosphate dehydrogenase (IMPDH) Investigational • Merimepodib[324] Inhibitor HCV • NS3/4A protease inhibitors Approved • Asunaprevir • Paritaprevir • Boceprevir • Simeprevir • Glecaprevir • Telaprevir • Grazoprevir • Voxilaprevir Investigational • Faldaprevir • Vedroprevir • NS5B polymerase inhibitors Approved • Dasabuvir • Sofosbuvir Investigational • Beclabuvir • Uprifosbuvir • NS5A inhibitors Approved • Elbasvir • Ombitasvir • Daclatasvir • Pibrentasvir • Ledipasvir • Velpatasvir Investigational • Odalasvir Ruzasvir • Ravidasvir • Interferon α/β receptor agonists Approved • Peginterferon α2a • Peginterferon α2b • Broad-spectrum activity Approved • Ribavirin HIV • Nucleoside and nucleotide reverse transcriptase Approved • Abacavir • Stavudine inhibitors (NRTI) • Didanosine • Tenofovir • Emtricitabine disoproxil • Lamivudine • Zalcitabine • Zidovudine Investigational • Elvucitabine • Racivir • Non-nucleoside reverse transcriptase inhibitors Approved • Delavirdine • Etravirine (NNRTI) • Doravirine • Nevirapine • Efavirenz • Rilpivirine Investigational • Atevirdine • Calanolide A • UC-781 • Protease inhibitors Approved • Amprenavir • Lopinavir • Atazanavir • Nelfinavir • Darunavir • Ritonavir • Fosamprenavir • Saquinavir • Indinavir • Tipranavir Investigational • TMC-310911 • Integrase inhibitors Approved • Bictegravir • Elvitegravir • Dolutegravir • Raltegravir • Fusion inhibitor Approved • Enfuvirtide • Entry inhibitors Approved • Maraviroc • Ibalizumab Investigational • Cenicriviroc • Leronlimab • Others Investigational • BMS-488043 • Lobucavir • Dexelvucitabine • Sorivudine • Fiacitabine (-) ssRNA RSV • Fusion inhibitor Approved • Palivizumab virus • Broad-spectrum activity Approved • Ribavirin Influenza • CAP endonuclease inhibitor Approved • Baloxavir marboxil virus • Neuraminidase inhibitors Approved • Oseltamivir • Zanamivir • Peramivir Investigational • Laninamivir • RNA synthesis inhibitors Approved • Rimantadine Experimental • Triazavirin • Fusion inhibitor Investigational • Umifenovir d Summary from DRUGBANK, category of Antiviral Agents (https://www.drugbank.ca/categories/DBCAT000066) when no other reference is provided.341 HIV among antiviral drugs can be confirmed in focus on directly targeting viruses, such as inhibitors another database: http://www.virusface.com/Drug/ of viral polymerases and proteases, or regulating cellu­ AntiviralDrug_Compound.html. No drugs can cure lar processes essential for viral replication[290]. For HIV and HCV infections completely, but they play example, most approved anti-HIV drugs are reverse a key role in extending the patient’s lifespan and transcriptase inhibitors (RTIs) which are divided into improving the quality of life. nucleoside and nucleotide RTIs (NRTIs) and non- Even though the concepts for the design of antiviral nucleoside RTIs (NNRTIs) [291,292]. Subsequently, drugs are constantly evolving, the central rules still protease inhibitors also account for a large proportion VIRULENCE 457 of antiviral agents[293]. In addition, we also find drugs and media approval [305,306]. In spite of good activ­ acting as integrase inhibitors (Elvitegravir, Raltegravir, ity against SARS-CoV in vitro[307], the reports of the etc.) to block the integration of viral genetic material high risk of chloroquine and hydroxychloroquine to into human chromosomes[294], fusion inhibitor the human cardiovascular system should not be (Enfuvirtide) to prevent viral entry [295] and entry ignored[308]. One review has summarized the treat­ inhibitor (Maraviroc) to antagonize the interaction ment strategies against SARS-CoV and MERS-CoV between HIV-1 gp120 and CCR5[296]. Similarly, most but most are drug combination[309]. Due to the approved anti-HCV drugs target specific HCV non- current COVID-19 pandemic, research in the field structural proteins (NS), including inhibitors against advances very rapidly and many novel strategies are NS3/4A protease, NS5B polymerase, and NS5A protein waiting for final approval. [59,297]. However, the available drugs to treat viruses that Discovery of antiviral active compounds lead to severe respiratory symptoms are very limited. It is demonstrated that the broad-spectrum anti-HCV According to the above discussed, we observe that drug, Ribavirin, which blocks nucleic acid synthesis, most developed antiviral compounds are nucleoside can be used to treat RSV[298]. Besides, the approval or nucleotide analogues, which mimic the natural of Palivizumab, a monoclonal antibody acting as nucleosides[310]. One of the main reasons why a fusion inhibitor against RSV, opens another path for these analogues are so effective is their ability to the development of alternative anti-RSV drugs based target viral replication. For some analogues, it is on the targeting of the F protein that mediates the their cleavage products, released as mono-, di- or virus-host fusion process. Meanwhile, other strategies triphosphorylated nucleosides that act as the active are considered, such as targeting RNA polymerases or compound[311]. For example, triphosphorylated nucleocapsid mRNAs through nucleoside analogues or forms of nucleoside or nucleotide analogues are com­ small-interfering RNAs (siRNAs) respectively to inhibit petitive RTIs and nearly all NRTIs are derivatives of viral replication. However, although infants are the sugar scaffold of natural nucleosides, which will most vulnerable group of risk for RSV disease, most cause polymerase chain termination due to the 3′- of the anti-RSV candidates that are under clinical trials OH modification[312]. Nucleoside (or nucleotide) have only been studied in adults [299,300]. analogues, including Sofosbuvir and Remdesivir, Until now, there are no approved drugs specifi­ also show activity against RdRp, indicating that cally designed against CoVs, but scientists are cur­ these analogues may work as effective inhibitors rently committed to develop anti-CoV therapeutic against CoV polymerases and can be candidates agents. One representative drug under investigation against COVID-19 [313,314]. is Remdesivir, a nucleoside analogue as well as Also, computational structure-based approaches a prodrug of GS-441,524 which has shown high have been of great help to select active compounds activity against various CoVs such as SARS-CoV, against viral protein or key cell receptors, which are MERS-CoV, MHV, and even SARS-CoV-2 either widely used to design a new generation of antiviral in vitro, in vivo, or in humans[301]. The mechanism drugs [315,316]. The programs can calculate the of Remdesivir is ascribed to the inhibition of the viral degree of interaction between a list of compounds RNA polymerase RdRp[302]. The nucleoside drug is from large databases and its target in three- also designed to block the error-proof exoribonu­ dimensional models and then the top-ranked com­ clease and reduce at most the capacity of viruses to pounds are chosen for further testing in vitro or acquire resistance, a property that put forward its in vivo to confirm its predicted activity[317]. We superiority to other agents[303]. Following recent find many successful examples of lead compounds trials at the National Institutes of Health, in the development of anti-ssRNA viral drugs[318]. Remdesivir, has recently obtained conditional Most recently, after screening 61 molecules with anti­ approval for the treatment of severe COVID-19 hos­ viral activity by molecular docking studies, it was pitalized patients in the EU and US[304]. On the found that all tested HIV protease inhibitors, other hand, traditional antimalarial drugs, chloro­ Lopinavir, Asunaprevir, Indinavir, and Ritonavir, quine and hydroxychloroquine, have also been pro­ showed significant binding interactions with posed to treat COVID-19, with controversial results COVID-19 enzymes[319]. Clinical trials also indicate following clinical trials, uncontrolled cases, or public that following administration of Lopinavir/Ritonavir, 458 J. LI AND E. BOIX or both, to COVID-19 patients, lower viral loads and virus itself to avoid toxicity and the emergence of better clinical symptoms are achieved[320]. However, resistance to antiviral drugs. As discussed before, an the real role of Lopinavir/Ritonavir in anti-COVID exhaustive knowledge of the interaction between the -19 treatment is still controversial[321]. Table 2 pro­ virus and the PRR signaling pathway can assist in the vides an overall summary of the currently available design of multiple antiviral therapies. Among them, we drugs, either approved or in clinical trials, to treat find the use of PRR agonists as adjuvants, drugs that viral infection by ssRNA enveloped viruses. target crucial host signaling or viral immunosuppres­ sive proteins[334]. For example, administration of modified interferons, like Peginterferon (PEG-IFN) Current perspectives alfa-2a and 2b, have been developed to stimulate an Unfortunately, it is still difficult to find drugs that innate immune response in combination with Ribavirin interfere with viral replication without damaging host and tested in clinical trials with thousands of HCV cells. The use of nucleoside or nucleotide analogues is patients[335]. Many Toll-like receptor agonists that of a potential danger due to the risk of intake by human activate the production of type I IFN are currently polymerases and incorporation into host RNA or DNA. under development[336]. A typical example is a mitochondrial toxicity in which Likewise, host defense proteins/peptides endowed nucleoside analogues interfere with mitochondrial with either direct antiviral activity, immune regulation DNA replication and thereby lead to reduced function function, or both, are promising therapeutic drugs. of mitochondria[322]. It is also observed that liver, Although the number of reported antiviral peptides kidney, or other tissue injuries are associated with is still very low, many natural and synthetic peptides antiviral therapies by using different types of drugs have shown effectivity against diverse RNA viruses [325–327]. Moreover, misfortune never comes alone; [337,338]. Among host defense proteins from nature, resistance to traditional antiviral drugs, either anti- RNases are both involved in the host defense immu­ HIV or anti-HCV, is continuously emerging nity system and display anti-infective activity [15,200]. [312,328,329]. WHO already warned in 2017 that The multifaceted properties of antimicrobial RNases, more than 10% of the patients receiving antiretroviral which combine among others, activation of the therapy have a strain that is resistant to some widely immune system, direct killing action, and the ability used anti-HIV drugs. Similarly, HCV mutations resis­ to inhibit the development of drug resistance encou­ tance to direct antiviral drug treatments has already rage further research in this field[339]. Besides, novel been well demonstrated[330]. methodologies such as the use of probiotics are devel­ A combination of antiviral drugs has been recom­ oped to offer a cheaper affordable high-scale produc­ mended to prevent the emergence of resistance. tion and overcome the present main drawback of Complementarily, combination therapies can reduce antimicrobial proteins pointed out by pharmaceutical the required amount of each drug to alleviate adverse companies[340]. In this review, we have described effects. On the other hand, drugs with multiple some representative RNases with demonstrated anti­ mechanisms can target different stages of the viral life viral activity. We find references of RNases that can cycle. Thus, if the virus develops resistance to one drug, directly block viral replication by their ribonuclease the others can still exert their antiviral effect. Also, activity or play a role in host PRR signaling pathways fixed-dose anti-HIV combinations are available on the or cell stress responses. We also pointed out the market that provide convenience for patients by taking advantages of host defense RNases in contrast to one pill a day[331]. Distinct combinations have been other drug candidates as effective antiviral agents. already under investigation against viral infections to Notwithstanding, protein-based drugs are encounter­ treat specific patients or to apply in case single drug ing difficulties in entering into the pharmaceutical treatments result is ineffective [329,332,333]. Particular market due to their poor bioavailability and high interest is provided by the combination of IFN with associated manufacturing costs. Currently, therapeutic antiviral drugs, such as Remdesivir, against CoVs[332]. proteins are well introduced in the treatment of The results are controversial and the treatment out­ chronic diseases in most developed countries, in con­ come is mostly dependent on the in vivo model, admin­ trast to infectious diseases that still remain non- istration route, disease stage, or IFN type. attractive to investors. Hopefully, the present sanitary Another approach that is gaining popularity is the emergency will soon decant the balance toward targeting of host signaling pathway rather than the a globally focused healthcare policy. VIRULENCE 459 Conclusions [3] World Health Organization. Summary of probable SARS cases with onset of illness from 1 November Among the diversity of existing viruses, ssRNA viruses play 2002 to 31 July 2003. WHO 2015. Available at: an important role in threatening human health. Enveloped https://www.who.int/csr/sars/country/table2004_04_ 21/en/. Accessed May 3, 2020 ssRNA viruses include not only HIV and HCV, which [4] World Health Organization. Middle East Respiratory cause chronic infections and do not have effective vaccines, Syndrome Coronavirus (MERS-CoV). WHO 2019. but also CoVs and RSV that induce acute respiratory Available at: https://www.who.int/emergencies/mers- symptoms and mostly threaten aged and infant population cov/en/. Accessed May 3, 2020. groups, respectively. There are many antiviral drugs avail­ [5] World Health Organization. Coronavirus disease (COVID-19) Situation Report-182. WHO Report, July able, but it is still challenging to design safe and effective 2020. antiviral drugs. One of the main difficulties encountered [6] Palacios Cruz M, Santos E, Velázquez Cervantes MA, lies in the fact that viruses use host cells to replicate them­ et al. COVID-19, a worldwide public health emer­ selves. Therefore, nowadays all research efforts of the phar­ gency. Rev Cln Esp. 2021; 221(1): 55–61. maceutical industry are joint to seek new antiviral targets [7] De Clercq E. Three decades of antiviral drugs. Nat Rev and new types of antiviral drugs. When infected by a virus, Drug Discov. 2007;6(12):941. the host cells will activate many signaling pathways and [8] Ilinskaya ON, Shah Mahmud R. Ribonucleases as anti­ viral agents. Mol Biol. 2014;48(5):615–623. secrete a variety of active factors to combat the foreign [9] Ferguson R, Subramanian V. The cellular uptake of invader. Therefore, host-derived components such as angiogenin, an angiogenic and neurotrophic factor is RNases may be ideal candidates for the design of new through multiple pathways and largely dynamin antiviral drugs with unique properties. The host secretory independent. PLoS One. 2018;13(2):e0193302. proteins are nontoxic and can exert their action both [10] Li S, Sheng J, Hu JK, et al. Ribonuclease 4 protects within cells and in the extracellular space to prevent viral neuron degeneration by promoting angiogenesis, neu­ replication as well as regulate innate immunity. Although rogenesis, and neuronal survival under stress NIH Public Access. Angiogenesis. 2013;16(2):387–404. recent successes for HCV and HIV suggest that direct- [11] Rademacher F, Dreyer S, Kopfnagel V, et al. The anti­ acting antiviral small molecules remain the gold standard microbial and immunomodulatory function of RNase 7 for antiviral drugs development, we cannot disregard the in skin. Front Immunol. 2019;10:2553. great potential of antimicrobial proteins and peptides with [12] Rudolph B, Podschun R, Sahly H, et al. Identification specific biological activities, such as RNases, as novel lead of RNase 8 as a novel human antimicrobial protein. candidates to design antiviral drugs. Antimicrob Agents Chemother. 2006;50(9):3194–3196. [13] Torrent M, Badia M, Moussaoui M, et al. Comparison of human RNase 3 and RNase 7 bactericidal action at the Gram-negative and Gram-positive bacterial cell Disclosure statement wall. FEBS J. 2010;277(7):1713–1725. The authors declare no conflict of interest. [14] Rosenberg HF, Domachowske JB. Eosinophils, eosino­ phil ribonucleases, and their role in host defense against respiratory virus pathogens. J Leukoc Biol. Funding 2001;70(5):691–698. [15] Lu L, Li J, Moussaoui M, et al. Immune modulation by This work was supported by Fundació La Marató de TV3 (ref. human secreted RNases at the extracellular space. 20180310) and Ministerio de Ciencia e Innovación, Spain, Front Immunol. 2018;9:1012. Grant (PID2019-106123GB-I00) to EB. JL is a China [16] Boix E, Acquati F, Leonidas D, et al. Editorial: role of Scholarship Council (CSC) predoctoral fellow. ribonucleases in immune response regulation during infection and cancer. Front Immunol. 2020;11:236. [17] Salganik RI, Batalina TA, Berdichevskaia LS, et al. ORCID Inhibition of RNA synthesis and reproduction of tick-encephalitis virus under the influence of Ester Boix http://orcid.org/0000-0003-1790-2142 ribonuclease. Dokl Akad Nauk SSSR. 1968;180 (6):1473–1475. [18] Rosenberg HF. Eosinophil-derived neurotoxin (EDN/ References RNase 2) and the mouse eosinophil-associated RNases (mEars): expanding roles in promoting host defense. [1] Oldstone MBA. History of Virology. In: Schmidt TM, Int J Mol Sci. 2015;16(7):15442–15455. editor. Encyclopedia of Microbiology (Fourth Edition). [19] Domachowske JB, Dyer KD, Bonville CA, et al. Academic Press; 2014. p. 608–612. Recombinant human eosinophil-derived neurotoxin/ [2] Hulo C, De Castro E, Masson P, et al. ViralZone: A RNase 2 functions as an effective antiviral agent against knowledge resource to understand virus diversity. respiratory syncytial virus. J Infect Dis. 1998;177 Nucleic Acids Res. 2011; 39: D576-D582. (6):1458–1464. 460 J. LI AND E. BOIX [20] Malathi K, Dong B, Gale M, et al. Small self-RNA [40] Lu G, Hu Y, Wang Q, et al. Molecular basis of binding generated by RNase L amplifies antiviral innate between novel human coronavirus MERS-CoV and its immunity. Nature. 2007;448(7155):816–819. receptor CD26. Nature. 2013;500(7461):227–231. [21] Ireland DDC, Stohlman SA, Hinton DR, et al. RNase [41] Li F. Structure, function, and evolution of coronavirus L mediated protection from virus induced spike proteins. Annu Rev Virol. 2016;3(1):237–261. demyelination. PLoS Pathog. 2009;5(10):e1000602. [42] Gallagher TM, Buchmeier MJ. Coronavirus spike pro­ [22] Lodish H, Berk A, Zipursky SL, et al. Viruses: struc­ teins in viral entry and pathogenesis. Virology. ture, Function, and Uses. In: Molecular cell biology. 2001;279(2):371–374. 4th edition. New York: WH Freeman; 2000. [43] Wan Y, Shang J, Sun S, et al. Molecular mechanism for [23] Gelderblom HR. Structure and Classification of antibody-dependent enhancement of coronavirus Viruses. Baron S, editor. Medical microbiology. 4th entry. J Virol. 2020;94(5):e02015–19. ed. Galveston: University of Texas Medical Branch at [44] Blanchard E, Belouzard S, Goueslain L, et al. Hepatitis Galveston; 1996. C virus entry depends on clathrin-mediated [24] Lucas W. Viral Capsids and Envelopes: structure and endocytosis. J Virol. 2006;80(14):6964–6972. Function. In: Encyclopedia of Life Sciences (ELS). [45] Douam F, Lavillette D, Cosset FL. The mechanism of Chichester: John Wiley & Sons, Ltd; 2010. HCV entry into host cells. In: Klasse PJ, editor. [25] Barocchi MA, Masignani V, Rappuoli R. Cell entry Progress in molecular biology and translational science. machines: A common theme in nature? Nat Rev Vol. 129. Academic Press; 2015. p. 63–107. Microbiol. 2005;3(4):349–358. [46] Helle F, Dubuisson J. Hepatitis C virus entry into host [26] Plemper RK. Cell entry of enveloped viruses. Curr cells. Cell Mol Life Sci. 2008;65(1):100–112. Opin Virol. 2011;1(2):92–100. [47] Ahlquist P, Noueiry AO, Lee W-M, et al. Host factors [27] Chou T. Stochastic entry of enveloped viruses: fusion in positive-strand RNA virus genome replication. versus endocytosis. Biophys J. 2007;93(4):1116–1123. J Virol. 2003;77(15):8181–8186. [28] Harrison SC. Viral membrane fusion. Virology. [48] Enjuanes L, Almazán F, Sola I, et al. Biochemical 2015;479-480:498–507. aspects of coronavirus replication and virus-host [29] White JM, Whittaker GR. Fusion of enveloped viruses interaction. Annu Rev Microbiol. 2006;60:211–230. in endosomes. Traffic. 2016;17(6):593–614. [49] Blanchard E, Roingeard P. Virus-induced [30] Philipson L. Replication. Transcription and Translation of double-membrane vesicles. Cell Microbiol. 2015;17 RNA Viruses. In: Lycke E, Norrby E, editors. Textbook of (1):45–50. Medical Virology. Butterworth-Heinemann; 1983. p. [50] Knoops K, Kikkert M, Van Den Worm SH, et al. 57–71. SARS-coronavirus replication is supported by [31] Fehr AR, Perlman S. Coronaviruses: an overview of a reticulovesicular network of modified endoplasmic their replication and pathogenesis. In: Maier H, reticulum. PLoS Biol. 2008;6(9):e226. Bickerton E, Britton P, editors. Coronaviruses. meth­ [51] Den Boon JA, Diaz A, Ahlquist P. Cytoplasmic viral repli­ ods in molecular biology. Vol. 1282. New York, NY: cation complexes. Cell Host Microbe. 2010;8(1):77–85. Humana Press; 2015. p. 1–23. [52] Deng X, Hackbart M, Mettelman RC, et al. [32] Catanese MT, Uryu K, Kopp M, et al. Ultrastructural Coronavirus nonstructural protein 15 mediates evasion analysis of hepatitis C virus particles. Proc Natl Acad of dsRNA sensors and limits apoptosis in Sci U S A. 2013;110(23):9505–9510. macrophages. Proc Natl Acad Sci U S A. 2017;114 [33] Petrosillo N, Viceconte G, Ergonul O, et al. COVID-19, (21):E4251–E4260. SARS and MERS: are they closely related? Clin [53] Hackbart M, Deng X, Baker SC. Coronavirus endor­ Microbiol Infect. 2020;26(6):729–734. ibonuclease targets viral polyuridine sequences to [34] Borgia SM, Hedskog C, Parhy B, et al. Identification of evade activating host sensors. Proc Natl Acad Sci a novel hepatitis C virus genotype from Punjab, India: U S A. 2020;117(14):8094–8103. expanding classification of hepatitis C virus into 8 [54] Chen Y, Cai H, Pan J, et al. Functional screen reveals genotypes. J Infect Dis. 2018;218(11):1722–1729. SARS coronavirus nonstructural protein nsp14 as [35] Belouzard S, Millet JK, Licitra BN, et al. Mechanisms of a novel cap N7 methyltransferase. Proc Natl Acad Sci coronavirus cell entry mediated by the viral spike U S A. 2009;106(9):3484–3489. protein. Viruses. 2012;4(6):1011–1033. [55] Ma Y, Wu L, Shaw N, et al. Structural basis and [36] Rey FA, Lok SM. Common features of enveloped functional analysis of the SARS coronavirus viruses and implications for immunogen design for nsp14-nsp10 complex. Proc Natl Acad Sci U S A. next-generation vaccines. Cell. 2018;172(6):1319–1334. 2015;112(30):9436–9441. [37] Li F, Li W, Farzan M, et al. Structure of SARS corona­ [56] Case JB, Li Y, Elliott R, et al. Murine hepatitis virus nsp14 virus spike receptor-binding domain complexed with exoribonuclease activity is required for resistance to innate receptor. Science. 2005;309(5742):1864–1868. immunity. J Virol. 2017;92(1):e01531–17. [38] Shang J, Ye G, Shi K, et al. Structural basis of receptor [57] Cottam EM, Maier HJ, Manifava M, et al. Coronavirus recognition by SARS-CoV-2. Nature. 2020;581 nsp6 proteins generate autophagosomes from the (7807):221–224. endoplasmic reticulum via an omegasome [39] Wang N, Shi X, Jiang L, et al. Structure of MERS-CoV intermediate. Autophagy. 2011;7(11):1335–1347. spike receptor-binding domain complexed with human [58] Dubuisson J. Hepatitis C virus proteins. World receptor DPP4. Cell Res. 2013;23(8):986–993. J Gastroenterol. 2007;13(17):2406–2415. VIRULENCE 461 [59] Geddawy A, Ibrahim YF, Elbahie NM, et al. Direct acting [77] Wilen CB, Tilton JC, Doms RW. HIV: cell binding and anti-hepatitis C virus drugs: clinical pharmacology and entry. Cold Spring Harb Perspect Med. 2012;2(8): future direction. J Transl Int Med. 2017;5(1):8–17. a006866. [60] Griffiths C, Drews SJ, Marchant DJ. Respiratory syncy­ [78] Miyauchi K, Kim Y, Latinovic O, et al. HIV enters cells tial virus: infection, detection, and new options for via endocytosis and dynamin-dependent fusion with prevention and treatment. Clin Microbiol Rev. endosomes. Cell. 2009;137(3):433–444. 2017;30(1):277–319. [79] Permanyer M, Ballana E, Esté JA. Endocytosis of HIV: [61] Vainionpää R, Hyypiä T. Biology of parainfluenza anything goes. Trends Microbiol. 2010;18(12):543–551. viruses. Clin Microbiol Rev. 1994;7(2):265–275. [80] Wilen CB, Tilton JC, Doms RW. Molecular mechan­ [62] Nair H, Nokes DJ, Gessner BD, et al. Global burden of isms of HIV entry. In: Rossmann M, Rao V, editors. acute lower respiratory infections due to respiratory Viral molecular machines. Vol. 726. Boston, MA: syncytial virus in young children: a systematic review Springer; 2012. p. 223–242. and meta-analysis. Lancet. 2010;375(9725):1545–1555. [81] German Advisory Committee Blood (Arbeitskreis Blut), [63] Pawełczyk M, Kowalski ML. The role of human parain­ Subgroup ‘Assessment of Pathogens Transmissible by fluenza virus infections in the immunopathology of the Blood’. Human Immunodeficiency Virus (HIV). respiratory tract. Curr Allergy Asthma Rep. 2017;17(3):16. Transfus Med Hemother. 2016;43(3):203–222. [64] Srinivasakumar N, Ogra PL, Flanagan TD. [82] Enjuanes L, Sola I, Almazan F, et al. Coronavirus Characteristics of fusion of respiratory syncytial virus derived expression systems. In: Lavi E, Weiss SR, with HEp-2 cells as measured by R18 fluorescence Hingley ST, editors. The nidoviruses. advances in dequenching assay. J Virol. 1991;65(8):4063–4069. experimental medicine and biology. Vol. 494. Boston, [65] Krzyzaniak MA, Zumstein MT, Gerez JA, et al. Host MA: Springer; 2001. p. 309–321. cell entry of respiratory syncytial virus involves macro­ [83] Ruelas DS, Greene WC. An integrated overview of pinocytosis followed by proteolytic activation of the F HIV-1 latency. Cell. 2013;155(3):519–529. protein. PLoS Pathog. 2013;9(4):e1003309. [84] Wu Y. HIV-1 gene expression: lessons from provirus [66] Smith EC, Popa A, Chang A, et al. Viral entry mechan­ and non-integrated DNA. Retrovirology. 2004;1:13. isms: the increasing diversity of paramyxovirus entry. [85] Jakobovits A, Smith DH, Jakobovits EB, et al. FEBS J. 2009;276(24):7217–7227. A discrete element 3′ of human immunodeficiency [67] McLellan JS, Ray WC, Peeples ME. Structure and func­ virus 1 (HIV-1) and HIV-2 mRNA initiation sites tion of respiratory syncytial virus surface glycoproteins. mediates transcriptional activation by an HIV trans In: Anderson L, Graham B, editors. Challenges and activator. Mol Cell Biol. 1988;8(6):2555–2561. opportunities for respiratory syncytial virus vaccines. [86] Boasso A, Shearer GM, Chougnet C. Immune dysregula­ Vol. 372. Berlin, Heidelberg: Springer; 2013. p. 83–104. tion in human immunodeficiency virus infection: know it, [68] Chaiwatpongsakorn S, Epand RF, Collins PL, et al. Soluble fix it, prevent it? J Intern Med. 2009;265(1):78–96. respiratory syncytial virus fusion protein in the fully cleaved, [87] Nardacci R, Ciccosanti F, Marsella C, et al. Role of pretriggered state is triggered by exposure to low-molarity autophagy in HIV infection and pathogenesis. buffer. J Virol. 2011;85(8):3968–3977. J Intern Med. 2017;281(5):422–432. [69] Hallak LK, Collins PL, Knudson W, et al. Iduronic [88] Beachboard DC, Horner SM. Innate immune evasion acid-containing glycosaminoglycans on target cells are strategies of DNA and RNA viruses. Curr Opin required for efficient respiratory syncytial virus Microbiol. 2016;32:113–119. infection. Virology. 2000;271(2):264–275. [89] Akira S, Uematsu S, Takeuchi O. Pathogen recognition [70] Ortín J, Martín-Benito J. The RNA synthesis machin­ and innate immunity. Cell. 2006;124(4):783–801. ery of negative-stranded RNA viruses. Virology. [90] Bowie AG, Unterholzner L. Viral evasion and subver­ 2015;479–480:532–544. sion of pattern-recognition receptor signalling. Nat Rev [71] Noton SL, Fearns R. Initiation and regulation of para­ Immunol. 2008;8(12):911–922. myxovirus transcription and replication. Virology. [91] Ma Z, Damania B. The cGAS-STING defense pathway 2015;479–480:545–554. and its counteraction by viruses. Cell Host Microbe. [72] Audsley MD, Moseley GW. Paramyxovirus evasion of 2016;19(2):150–158. innate immunity: diverse strategies for common [92] Nikonov A, Mölder T, Sikut R, et al. RIG-I and MDA-5 targets. World J Virol. 2013;2(2):57–70. detection of viral RNA-dependent RNA polymerase [73] Sakuragi JI. Morphogenesis of the infectious HIV-1 activity restricts positive-strand RNA virus virion. Front Microbiol. 2011;2:242. replication. PLoS Pathog. 2013;9(9):e1003610. [74] Taveira N. HIV-2 envelope: structure, diversity, and evolu­ [93] Kawasaki T, Kawai T. Toll-like receptor signaling tion. In: Hope T, Stevenson M, Richman D, editors. pathways. Front Immunol. 2014;5:461. Encyclopedia of AIDS. New York, NY: Springer; 2013. p. [94] Jensen S, Thomsen AR. Sensing of RNA viruses: 1–6. A review of innate immune receptors involved in [75] Zheng NN, Kiviat NB, Sow PS, et al. Comparison of recognizing RNA virus invasion. J Virol. 2012;86 human immunodeficiency virus (HIV)-specific T-Cell (6):2900–2910. responses in HIV-1- and HIV-2-infected individuals in [95] Brisse M, Ly H. Comparative structure and function Senegal. J Virol. 2004;78(24):13934–13942. analysis of the RIG-I-like receptors: RIG-I and MDA5. [76] Doms RW, Trono D. The plasma membrane as Front Immunol. 2019;10:1586. a combat zone in the HIV battlefield. Genes Dev. [96] Henrick BM, Yao XD, Rosenthal KL. HIV-1 structural 2000;14(21):2677–2688. proteins serve as PAMPs for TLR2 heterodimers 462 J. LI AND E. BOIX significantly increasing infection and innate immune [114] Han X, Li X, Yue SC, et al. Epigenetic regulation of activation. Front Immunol. 2015;6:426. tumor necrosis factor α (TNFα) release in human [97] Loo YM, Gale M. Immune signaling by RIG-I-like macrophages by HIV-1 single-stranded RNA (ssRNA) receptors. Immunity. 2011;34(5):680–692. is dependent on TLR8 signaling. J Biol Chem. 2012;287 [98] Said EA, Tremblay N, Al-Balushi MS, et al. Viruses (17):13778–13786. seen by our cells: the role of viral RNA sensors. [115] Bernard MA, Zhao H, Yue SC, et al. Novel HIV-1 J Immunol Res. 2018;2018:9480497. miRNAs stimulate TNFα release in human macro­ [99] Lee HC, Chathuranga K, Lee JS. Intracellular sensing of phages via TLR8 signaling pathway. PLoS One. 2014;9 viral genomes and viral evasion. Exp Mol Med. 2019;51 (9):e106006. (12):1–13. [116] Chan YK, Gack MU. Viral evasion of intracellular [100] Sadler AJ, Williams BRG. Interferon-inducible antiviral DNA and RNA sensing. Nat Rev Microbiol. 2016;14 effectors. Nat Rev Immunol. 2008;8(7):559–568. (6):360–373. [101] García MA, Gil J, Ventoso I, et al. Impact of protein [117] Yin X, Langer S, Zhang Z, et al. Sensor sensibility— kinase PKR in cell biology: from antiviral to antiproli­ HIV-1 and the innate immune response. Cells. 2020;9 ferative action. Microbiol Mol Biol Rev. 2006;70 (1):254. (4):1032–1060. [118] Freund I, Eigenbrod T, Helm M, et al. RNA modifica­ [102] Roth-Cross JK, Bender SJ, Weiss SR. Murine corona­ tions modulate activation of innate toll-like receptors. virus mouse hepatitis virus is recognized by MDA5 and Genes (Basel). 2019;10(2):92. induces Type I interferon in brain macrophages/micro­ [119] Ayadi L, Galvanin A, Pichot F, et al. RNA ribose glia. J Virol. 2008;82(20):9829–9838. methylation (2′-O-methylation): occurrence, biosynth­ [103] Mazaleuskaya L, Veltrop R, Ikpeze N, et al. Protective esis and biological functions. Biochim Biophys Acta role of Toll-like receptor 3-induced type I interferon in Gene Regul Mech. 2019;1862(3):253–269. murine coronavirus infection of macrophages. Viruses. [120] Züst R, Cervantes-Barragan L, Habjan M, et al. Ribose 2012;4(5):901–923. 2′-O-methylation provides a molecular signature for [104] Wang Y, Liu L. The membrane protein of severe acute the distinction of self and non-self mRNA dependent respiratory syndrome coronavirus functions as a novel on the RNA sensor Mda5. Nat Immunol. 2011;12 cytosolic pathogen-associated molecular pattern to (2):137–143. promote beta interferon induction via a toll-like- [121] Schuberth-Wagner C, Ludwig J, Bruder AK, et al. receptor-related TRAF3-independent mechanism. A conserved histidine in the RNA sensor RIG-I con­ mBio. 2016;7(1):e01872–15. trols immune tolerance to N1-2′O-methylated self [105] Chigbu DI, Loonawat R, Sehgal M, et al. Hepatitis RNA. Immunity. 2015;43(1):41–51. C virus infection: host-virus interaction and mechan­ [122] Menachery VD, Yount BL, Josset L, et al. isms of viral persistence. Cells. 2019;8(4):376. Attenuation and restoration of severe acute respira­ [106] Rudd BD, Burstein E, Duckett CS, et al. Differential tory syndrome coronavirus mutant lacking 2′- role for TLR3 in respiratory syncytial virus-induced O-methyltransferase activity. J Virol. 2014;88 chemokine expression. J Virol. 2005;79(6):3350–3357. (8):4251–4264. [107] Loo Y-M, Fornek J, Crochet N, et al. Distinct [123] Ringeard M, Marchand V, Decroly E, et al. FTSJ3 is an RIG-I and MDA5 signaling by RNA viruses in innate RNA 2′-O-methyltransferase recruited by HIV to avoid immunity. J Virol. 2008;82(1):335–345. innate immune sensing. Nature. 2019;565 [108] Kurt-Jones EA, Popova L, Kwinn L, et al. (7740):500–504. Pattern-recognition receptors TLR4 and CD14 mediate [124] Gokhale NS, Horner SM. RNA modifications go viral. response to respiratory syncytial virus. Nat Immunol. PLoS Pathog. 2017;13(3):e1006188. 2000;1(5):398–401. [125] Arguello AE, Deliberto AN, Kleiner RE, et al. RNA [109] Sabbah A, Chang TH, Harnack R, et al. Activation of chemical proteomics reveals the N6-methyladenosine innate immune antiviral responses by Nod2. Nat (m6A)-regulated protein-RNA interactome.. J Am Immunol. 2009;10(10):1073–1080. Chem Soc. 2017;139(48):17249–17252. [110] Beignon AS, McKenna K, Skoberne M, et al. [126] Kong W, Rivera-Serrano EE, Neidleman JA, et al. HIV- Endocytosis of HIV-1 activates plasmacytoid dendritic 1 replication benefits from the RNA epitranscriptomic cells via Toll-like receptor-viral RNA interactions. code. J Mol Biol. 2019;431(24):5032–5038. J Clin Invest. 2005;115(11):3265–3275. [127] Lu M, Zhang Z, Xue M, et al. N6-methyladenosine [111] Hardy AW, Graham DR, Shearer GM, et al. HIV turns modification enables viral RNA to escape recognition plasmacytoid dendritic cells (pDC) into TRAIL- by RNA sensor RIG-I. Nat Microbiol. 2020;5 expressing killer pDC and down-regulates HIV core­ (4):584–598. ceptors by Toll-like receptor 7-induced IFN-α. Proc [128] Shulman Z, Stern-Ginossar N. The RNA modification Natl Acad Sci U S A. 2007;104(44):17453–17458. N6-methyladenosine as a novel regulator of the [112] Silvin A, Manel N. Innate immune sensing of HIV immune system. Nat Immunol. 2020;21(5):501–512. infection. Curr Opin Immunol. 2015;32:54–60. [129] Tsai K, Jaguva Vasudevan AA, Martinez Campos C, [113] Guo H, Gao J, Taxman DJ, et al. HIV-1 infection et al. Acetylation of cytidine residues boosts HIV-1 induces interleukin-1β production via TLR8 protein- gene expression by increasing viral RNA stability. Cell dependent and NLRP3 inflammasome mechanisms in Host Microbe. 2020;28(2):306–312. human monocytes. J Biol Chem. 2014;289 [130] Eddy SR. Non-coding RNA genes and the modern (31):21716–21726. RNA world. Nat Rev Genet. 2001;2(12):919–929. VIRULENCE 463 [131] Damas ND, Fossat N, Scheel TKH. Functional inter­ of suppressor of cytokine signaling-3. FASEB J. 2003;17 play between RNA viruses and non-coding RNA in (3):488–490. mammals. Noncoding RNA. 2019;5(1):7. [149] Romero-Brey I, Bartenschlager R. Membranous repli­ [132] Wang Z, Zhao Y, Zhang Y. Viral lncRNA: A regulatory cation factories induced by plus-strand RNA viruses. molecule for controlling virus life cycle. Non-coding Viruses. 2014;6(7):2826–2857. RNA Res. 2017;2(1):38–44. [150] Angelini MM, Akhlaghpour M, Neuman BW, et al. [133] Li Y, Wang C, Miao Z, et al. ViRBase: A resource for Severe acute respiratory syndrome coronavirus nonstruc­ virus-host ncRNA-associated interactions. Nucleic tural proteins 3, 4, and 6 induce double-membrane Acids Res. 2015;43(D1):D578–D582. vesicles. mBio. 2013;4(4):e00524–13. [134] Tycowski KT, Guo YE, Lee N, et al. Viral noncoding [151] Oudshoorn D, Rijs K, Limpens RWAL, et al. RNAs: more surprises. Genes Dev. 2015;29(6):567–584. Expression and cleavage of middle east respiratory [135] Ouyang J, Hu J, Chen J-L. lncRNAs regulate the innate syndrome coronavirus nsp3-4 polyprotein induce the immune response to viral infection. Wiley Interdiscip formation of double-membrane vesicles that mimic Rev RNA. 2016;7(1):129–143. those associated with coronaviral RNA replication. [136] Wu W, Choi EJ, Lee I, et al. Non-coding RNAs and mBio. 2017;8(6):e01658–17. their role in respiratory syncytial virus (RSV) and [152] Wang H, Tai AW. Mechanisms of cellular membrane human metapneumovirus (hMPV) infections. Viruses. reorganization to support hepatitis C virus replication. 2020;12(3):345. Viruses. 2016;8(5):142. [137] Carnero E, Barriocanal M, Prior C, et al. Long non­ [153] Collins PL, Fearns R, Graham BS. Respiratory syncytial coding RNA EGOT negatively affects the antiviral virus: virology, reverse genetics, and pathogenesis of response and favors HCV replication. EMBO Rep. disease. In: Anderson L, Graham B, editors. Challenges 2016;17(7):1013–1028. and opportunities for respiratory syncytial virus vac­ [138] Amador-Cañizares Y, Panigrahi M, Huys A, et al. miR- cines. Vol. 372. Berlin, Heidelberg: Springer; 2013. p. 122, small RNA annealing and sequence mutations 3–38. alter the predicted structure of the Hepatitis C virus 5 [154] Kikkert M. Innate immune evasion by human respira­ UTR RNA to stabilize and promote viral RNA tory RNA viruses. J Innate Immun. 2020;12(1):4–20. accumulation. Nucleic Acids Res. 2018;46 [155] Anderson P, Kedersha N. Stress granules: the Tao of (18):9776–9792. RNA triage. Trends Biochem Sci. 2008;33(3):141–150. [139] Colussi TM, Costantino DA, Hammond JA, et al. The [156] Raaben M, Groot Koerkamp MJ, Rottier PJM, et al. structural basis of transfer RNA mimicry and confor­ Mouse hepatitis coronavirus replication induces host mational plasticity by a viral RNA. Nature. 2014;511 translational shutoff and mRNA decay, with concomi­ (7509):366–369. tant formation of stress granules and processing [140] Hammond JA, Rambo RP, Filbin ME, et al. bodies. Cell Microbiol. 2007;9(9):2218–2229. Comparison and functional implications of the 3D [157] Lindquist ME, Lifland AW, Utley TJ, et al. Respiratory architectures of viral tRNA-like structures. RNA. syncytial virus induces host RNA stress granules to 2009;15(2):294–307. facilitate viral replication. J Virol. 2010;84 [141] Dreher TW. Role of tRNA-like structures in controlling (23):12274–12284. plant virus replication. Virus Res. 2009;139(2):217–229. [158] Zhang Q, Sharma NR, Zheng ZM, et al. Viral regula­ [142] Larsen KP, Mathiharan YK, Kappel K, et al. tion of RNA granules in infected cells. Virol Sin. Architecture of an HIV-1 reverse transcriptase initia­ 2019;34(2):175–191. tion complex. Nature. 2018;557(7703):118–122. [159] Miller CL. Stress granules and virus replication. Future [143] Nan Y, Nan G, Zhang YJ. Interferon induction by RNA Virol. 2011;6(11):1329–1338. viruses and antagonism by viral pathogens. Viruses. [160] Hashimoto S, Yamamoto S, Ogasawara N, et al. 2014;6(12):4999–5027. Mumps virus induces protein-kinase-R-dependent [144] Kindler E, Gil-Cruz C, Spanier J, et al. Early stress granules, partly suppressing type III interferon endonuclease-mediated evasion of RNA sensing production. PLoS One. 2016;11(8):e0161793. ensures efficient coronavirus replication. PLoS [161] Thomas MG, Loschi M, Desbats MA, et al. RNA gran­ Pathog. 2017;13(2):e1006195. ules: the good, the bad and the ugly. Cell Signal. [145] Totura AL, Baric RS. SARS coronavirus pathogenesis: 2011;23(2):324–334. host innate immune responses and viral antagonism of [162] Garaigorta U, Heim MH, Boyd B, et al. Hepatitis interferon. Curr Opin Virol. 2012;2(3):264–275. C virus (HCV) induces formation of stress granules [146] Boyapalle S, Wong T, Garay J, et al. Respiratory syn­ whose proteins regulate HCV RNA replication and cytial virus NS1 protein colocalizes with mitochondrial virus assembly and egress. J Virol. 2012;86 antiviral signaling protein MAVS following infection. (20):11043–11056. PLoS One. 2012;7(2):e29386. [163] Onomoto K, Jogi M, Yoo JS, et al. Critical role of an [147] Sandstrom TS, Ranganath N, Angel JB. Impairment of antiviral stress granule containing RIG-I and PKR in the type I interferon response by HIV-1: potential viral detection and innate immunity. PLoS One. 2012;7 targets for HIV eradication. Cytokine Growth Factor (8):e43031. Rev. 2017;37:1–16. [164] Onomoto K, Yoneyama M, Fung G, et al. Antiviral [148] Bode JG, Ludwig S, Ehrhardt C, et al. IFN-α antago­ innate immunity and stress granule responses. Trends nistic activity of HCV core protein involves induction Immunol. 2014;35(9):420–428. 464 J. LI AND E. BOIX [165] Yoo JS, Takahasi K, Ng CS, et al. DHX36 enhances [180] Choi Y, Bowman JW, Jung JU. Autophagy during viral RIG-I signaling by facilitating PKR-mediated antiviral infection - A double-edged sword. Nat Rev Microbiol. stress granule formation. PLoS Pathog. 2014;10(3): 2018;16(6):341–354. e1004012. [181] Cottam EM, Whelband MC, Wileman T. Coronavirus [166] McCormick C, Khaperskyy DA. Translation inhibition NSP6 restricts autophagosome expansion. Autophagy. and stress granules in the antiviral immune response. 2014;10(8):1426–1441. Nat Rev Immunol. 2017;17(10):647–660. [182] Gassen NC, Niemeyer D, Muth D, et al. SKP2 attenu­ [167] Poblete-Durán N, Prades-Pérez Y, Vera-Otarola J, et al. ates autophagy through Beclin1-ubiquitination and its Who regulates whom? An overview of RNA granules inhibition reduces MERS-Coronavirus infection. Nat and viral infections. Viruses. 2016;8(7):180. Commun. 2019;10(1):5770. [168] Nakagawa K, Narayanan K, Wada M, et al. Inhibition [183] Yang N, Shen HM. Targeting the endocytic pathway of stress granule formation by middle east respiratory and autophagy process as a novel therapeutic strategy syndrome coronavirus 4a accessory protein facilitates in COVID-19. Int J Biol Sci. 2020;16(10):1724–1731. viral translation, leading to efficient virus replication. [184] Prentice E, Jerome WG, Yoshimori T, et al. J Virol. 2018;92(20):e00902-18. Coronavirus replication complex formation utilizes [169] Valiente-Echeverría F, Melnychuk L, Vyboh K, et al. components of cellular autophagy. J Biol Chem. EEF2 and Ras-GAP SH3 domain-binding protein 2004;279(11):10136–10141. (G3BP1) modulate stress granule assembly during [185] Li M, Li J, Zeng R, et al. Respiratory syncytial virus HIV-1 infection. Nat Commun. 2014;5:4819. replication is promoted by autophagy-mediated inhibi­ [170] Cinti A, Le Sage V, Ghanem M, et al. HIV-1 gag tion of apoptosis. J Virol. 2018;92(8):e02193–17. blocks selenite-induced stress granule assembly by [186] Chan ST, Ou JJ. Hepatitis C virus-induced autophagy and altering the mRNA cap-binding complex. mBio. host innate immune response. Viruses. 2017;9(8):224. 2016;7(2):e00329. [187] Kvansakul M. Viral infection and apoptosis. Viruses. [171] Abrahamyan LG, Chatel-Chaix L, Ajamian L, et al. 2017;9(12):356. Novel Staufen1 ribonucleoproteins prevent formation [188] Chan CM, Ma CW, Chan WY, et al. The of stress granules but favour encapsidation of HIV-1 SARS-coronavirus membrane protein induces apopto­ genomic RNA. J Cell Sci. 2010;123(3):369–383. sis through modulating the Akt survival pathway. Arch [172] Rao S, Cinti A, Temzi A, et al. HIV-1 NC-induced Biochem Biophys. 2007;459(2):197–207. stress granule assembly and translation arrest are [189] Tan Y-X, Tan THP, Lee MJ-R, et al. Induction of inhibited by the dsRNA binding protein Staufen1. apoptosis by the severe acute respiratory syndrome RNA. 2018;24(2):219–236. coronavirus 7a protein is dependent on its interaction [173] Lindquist ME, Mainou BA, Dermody TS, et al. with the Bcl-XL protein. J Virol. 2007;81 Activation of protein kinase R is required for induction (12):6346–6355. of stress granules by respiratory syncytial virus but [190] Chu H, Zhou J, Wong BHY, et al. Middle east respira­ dispensable for viral replication. Virology. 2011;413 tory syndrome coronavirus efficiently infects human (1):103–110. primary T lymphocytes and activates the extrinsic [174] Fricke J, Koo LY, Brown CR, et al. p38 and OGT and intrinsic apoptosis pathways. J Infect Dis. sequestration into viral inclusion bodies in cells 2016;213(6):904–914. infected with human respiratory syncytial virus sup­ [191] Sun Y, López CB. The innate immune response to presses MK2 activities and stress granule assembly. RSV: advances in our understanding of critical viral J Virol. 2013;87(3):1333–1347. and host factors. Vaccine. 2017;35(3):481–488. [175] Wang Q, Lee I, Ren J, et al. Identification and func­ [192] Mehrbod P, Ande SR, Alizadeh J, et al. The roles of tional characterization of tRNA-derived RNA frag­ apoptosis, autophagy and unfolded protein response in ments (tRFs) in respiratory syncytial virus infection. arbovirus, influenza virus, and HIV infections. Mol Ther. 2013;21(2):368–379. Virulence. 2019;10(1):376–413. [176] Ivanov P. Emerging roles of tRNA-derived fragments [193] Bantel H, Schulze-Osthoff K. Apoptosis in hepatitis in viral infections: the case of respiratory syncytial C virus infection. Cell Death Differ. 2003;10(S1):S48–S58. virus. Mol Ther. 2015;23(10):1557–1558. [194] Kanwar S. Ribonucleases and their applications. J Adv [177] Emara MM, Ivanov P, Hickman T, et al. Angiogenin- Biotechnol Bioeng. 2016;4(1):17–26. induced tRNA-derived stress-induced RNAs promote [195] Deutscher MP, Li Z. Exoribonucleases and their multi­ stress-induced stress granule assembly. J Biol Chem. ple roles in RNA metabolism. Prog Nucleic Acid Res 2010;285(14):10959–10968. Mol Biol. 2000;66:67–105. [178] Saikia M, Hatzoglou M. The many virtues of [196] Fang EF, Ng TB. Ribonucleases of different origins tRNA-derived stress-induced RNAs (tiRNAs): disco­ with a wide spectrum of medicinal applications. vering Novel mechanisms of stress response and effect Biochim Biophys Acta (BBA)-Reviews Cancer. on human health. J Biol Chem. 2015;290 2011;1815(1):65–74. (50):29761–29768. [197] Schein CH. From housekeeper to microsurgeon: the [179] Olasunkanmi OI, Chen S, Mageto J, et al. Virus- diagnostic and therapeutic potential of ribonucleases. induced cytoplasmic aggregates and inclusions are cri­ Nat Biotechnol. 1997;15(6):529–536. tical cellular regulatory and antiviral factors. Viruses. [198] Sorrentino S. The eight human “canonical” ribonu­ 2020;12(4):399. cleases: molecular diversity, catalytic properties, and VIRULENCE 465 special biological actions of the enzyme proteins. FEBS [214] Lindsley AW, Schwartz JT, Rothenberg ME. Eosinophil Lett. 2010;584(11):2194–2200. responses during COVID-19 infections and corona­ [199] Gupta SK, Haigh BJ, Griffin FJ, et al. The mammalian virus vaccination. J Allergy Clin Immunol. 2020;146 secreted RNases: mechanisms of action in host defence. (1):1–7. Innate Immun. 2013;19(1):86–97. [215] Lippi G, Henry BM. Eosinophil count in severe coro­ [200] Boix E, Nogués MV. Mammalian antimicrobial pro­ navirus disease 2019 (COVID-19). QJM. 2020;133 teins and peptides: overview on the RNase (7):511–512. A superfamily members involved in innate host [216] Liu F, Xu A, Zhang Y, et al. Patients of COVID-19 may defence. Mol Biosyst. 2007;3(5):317–335. benefit from sustained Lopinavir-combined regimen [201] Rosenberg HF. RNase A ribonucleases and host and the increase of eosinophil may predict the outcome defense: an evolving story. J Leukoc Biol. 2008;83 of COVID-19 progression. Int J Infect Dis. (5):1079–1087. 2020;95:183–191. [202] Ostendorf T, Zillinger T, Andryka K, et al. Immune [217] Dosanjh A. Eosinophil-derived neurotoxin and respira­ sensing of synthetic, bacterial, and protozoan RNA by tory tract infection and inflammation: implications for Toll-like receptor 8 requires coordinated processing by COVID-19 management. J Interferon Cytokine Res. RNase T2 and RNase 2. Immunity. 2020;52(4):591– 2020;40(9):443–445. 605. [218] Rodrigo-Muñoz JM, Sastre B, Cañas JA, et al. [203] Koczera P, Martin L, Marx G, et al. The ribonuclease Eosinophil response against classical and emerging a superfamily in humans: canonical RNases as the respiratory viruses: COVID-19. J Investig Allergol buttress of innate immunity. Int J Mol Sci. 2016;17 Clin Immunol. 2020;31. (8):1278. [219] Sheng J, Xu Z. Three decades of research on angio­ [204] Lee-Huang S, Huang PL, Sun Y, et al. Lysozyme and genin: A review and perspective. Acta Biochim Biophys RNases as anti-HIV components in-core preparations Sin (Shanghai).. 2016;48(5):399–410. of human chorionic gonadotropin. Proc Natl Acad Sci [220] Cocchi F, DeVico AL, Lu W, et al. Soluble factors from U S A. 1999;96(6):2678–2681. T cells inhibiting X4 strains of HIV are a mixture of β [205] Rugeles MT, Trubey CM, Bedoya VI, et al. chemokines and RNases. Proc Natl Acad Sci U S A. Ribonuclease is partly responsible for the HIV-1 inhi­ 2012;109(14):5411–5416. bitory effect activated by HLA alloantigen recognition. [221] Akiyama Y, Kharel P, Abe T, et al. Isolation and initial AIDS. 2003;17(4):481–486. structure-functional characterization of endogenous [206] Rosenberg HF. Eosinophil-derived neurotoxin/RNase tRNA-derived stress-induced RNAs. RNA Biol. 2: connecting the past, the present and the future. 2020;17(8):1116–1124. Curr Pharm Biotechnol. 2008;9(3):135–140. [222] Lyons SM, Fay MM, Ivanov P. The role of RNA mod­ [207] Kim C-K, Seo JK, Ban SH, et al. Eosinophil-derived ifications in the regulation of tRNA cleavage. FEBS neurotoxin levels at 3 months post-respiratory syncy­ Lett. 2018;592(17):2828–2844. tial virus bronchiolitis are a predictive biomarker of [223] Bedoya VI, Boasso A, Hardy AW, et al. Ribonucleases recurrent wheezing. Biomarkers. 2013;18(3):230–235. in HIV type 1 inhibition: effect of recombinant RNases [208] Domachowske JB, Dyer KD, Adams AG, et al. on infection of primary T cells and immune Eosinophil cationic protein/RNase 3 is another RNase activation-induced RNase gene and protein A-family ribonuclease with direct antiviral activity. expression. AIDS Res Hum Retroviruses. 2006;22 Nucleic Acids Res. 1998;26(14):3358–3363. (9):897–907. [209] Lu L, Wei RL, Prats-Ejarque G, et al. Human RNase3 [224] Youle RJ, Wu YN, Mikulski SM, et al. RNase inhibition immune modulation by catalytic-dependent and inde­ of human immunodeficiency virus infection of H9 pendent modes in a macrophage-cell line infection cells. Proc Natl Acad Sci U S A. 1994;91 model. Cell Mol Life Sci. 2020. DOI:10.1007/s00018- (13):6012–6016. 020-03695-5. [225] Schein CH, Haugg M, Benner SA. Interferon-γ acti­ [210] Paganelli R, Fanales-Belasio E, Scala E, et al. Serum vates the cleavage of double-stranded RNA by bovine eosinophil cationic protein (ECP) in human immuno­ seminal ribonuclease. FEBS Lett. 1990;270 deficiency virus (HIV) infection. J Allergy Clin (1–2):229–232. Immunol. 1991;88(3):416–418. [226] Schein CH, Haugg M. Deletions at the C-terminus of [211] Onorato J, Esposito S, Scovena E, et al. Eosinophil interferon γ reduce RNA binding and activation of involvement and serum IgE level in HIV-1-infected double-stranded-RNA cleavage by bovine seminal children. J Allergy Clin Immunol. 1999;104 ribonuclease. Biochem J. 1995;307(1):123–127. (1):245–247. [227] Ardelt W, Mikulski SM, Shogen K. Amino acid [212] Sikriwal D, Seth D, Parveen S, et al. An insertion in sequence of an anti-tumor protein from Rana pipiens loop L7 of human eosinophil-derived neurotoxin is oocytes and early embryos. Homology to pancreatic crucial for its antiviral activity. J Cell Biochem. ribonucleases. J Biol Chem. 1991;266(1):245–251. 2012;113(10):3104–3112. [228] Saxena SK, Shogen K, Ardelt W. ONCONASE® and its [213] Lee YS, Chen CH, Chao A, et al. Molecular signature of therapeutic potential. Lab Med. 2003;34(5):380–387. clinical severity in recovering patients with severe acute [229] Saxena A, Saxena SK, Shogen K. Effect of onconase on respiratory syndrome coronavirus (SARS-CoV). BMC double-stranded RNA in vitro. Anticancer Res. 2009;29 Genomics. 2005;6:132. (4):1067–1072. 466 J. LI AND E. BOIX [230] Saxena SK, Gravell M, Wu YN, et al. Inhibition of [246] Behera AK, Kumar M, Lockey RF, et al. 2′-5′ oligoade­ HIV-1 production and selective degradation of viral nylate synthetase plays a critical role in interferon-γ RNA by an amphibian ribonuclease. J Biol Chem. inhibition of respiratory syncytial virus infection of 1996;271(34):20783–20788. human epithelial cells. J Biol Chem. 2002;277 [231] Suhasini AN, Sirdeshmukh R. Onconase action on (28):25601–25608. tRNALys3, the primer for HIV-1 reverse transcription. [247] Chakrabarti A, Ghosh PK, Banerjee S, et al. RNase Biochem Biophys Res Commun. 2007;363(2):304–309. L triggers autophagy in response to viral infections. [232] Vert A, Castro J, Ribó M, et al. Activating transcription J Virol. 2012;86(20):11311–11321. factor 3 is crucial for antitumor activity and to [248] Bisbal C, Silverman RH. Diverse functions of RNase strengthen the antiviral properties of Onconase. L and implications in pathology. Biochimie. 2007;89 Oncotarget. 2017;8(7):11692–11707. (6–7):789–798. [233] Shu M, Du T, Zhou G, et al. Role of activating tran­ [249] Comar CE, Goldstein SA, Li Y, et al. Antagonism of scription factor 3 in the synthesis of latency-associated dsRNA-induced innate immune pathways by NS4a and transcript and maintenance of herpes simplex virus 1 NS4b accessory proteins during MERS coronavirus in latent state in ganglia. Proc Natl Acad Sci U S A. infection. mBio. 2019;10(2):e00319-19. 2015;112(39):E5420–E5426. [250] Zhao L, Jha BK, Wu A, et al. Antagonism of the [234] Lu L, Arranz-Trullén J, Prats-Ejarque G, et al. Human interferon-induced OAS-RNase L pathway by murine antimicrobial RNases inhibit intracellular bacterial coronavirus ns2 protein is required for virus replica­ growth and induce autophagy in tion and liver pathology. Cell Host Microbe. 2012;11 mycobacteria-infected macrophages. Front Immunol. (6):607–616. 2019;10:1500. [251] Thornbrough JM, Jha BK, Yount B, et al. Middle east [235] Fiorini C, Gotte G, Donnarumma F, et al. Bovine respiratory syndrome coronavirus NS4b protein inhibits seminal ribonuclease triggers Beclin1-mediated autop­ host RNase L activation. mBio. 2016;7(2):e00258-16. hagic cell death in pancreatic cancer cells. Biochim [252] Schröder HC, Ugarković D, Wenger R, et al. Binding Biophys Acta (BBA)-Molecular Cell Res. 2014;1843 of Tat protein to TAR region of human immunodefi­ (5):976–984. ciency virus type 1 blocks TAR-mediated activation of [236] Navarro S, Aleu J, Jiménez M, et al. The cytotoxicity of (2′-5′)oligoadenylate synthetase. AIDS Res Hum eosinophil cationic protein/ribonuclease 3 on eukaryo­ Retroviruses. 1990;6(5):659–672. tic cell lines takes place through its aggregation on the [253] Martinand C, Montavon C, Salehzada T, et al. RNase cell membrane. Cell Mol Life Sci. 2008;65(2):324–337. L inhibitor is induced during human immunodefi­ [237] Chang KC, Lo CW. Fan T chi, et al. TNF-α mediates ciency virus type 1 infection and down regulates the eosinophil cationic protein-induced apoptosis in 2-5A/RNase L pathway in human T cells. J Virol. BEAS-2B cells. BMC Cell Biol. 2010;11:6. 1999;73(1):290–296. [238] Turcotte RF, Raines RT. Design and characterization of [254] Han J-Q, Townsend HL, Jha BK, et al. A phylogenetically an HIV-specific ribonuclease zymogen. AIDS Res Hum conserved RNA structure in the poliovirus open reading Retroviruses. 2008;24(11):1357–1363. frame inhibits the antiviral endoribonuclease RNase L. [239] Silverman RH. Viral encounters with 2′,5′- J Virol. 2007;81(11):5561–5572. oligoadenylate synthetase and RNase L during the [255] Drappier M, Jha BK, Stone S, et al. A novel mechanism interferon antiviral response. J Virol. 2007;81 of RNase L inhibition: theiler’s virus L* protein pre­ (23):12720–12729. vents 2-5A from binding to RNase L. PLoS Pathog. [240] Banerjee S, Gusho E, Gaughan C, et al. OAS-RNase 2018;14(4):e1006989. L innate immune pathway mediates the cytotoxicity of [256] Di Maria E, Latini A, Borgiani P, et al. Genetic variants a DNA-demethylating drug. Proc Natl Acad Sci U S A. of the human host influencing the 2019;116(11):5071–5076. coronavirus-associated phenotypes (SARS, MERS and [241] Drappier M, Michiels T. Inhibition of the OAS/RNase COVID-19): rapid systematic review and field L pathway by viruses. Curr Opin Virol. 2015;15:19–26. synopsis. Hum Genomics. 2020;14(1):30. [242] Manivannan P, Siddiqui MA, Malathi K. RNase [257] Pairo-Castineira E, Clohisey S, Klaric L, et al. Genetic L amplifies Interferon signaling by inducing mechanisms of critical illness in Covid-19. Nature PKR-mediated antiviral stress granules. J Virol. 2020. 2020;94(13):e00205–20. [258] Macintosh GC. RNase T2 family: enzymatic properties, [243] Ezelle HJ, Malathi K, Hassel BA. The roles of functional diversity, and evolution of ancient ribonu­ RNase-L in antimicrobial immunity and the cleases. In: Nicholson A, editor. Ribonucleases. Nucleic cytoskeleton-associated innate response. Int J Mol Sci. Acids and Molecular Biology. Berlin, Heidelberg: 2016;17(1):74. Springer; 2011. p. 89–114. [244] Malathi K, Saito T, Crochet N, et al. RNase L releases [259] Luhtala N, Parker R. T2 Family ribonucleases: ancient a small RNA from HCV RNA that refolds into a potent enzymes with diverse roles. Trends Biochem Sci. PAMP. RNA. 2010;16(11):2108–2119. 2010;35(5):253–259. [245] Maitra RK, Silverman RH. Regulation of human [260] Henneke M, Diekmann S, Ohlenbusch A, et al. immunodeficiency virus replication by 2′,5′- RNASET2-deficient cystic leukoencephalopathy resem­ oligoadenylate-dependent RNase L. J Virol. 1998;72 bles congenital cytomegalovirus brain infection. Nat (2):1146–1152. Genet. 2009;41(7):773–775. VIRULENCE 467 [261] Thorn A, Steinfeld R, Ziegenbein M, et al. Structure [277] Samuel MA, Whitby K, Keller BC, et al. PKR and and activity of the only human RNase T2. Nucleic RNase L contribute to protection against lethal West Acids Res. 2012;40(17):8733–8742. Nile virus infection by controlling early viral spread in [262] Greulich W, Wagner M, Gaidt MM, et al. TLR8 is the periphery and replication in neurons. J Virol. a sensor of RNase T2 degradation products. Cell. 2006;80(14):7009–7019. 2019;179(6)1264–1275:e13. [278] Lin R-J, Chu J-S, Chien H-L, et al. MCPIP1 suppresses [263] Lin RJ, Chien HL, Lin SY, et al. MCPIP1 ribonuclease hepatitis C virus replication and negatively regulates exhibits broad-spectrum antiviral effects through viral virus-induced proinflammatory cytokine responses. RNA binding and degradation. Nucleic Acids Res. J Immunol. 2014;193(8):4159–4168. 2013;41(5):3314–3326. [279] Tamkovich N, Koroleva L, Kovpak M, et al. Design, [264] Nadal A, Martell M, Robin Lytle J, et al. Specific RNA cleavage and antiviral activity of new artificial cleavage of hepatitis C virus RNA genome by human ribonucleases derived from mono-, di- and tripeptides RNase P. J Biol Chem. 2002;277(34):30606–30613. connected by linkers of different hydrophobicity. [265] Shah Mahmud R, Müller C, Romanova Y, et al. Bioorg Med Chem. 2016;24(6):1346–1355. Ribonuclease from bacillus acts as an antiviral agent [280] Scarborough RJ, Gatignol A. HIV and ribozymes. Adv against negative- and positive-sense single stranded Exp Med Biol. 2015;848:97–116. human respiratory RNA viruses. Biomed Res Int. [281] Van Nostrand EL, Pratt GA, Yee BA, et al. Principles of 2017;2017:5279065. RNA processing from analysis of enhanced CLIP maps for [266] Shah Mahmud R, Mostafa A, Müller C, et al. Bacterial 150 RNA binding proteins. Genome Biol. 2020;21(1):90. ribonuclease binase exerts an intra-cellular anti-viral [282] Chahal J, Gebert LFR, Gan HH, et al. mIR-122 and mode of action targeting viral RNAs in influenza a Ago interactions with the HCV genome alter the struc­ virus-infected MDCK-II cells. Virol J. 2018;15(1):5. ture of the viral 5′ terminus. Nucleic Acids Res. 2019;47 [267] Müller C, Ulyanova V, Ilinskaya O, et al. A novel (10):5307–5324. antiviral strategy against MERS-CoV and HCoV-229E [283] Li YP, Van Pham L, Uzcategui N, et al. Functional using Binase to target viral genome replication. analysis of microRNA-122 binding sequences of hepa­ Bionanoscience. 2017;7(2):294–299. titis C virus and identification of variants with high [268] Flodström-Tullberg M, Hultcrantz M, Stotland A, et al. resistance against a specific antagomir. J Gen Virol. RNase L and double-stranded RNA-dependent protein 2016;97(6):1381–1394. kinase exert complementary roles in islet cell defense [284] Rangel-Muñoz N, Suarez-Arnedo A, Anguita R, et al. during coxsackievirus infection. J Immunol. 2005;174 Magnetite nanoparticles functionalized with RNases (3):1171–1177. against intracellular infection of pseudomonas [269] Li X-L, Blackford JA, Hassel BA. RNase L mediates the aeruginosa. Pharmaceutics. 2020;12(7):631. antiviral effect of interferon through a selective reduc­ [285] Kordalivand N, Li D, Beztsinna N, et al. tion in viral RNA during encephalomyocarditis virus Polyethyleneimine coated nanogels for the intracellular infection. J Virol. 1998;72(4):2752–2759. delivery of RNase A for cancer therapy. Chem Eng J. [270] Li G, Xiang Y, Sabapathy K, et al. An apoptotic signal­ 2018;340:32–41. ing pathway in the interferon antiviral response [286] Lomax JE, Eller CH, Raines RT. Rational design and mediated by RNase L and c-Jun NH2-terminal evaluation of mammalian ribonuclease cytotoxins. In: Kinase. J Biol Chem. 2004;279(2):1123–1131. Wittrup KD, Verdine GL, editors. Methods in [271] Han JQ, Wroblewski G, Xu Z, et al. Sensitivity of Enzymology. Vol. 502. Academic Press; 2012. p. 273–290. hepatitis C virus RNA to the antiviral enzyme ribonu­ [287] De Clercq E, Li G. Approved antiviral drugs over the clease L is determined by a subset of efficient cleavage past 50 years. Clin Microbiol Rev. 2016;29(3):695–747. sites. J Interferon Cytokine Res. 2004;24(11):664–676. [288] Vardanyan R, Hruby V. Antiviral Drugs. In: [272] Cooper DA, Banerjee S, Chakrabarti A, et al. RNase Vardanyan R, Hruby V, editors. Synthesis of best- L targets distinct sites in influenza A virus RNAs. seller drugs. Academic Press; 2016. p. 687–736. J Virol. 2015;89(5):2764–2776. [289] Fu DY, Xue YR, Yu X, et al. Anti-virus reagents target­ [273] Siddiqui MA, Malathi K. RNase L induces autophagy ing the capsid protein assembly. J Mater Chem B. via c-Jun N-terminal kinase and double-stranded 2019;7(21):3331–3340. RNA-dependent protein kinase signaling pathways. [290] Richman DD, Nathanson N. Antiviral Therapy. In: J Biol Chem. 2012;287(52):43651–43664. Katze MG, Korth MJ, Law GL, et al., editors. Viral [274] Ryman KD, White LJ, Johnston RE, et al. Effects of Pathogenesis (Third Edition). Academic Press; 2016. PKR/RNase L-dependent and alternative antiviral p. 271–287. pathways on alphavirus replication and pathogenesis. [291] Cihlar T, Ray AS. Nucleoside and nucleotide HIV Viral Immunol. 2002;15(1):53–76. reverse transcriptase inhibitors: 25 years after [275] Sawicki DL, Silverman RH, Williams BR, et al. zidovudine. Antiviral Res. 2010;85(1):39–58. Alphavirus minus-strand synthesis and persistence in [292] Sluis-Cremer N, Tachedjian G. Mechanisms of inhibi­ mouse embryo fibroblasts derived from mice lacking tion of HIV replication by non-nucleoside reverse tran­ RNase L and protein kinase R. J Virol. 2003;77 scriptase inhibitors. Virus Res. 2008;134(1–2):147–156. (3):1801–1811. [293] Ghosh AK, Osswald HL, Prato G. Recent progress in [276] Scherbik SV, Paranjape JM, Stockman BM, et al. RNase the development of HIV-1 protease inhibitors for the L plays a role in the antiviral response to West Nile treatment of HIV/AIDS. J Med Chem. 2016;59 virus. J Virol. 2006;80(6):2987–2999. (11):5172–5208. 468 J. LI AND E. BOIX [294] Wong E, Trustman N, Yalong A. HIV pharmacother­ [311] Jordheim LP, Durantel D, Zoulim F, et al. Advances in apy: A review of integrase inhibitors. JAAPA. 2016;29 the development of nucleoside and nucleotide analo­ (2):36–40. gues for cancer and viral diseases. Nat Rev Drug [295] Kitchen CMR, Nuño M, Kitchen SG, et al. Enfuvirtide Discov. 2013;12(6):447–464. antiretroviral therapy in HIV-1 infection. Ther Clin [312] Menéndez-Arias L. Mechanisms of resistance to Risk Manag. 2008;4(2):433–439. nucleoside analogue inhibitors of HIV-1 reverse [296] Woollard SM, Kanmogne GD. Maraviroc: A review of transcriptase. Virus Res. 2008;134(1–2):124–146. its use in HIV infection and beyond. Drug Des Devel [313] Elfiky AA. Ribavirin, Remdesivir, Sofosbuvir, Galidesivir, Ther. 2015;9:5447–5468. and Tenofovir against SARS-CoV-2 RNA dependent RNA [297] Chatel-Chaix L, Baril M, Lamarre D. Hepatitis C virus polymerase (RdRp): a molecular docking study.. Life Sci. NS3/4A protease inhibitors: a Light at the end of the 2020;253:117592. tunnel. Viruses. 2010;2(8):1752–1765. [314] Ju J, Li X, Kumar S, et al. Nucleotide analogues as [298] Pradat P, Virlogeux V, Gagnieu M-C, et al. Ribavirin at inhibitors of SARS-CoV polymerase. Pharmacol Res the era of novel direct antiviral agents for the treatment Perspect. 2020;8(6):e00674. of hepatitis C virus infection: relevance of pharmaco­ [315] Ganesan A, Barakat K. Applications of computer-aided logical monitoring. Adv Hepatol. 2014;2014. approaches in the development of hepatitis C antiviral [299] Nicholson EG, Munoz FM. A review of therapeutics in agents. Expert Opin Drug Discov. 2017;12(4):407–425. clinical development for respiratory syncytial virus and [316] Boopathi S, Poma AB, Kolandaivel P. Novel 2019 corona­ influenza in children. Clin Ther. 2018;40(8):1268–1281. virus structure, mechanism of action, antiviral drug pro­ [300] Xing Y, Proesmans M. New therapies for acute RSV mises and rule out against its treatment. J Biomol Struct infections: where are we? Eur J Pediatr. 2019;178 Dyn. 2020;1–10. (2):131–138. [317] Parks JM, Smith JC. How to discover antiviral drugs [301] Amirian ES, Levy JK. Current knowledge about the quickly. N Engl J Med. 2020;382(23):2261–2264. antivirals remdesivir (GS-5734) and GS-441524 as ther­ [318] Murgueitio MS, Bermudez M, Mortier J, et al. In silico apeutic options for coronaviruses. One Health. virtual screening approaches for anti-viral drug 2020;9:100128. discovery. Drug Discov Today Technol. 2012;9(3): [302] Gordon CJ, Tchesnokov EP, Feng JY, et al. The anti­ e219–e225. viral compound remdesivir potently inhibits [319] Shah B, Modi P, Sagar SR. In silico studies on thera­ RNA-dependent RNA polymerase from Middle East peutic agents for COVID-19: drug repurposing respiratory syndrome coronavirus. J Biol Chem. approach. Life Sci. 2020;252:117652. 2020;295(15):4773–4779. [320] Lim J, Jeon S, Shin HY, et al. Case of the index patient who [303] Agostini ML, Andres EL, Sims AC, et al. Coronavirus caused tertiary transmission of coronavirus disease 2019 susceptibility to the antiviral remdesivir (GS-5734) is in Korea: the application of lopinavir/ritonavir for the mediated by the viral polymerase and the proofreading treatment of COVID-19 pneumonia monitored by quan­ exoribonuclease. mBio. 2018;9(2):e00221-18. titative RT-PCR. J Korean Med Sci. 2020;35(6):e79. [304] Grein J, Ohmagari N, Shin D, et al. Compassionate use [321] Cao B, Wang Y, Wen D, et al. A trial of of remdesivir for patients with severe Covid-19. N Engl lopinavir-ritonavir in adults hospitalized with severe J Med. 2020;382(24):2327–2336. covid-19. N Engl J Med. 2020;382(19):1787–1799. [305] Borba MGS, Val FFA, Sampaio VS, et al. Effect of high [322] Brinkman K, ter Hofstede HJ, Burger DM, et al.. vs low doses of chloroquine diphosphate as adjunctive Adverse effects of reverse transcriptase inhibitors: therapy for patients hospitalized with severe acute mitochondrial toxicity as common pathway. AIDS respiratory syndrome coronavirus 2 (SARS-CoV-2) 1998; 12(14): 1735–1744. infection: A randomized clinical trial. JAMA Network [323] Cai Q, Yang M, Liu D, et al. Experimental treatment Open. 2020;3(4):e208857. with favipiravir for COVID-19: an open-label control [306] Pastick KA, Okafor EC, Wang F, et al. Review: hydro­ study. Engineering(Beijing). 2020;6(10):1192–1198. xychloroquine and chloroquine for treatment of [324] Bukreyeva N, Sattler RA, Mantlo EK et al. The IMPDH SARS-CoV-2 (COVID-19). Open Forum Infect Dis. inhibitor merimepodib provided in combination with 2020;7(4):ofaa130. the adenosine analogue remdesivir reduces SARS-CoV- [307] Vincent MJ, Bergeron E, Benjannet S, et al. 2 replication to undetectable levels in vitro. Chloroquine is a potent inhibitor of SARS coronavirus F1000Research. 2020, 9:361 infection and spread. Virol J. 2005;2:69. [325] Gitman MD, Hirschwerk D, Baskin CH, et al. [308] Erickson TB, Chai PR, Boyer EW. Chloroquine, hydro­ Tenofovir-induced kidney injury. Expert Opin Drug xychloroquine and COVID-19. Toxicol Commun. Saf. 2007;6(2):155–164. 2020;4:40–42. [326] Soriano V, Puoti M, Garcia-Gascó P, et al. Antiretroviral [309] Zumla A, Chan JF. Azhar EI, et al. Coronaviruses — drugs and liver injury. AIDS. 2008;22(1):1–13. drug discovery and therapeutic options. Nat Rev Drug [327] Neuman MG, Schneider M, Nanau RM, et al. HIV- Discov. 2016;15:327–347. antiretroviral therapy induced liver, gastrointestinal, [310] Seley-Radtke KL, Yates MK. The evolution of nucleo­ and pancreatic injury. Int J Hepatol. 2012;2012:760706. side analogue antivirals: A review for chemists and [328] Irwin KK, Renzette N, Kowalik TF, et al. Antiviral drug non-chemists. Part 1: early structural modifications to resistance as an adaptive process. Virus Evol. 2016;2(1): the nucleoside scaffold. Antiviral Res. 2018;154:66–86. vew014. VIRULENCE 469 [329] Wyles DL, Luetkemeyer AF. Understanding hepatitis patients with chronic hepatitis C: a retrospective C virus drug resistance: clinical implications for current study using the japanese interferon database. Drug and future regimens. Top Antivir Med. 2017;25(3):103–109. Des Devel Ther. 2014;9:283–290. [330] Kim S, Han KH, Ahn SH. Hepatitis C virus and anti­ [336] Horscroft NJ, Pryde DC, Bright H. Antiviral applica­ viral drug resistance. Gut Liver. 2016;10(6):890–895. tions of Toll-like receptor agonists. J Antimicrob [331] De Clercq E. Anti-HIV drugs: 25 compounds approved Chemother. 2012;67(4):789–801. within 25 years after the discovery of HIV. [337] Mulder KCL, Lima LA, Miranda VJ, et al. Current scenario Int J Antimicrob Agents. 2009;33(4):307–320. of peptide-based drugs: the key roles of cationic antitumor [332] Sheahan TP, Sims AC, Leist SR, et al. Comparative and antiviral peptides. Front Microbiol. 2013;4:321. therapeutic efficacy of remdesivir and combination [338] Vilas Boas LCP, Campos ML, Berlanda RLA, et al. lopinavir, ritonavir, and interferon beta against Antiviral peptides as promising therapeutic drugs. MERS-CoV. Nat Commun. 2020;11(1):222. Cell Mol Life Sci. 2019;76(18):3525–3542. [333] Chávez-Bueno S, Mejías A, Merryman RA, et al. [339] Prats-Ejarque G, Li J, Ait-Ichou F, et al. Testing Intravenous palivizumab and ribavirin combination for a human antimicrobial RNase chimera against bacterial respiratory syncytial virus disease in high-risk pediatric resistance. Front Microbiol. 2019;10:1357. patients. Pediatr Infect Dis J. 2007;26(12):1089–1093. [340] Gifre-Renom L, Cano-Garrido O, Fàbregas F, et al. [334] Unterholzner L, Bowie AG. The interplay between A new approach to obtain pure and active proteins viruses and innate immune signaling: recent insights from Lactococcus lactis protein aggregates. Sci Rep. and therapeutic opportunities. Biochem Pharmacol. 2018;8(1):13917. 2008;75(3):589–602. [341] Wishart DS, Feunang YD, Guo AC, et al. DrugBank 5.0: [335] Sato I, Shimbo T, Kawasaki Y, et al. Comparison of a major update to the DrugBank database for 2018. peginterferon alfa-2a and alfa-2b for treatment of Nucleic Acids Res. 2018;46(D1):D1074–D1082.