REVIEW published: 16 March 2021 doi: 10.3389/fmicb.2021.606087 Impact of Synonymous Genome Recoding on the HIV Life Cycle Ana Jordan-Paiz † , Sandra Franco and Miguel Angel Martínez* IrsiCaixa, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona (UAB), Badalona, Spain Synonymous mutations within protein coding regions introduce changes in DNA or messenger (m) RNA, without mutating the encoded proteins. Synonymous recoding of virus genomes has facilitated the identification of previously unknown virus biological features. Moreover, large-scale synonymous recoding of the genome of human Edited by: immunodeficiency virus type 1 (HIV-1) has elucidated new antiviral mechanisms within Cara Carthel Burns, the innate immune response, and has improved our knowledge of new functional Centers for Disease Control and Prevention (CDC), United States virus genome structures, the relevance of codon usage for the temporal regulation of Reviewed by: viral gene expression, and HIV-1 mutational robustness and adaptability. Continuous Antoinette Van Der Kuyl, improvements in our understanding of the impacts of synonymous substitutions on University of Amsterdam, Netherlands Zuben E. Sauna, virus phenotype – coupled with the decreased cost of chemically synthesizing DNA United States Food and Drug and improved methods for assembling DNA fragments – have enhanced our ability Administration, United States to identify potential HIV-1 and host factors and other aspects involved in the infection *Correspondence: process. In this review, we address how silent mutagenesis impacts HIV-1 phenotype Miguel Angel Martínez mmartinez@irsicaixa.es and replication capacity. We also discuss the general potential of synonymous recoding † Present address: of the HIV-1 genome to elucidate unknown aspects of the virus life cycle, and to identify Ana Jordan-Paiz, new therapeutic targets. Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Keywords: HIV-1, synonymous, mutations, virus, phenotype Hamburg, Germany Specialty section: INTRODUCTION This article was submitted to Virology, Despite the relatively recent introduction of HIV-1 within the human population, this virus a section of the journal has already exhibited enormous diversification. This high genetic diversity results from its fast Frontiers in Microbiology replication cycle, with the generation of about 1010 virions daily in an infected individual, coupled Received: 14 September 2020 with its high mutation rate of approximately 3 × 10−5 per nucleotide base per replication cycle, Accepted: 25 February 2021 and the recombinogenic properties of its reverse transcriptase (RT) (Coffin and Swanstrom, 2013). Published: 16 March 2021 The HIV-1 RNA genome comprises an above-average proportion of adenine (A) nucleotides, while Citation: being extremely poor in cytosine (C) (van der Kuyl and Berkhout, 2012). Interestingly, despite the Jordan-Paiz A, Franco S and high variability of the HIV-1 genome, its base composition is surprisingly stable over time, varying Martínez MA (2021) Impact of Synonymous Genome Recoding on by <1% per base per isolate regardless of whether it originates from the early or later years of the HIV Life Cycle. the epidemic (van der Kuyl and Berkhout, 2012). This stability of the peculiar base composition Front. Microbiol. 12:606087. of the HIV-1 genome strongly impacts its synonymous codon and codon pair usage, as well as doi: 10.3389/fmicb.2021.606087 dinucleotide frequencies. Frontiers in Microbiology | www.frontiersin.org 1 March 2021 | Volume 12 | Article 606087 Jordan-Paiz et al. HIV Synonymous Genome Recoding All organisms share the same genetic code, in which four Remarkably relevant is how HIV-1 synonymous genome different nucleotides generate codon triplets – such that 43 , or recoding has allowed the description of previous unknown 64, different codons are possible. These 64 codons encode 20 functions of cell factors involved in the innate immune response different amino acids and 3 translation stop codons. Of these (Li et al., 2012; Takata et al., 2017). 20 amino acids, 18 are encoded by more than one synonymous codon, and only methionine and tryptophan are encoded by a unique triplet codon. The ratios of synonymous codons are SYNONYMOUS SUBSTITUTIONS AND highly non-random, i.e., some synonymous codons appear more HIV-1 REPLICATION CAPACITY frequent than others (Grantham et al., 1980), a phenomenon termed codon usage bias. Codon usage differs among different A well-known and common application of synonymous species, strongly suggesting that codon usage is an adaptive trait nucleotide recoding is synonymous codon optimization to affected by selective pressure and random drift. Another bias increase protein expression in various systems (Mauro and that can be observed in organism genomes is that codon pair Chappell, 2014). On the other hand, an interesting and less frequencies are not random, which is termed codon-pair bias. known application of synonymous nucleotide recoding is to The frequencies of codon-pairs can be different from what would synonymously deoptimize codon usage, codon-pair usage, be expected based on the individual codon usage bias of a given or dinucleotide frequencies to reduce protein expression genome, as reviewed by Alexaki et al. (2019). Indeed, codon- and attenuate virus replication capacity, which has been pair bias have been also described in other organisms, including described for several RNA viruses (Figure 3; Martínez et al., HIV-1 (Martrus et al., 2013). 2016, 2019). Recoding viral genomes through numerous In addition to the translation and abundance of isoaccepting synonymous but suboptimal substitutions represents a new tRNAs, synonymous codon mutations can impact many other source of live attenuated vaccine candidates. In pioneer molecular phenotypes, including transcription modifications research with poliovirus, the introduction of 542 synonymous (Zhou et al., 2016; Findlay et al., 2018), translation initiation substitutions among the 2,555 nucleotides of the virus capsid (Kudla et al., 2009; Goodman et al., 2013; Stergachis et al., 2013), region reduced the virus replication capacity by up to 98% in translation elongation (Sorensen et al., 1989; Boel et al., 2016), HeLa cells (Burns et al., 2006). A similar approach involving translation accuracy (Akashi, 1994; Drummond and Wilke, synonymous deoptimization of the poliovirus capsid coding 2008), RNA stability (Presnyak et al., 2015), RNA structure and region generated a virus that exhibited a neuro-attenuated folding (Shabalina et al., 2006; Kudla et al., 2009), RNA splicing phenotype in transgenic mice (Mueller et al., 2006). Large- (Pagani et al., 2005; Takata et al., 2018), RNA toxicity (Mittal scale synonymous codon usage recoding has been used to et al., 2018), cotranslational folding (Pechmann and Frydman, generate prototypes of live attenuated vaccines for several RNA 2013), chromatin organization (Warnecke et al., 2009), enhancer viruses, including poliovirus (Burns et al., 2006; Mueller et al., functions (Lin et al., 2011; Birnbaum et al., 2012), and microRNA 2006), influenza virus (Mueller et al., 2010; Yang et al., 2013), targeting (Brest et al., 2011; Birnbaum et al., 2012). These impacts respiratory syncytial virus (RSV) (Nouën et al., 2014), vesicular of synonymous mutations on cell phenotype further indicate that stomatitis virus (Wang et al., 2015), porcine reproductive and the distribution of synonymous substitutions throughout genes respiratory syndrome virus (Ni et al., 2014), dengue virus and genomes is neither random nor neutral, and is thus subjected (Shen et al., 2015), zika virus (Li et al., 2018), echovirus 7 to selective forces. As mentioned above, HIV-1 is a good example (Fros et al., 2017), foot and mouth disease virus (Diaz-San since, although the genome is highly variable, the genomic base Segundo et al., 2021), and the plant cucumber mosaic virus composition has been tremendously stable over time. (Mochizuki et al., 2018); arboviruses, such as Chikungunya Forty years ago, the invention of PCR and the chemical virus (Nougairede et al., 2013) and tick-borne encephalitis virus synthesis of DNA oligonucleotides opened the door to synthetic (de Fabritus et al., 2015); and DNA viruses, such as Marek’s genomics. Nowadays, DNA chemical synthesis enables the disease herpesvirus (Conrad et al., 2018; Eschke et al., 2018). synthesis of 200-nucleotide-long oligonucleotides (Hughes Clinical trials have been performed using codon-deoptimized and Ellington, 2017). Overlapping single-stranded DNA type 2 poliovirus. These trials found the vaccine candidate to oligonucleotides of 100–200 nucleotides in length can easily be safe and immunogenic in infants and toddlers (Van Damme be assembled by PCR or isothermal amplification to generate et al., 2019; Konopka-Anstadt et al., 2020; De Coster et al., 2021; DNA fragments of 1,000–2,000 base pairs (bps) (Figure 1). Sáez-Llorens et al., 2021). Conventional cloning methods can be applied to clone these The HIV-1 genome has been also synonymously deoptimized synthetic DNA fragments into a bacterial plasmid vector, and (Martrus et al., 2013; Klaver et al., 2017). In one study, up to 118 individual clones can be isolated and sequenced by Sanger substitutions were introduced in the 1,508-nucleotide structural sequencing. Virus genome recoding is a recent tool that is Gag-coding region, generating viruses with lower replicative enabling us to elucidate fundamental aspects of virus biology capacities in an established MT-4 cell line and in peripheral blood (Figure 2). Synthetic recoding can also help us to develop better mononuclear cells (PBMCs) from uninfected donors (Martrus therapeutic tools, such as new synthetic vaccines and virus-based et al., 2013). The replication capacity of these recoded variants gene therapy vectors. Next, we will discuss how synthetic HIV-1 was reduced up to 39 and 85% in MT-4 cells and PBMCs, synonymous genome recoding (Table 1) is uncovering HIV-1 respectively (Martrus et al., 2013). Similarly, the introduction of biology, and opening the door to new therapeutic opportunities. 41 substitutions in protease coding region also generated a virus Frontiers in Microbiology | www.frontiersin.org 2 March 2021 | Volume 12 | Article 606087 Jordan-Paiz et al. HIV Synonymous Genome Recoding FIGURE 1 | Synthetic HIV-1 DNA oligonucleotide assembly and cell transfection. Overlapping oligonucleotides encoding a full-length HIV-1 genome DNA duplex are successively assembled via PCR or isothermal amplification. The designed HIV-1 DNA duplex can then be directly transfected into susceptible cells (e.g., MT-4 and PBMCs) to yield infectious virus (Fujita et al., 2013). Alternatively, synthetic HIV-1 DNA can be cloned in bacteria or other vectors for further manipulation. FIGURE 2 | Methods for genome synonymous recoding. Four main strategies have been used to synonymously recode virus genome sequences: codon usage modification, codon-pair usage modification, CpG content modification, and modification of codons that can generate stop mutations after a single nucleotide substitution. with reduced replication. To test the phenotypic stability of these that several deoptimizing synonymous substitutions had reverted protease and gag variants, the viruses were serially propagated to WT; many additional synonymous and non-synonymous in MT-4 cells, revealing that all deoptimized viruses recovered mutations were also detected. The clinical development of an wild-type (WT) replication capacity after 60 days of cell culture attenuated HIV-1 vaccine is thus improbable. However, these propagation (Martrus et al., 2013). Individual virus clones were experiments confirm the rapid evolution of an RNA virus, and obtained after cell culture passages, and sequencing revealed the necessity of rigorous experiments to evaluate the stability of Frontiers in Microbiology | www.frontiersin.org 3 March 2021 | Volume 12 | Article 606087 Jordan-Paiz et al. HIV Synonymous Genome Recoding TABLE 1 | Examples of HIV-1/SIV phenotype modification by genome synonymous substitutions. Targeted Gene Recoding method Implicated host factor Resulting phenotype References gag Codon usage Innate response (SLFN11) Translation inhibition (lethality) Li et al., 2012 gag, pol Codon-pair usage ND Translation inhibition (attenuation, lethality) Martrus et al., 2013 env Codon usage Innate response (Type 1 interferon) Transcription inhibition (attenuation) Vabret et al., 2014 gag, pol, env CpG content Innate response (ZAP) Transcription inhibition (attenuation, lethality) Takata et al., 2017 pol Codon-pair usage None Sequence space modification (none) Nevot et al., 2018 gag, pol, env CpG content mRNA splicing Suppression of splice sites (attenuation, lethality) Takata et al., 2018 env Codon usage mRNA splicing Suppression of essential mRNA structures Jordan-Paiz et al., 2020 (attenuation, lethality) ND, not determined. FIGURE 3 | Viral population diversity and evolvability of a wild-type sequence and a synonymously recoded sequence. (A) Synonymous recodification of a viral sequence leads to a different and limited mutant spectra which may result in the generation of stop codons. The generation of stop codons in the mutant spectra results in virus attenuation (Lauring et al., 2012; Moratorio et al., 2017). (B) Replication of a wild-type (WT) virus and a synonymous-recoded virus under a constant environment may lead to different mutant spectra, but not necessarily to generation of stop codons or viral attenuation. Under the presence of a viral inhibitor, the number of mutations found in the synonymous-recoded virus is higher than those generated in the WT virus. Moreover, these mutations differ from both viral mutant spectra, indicating that the sequence space influences the development of inhibitor resistances (Nevot et al., 2018). all candidates for new attenuated virus vaccines based on genome of its replication capacity (Vabret et al., 2014). Interestingly, the synonymous recoding. recoded virus exhibited a reduced ability to stimulate type I In another lentivirus, simian immunodeficiency virus (SIV), interferon, which may have attenuated its pathogenic potential. the introduction of 169 synonymous nucleotide optimizing Analogously, synonymous deoptimization of the Streptococcus mutations in gag and pol yielded a virus with a 100-fold decrease pneumoniae pneumolysin gene with underrepresented codon Frontiers in Microbiology | www.frontiersin.org 4 March 2021 | Volume 12 | Article 606087 Jordan-Paiz et al. HIV Synonymous Genome Recoding pairs resulted in an attenuated phenotype in a mouse pulmonary 2013). Research with polio, coxsackie B3, and influenza A infection model, which was associated with a markedly reduced viruses has revealed that synonymous recoding of the virus inflammatory response in the lungs (Coleman et al., 2011). genome can change its starting position in sequence space and Reports have also described cis-acting functions in the HIV-1 limit its access to mutational neighborhoods (Lauring et al., coding sequence for viral gene expression. Synonymous adaptive 2012; Moratorio et al., 2017), potentially resulting in virus mutations in the HIV-1 3’ pol gene result in parallel increases attenuation. Mutational neighborhoods refer to a network of or decreases in the expression levels of late viral proteins and in variants organized in sequence space around a single master viral replication capacity (Nomaguchi et al., 2014). These findings sequence (Domingo, 2020). In the cases of coxsackie B3 and suggest that viral fitness is altered through nucleotide-dependent influenza A viruses, leucine and serine codons were recoded modulation of the expression pattern of viral mRNAs. A global to favor the possibility of nonsense mutations resulting in silent mutagenesis experiment was performed to identify new cis- stop codons. The virus variants were attenuated in vivo and acting RNA elements in the HIV-1 genome that are important for exhibited increased numbers of stop codons. These findings virus replication (Takata et al., 2018). Sixteen mutant proviruses suggested the possibility of changing a virus’ starting position were designed and synthesized, which contained clusters of in sequence space, and redirecting it toward detrimental ∼50 to ∼200 synonymous mutations spanning nearly the entire mutational neighborhoods to generate self-limiting vaccine HIV-1 protein coding sequence. These mutant viruses were strains (Moratorio et al., 2017). Similarly, a synonymously analyzed and categorized into three phenotypic groups: (1) recoded poliovirus exhibited unique mutant spectra, showing mutants exhibiting near WT replication, (2) mutants exhibiting significantly different distributions of polymorphic amino replication defects accompanied by perturbed RNA splicing, acid substitutions in the capsid (Lauring et al., 2012). This and (3) mutants exhibiting replication defects without obvious recoded virus exhibited normal replication capacity in tissue splicing perturbation (Takata et al., 2017). Mutants of the culture, but displayed an attenuated phenotype in an animal second group generally contained point mutations that reduced model of infection, demonstrating the importance of mutant proximal splice site utilization. Mapping the changes responsible neighborhoods in determining viral pathogenesis. for splicing perturbations in these viruses revealed several RNA To explore whether the synonymous sequence space sequences that apparently suppressed the use of cryptic or influences the development of HIV-1 protease inhibitor (PI) canonical splice sites. These findings indicated complex negative resistance, WT HIV-1 was compared to a variant carrying a regulation of HIV-1 splicing via RNA elements in various regions protease gene with 38 synonymous mutations (13% of the of the HIV-1 genome, which maintained a balance between protease sequence) (Nevot et al., 2018). The 38 synonymous splicing and viral replication (Takata et al., 2018). Overall, these substitutions were scattered throughout the protease coding experiments demonstrated that synonymous HIV-1 recoding region, and were selected to improve protease gene codon-pair may provide insights into uncharacterized elements in the HIV-1 bias without modifying the codon bias or folding free energy genome that determine the fate and splicing of HIV-1 RNA and (Martrus et al., 2013). Importantly, replication in MT-4 cells or thus the ability of HIV-1 to replicate. PBMCs was indistinguishable between the recoded variant and the WT virus. Similar to the studies performed with poliovirus and coxsackie virus, this investigation was designed to explore SYNONYMOUS SEQUENCE SPACE how HIV-1 evolvability was influenced by the natural position in sequence space. In contrast to previous work, this study One unknown aspect of the genetic architecture of RNA viruses, explored how synonymous substitutions affected the specific including HIV-1, is how codon choice influences population selection pressure targeting a precise HIV-1 gene. Interestingly, diversity and evolvability. Early comparisons of the nucleotide upon PI treatment, the recoded virus displayed different patterns sequences of homologous genes revealed higher numbers of resistance mutations, demonstrating that sequence space of synonymous substitutions compared to non-synonymous position affects evolvability. However, although the WT and mutations, promoting an initial assumption that synonymous recoded proteases occupied different sequence spaces, they mutations were selectively neutral. This postulation contributed showed similar levels of development of phenotypic resistance to the foundations of the neutral theory of molecular evolution, to PIs – i.e., the recoded and WT virus exhibited the same in which organisms evolve mainly through the random drift robustness to overcome a specific selective pressure (Nevot of genomes carrying neutral or quasi-neutral mutations (King et al., 2018). Interestingly, the recoded virus showed significantly and Jukes, 1969; Kimura, 1983). However, although genome higher population diversity in the recoded and targeted gene random drift may play an important role in molecular evolution, following propagation in both the absence and presence of PIs. the currently available evidence indicates that synonymous It is tempting to speculate that the recoded virus was subjected mutations are not neutral (Martínez et al., 2019; Domingo, 2020). to greater pressure to change or revert to a WT-synonymous Synonymous substitutions can determine the evolutionary background. As discussed above, positioning a virus in a trajectory of a genome. Different codons that encode the same detrimental sequence space to reduce its capacity to produce fit amino acid can have different evolutionary potential in terms progeny may be a new strategy for attenuated poliovirus vaccine of the amino acids that they can access through a point development (Lauring et al., 2012). However, the research mutation, which can determine their likelihood of reaching involving the HIV-1 protease (Nevot et al., 2018) suggests that beneficial mutations that facilitate adaptation (Lauring et al., this approach must be cautiously developed, with careful testing Frontiers in Microbiology | www.frontiersin.org 5 March 2021 | Volume 12 | Article 606087 Jordan-Paiz et al. HIV Synonymous Genome Recoding of the long-term stability of synonymously recoded individual of the Env protein gp120 coding region did not significantly candidate viruses. affect virus replication capacity, despite the introduction of 15 new CpG dinucleotides (see in the next section the relevance of CpGs). In contrast, changing a single codon (AGG to CGU) SYNONYMOUS SUBSTITUTIONS AND within the gp41 coding region (HXB2 env position 2,125–2,127), HIV-1 RNA SECONDARY STRUCTURE which is located in the intronic splicing silencer (ISS), completely abolished virus replication and Env expression (Jordan-Paiz et al., RNA virus genomes contain multiple functional RNA elements 2020). Computational analyses of this mutant revealed severe that are required for translation or RNA replication. Synonymous disruption of the ISS RNA secondary structure. Moreover, a genome recoding has been exploited to identify specific RNA variant that restored the ISS secondary RNA structure also re- structures required for virus replication. Recoding does not established Env production and virus viability. These findings affect virus growth unless it destroys the sequence/structure of a indicate that external ISS loop disruption strongly affected HIV- functional RNA element (Song et al., 2012). SHAPE experiments 1 replication and Env translation – again highlighting the have revealed that in HIV-1 RNA, individual nucleotides exhibit relevance of synonymous recoding in maintaining biologically widely divergent tendencies to be base-paired (Wilkinson et al., relevant RNA structures. 2008; Watts et al., 2009). HIV-1 RNA secondary structures are conserved between strains, and thus might have a function in HIV-1 replication. As previously discussed, synonymous HIV-1 DINUCLEOTIDE FREQUENCIES recoding of the HIV-1 genome has elucidated the presence of AND INNATE IMMUNE RESPONSE unknown RNA cis-regulatory elements that influence balanced splicing and viral replication (Takata et al., 2018). Using As previously discussed, lentiviral RNA genomes (e.g., HIV-1 and dimethyl sulfate mutational profiling and sequencing (DMS- SIV) exhibit a strong bias in their nucleotide composition, with MaPseq) to investigate the HIV-1 RNA structure in cells has high frequencies of A and low content of C (van der Kuyl and revealed that the same RNA sequence can assume alternative Berkhout, 2012). In accordance with the nucleotide composition, conformations (Tomezsko et al., 2020). These findings have a biased dinucleotide frequency is also observed in HIV-1, revealed heterogeneous regions of RNA structure across the with the most frequent occurrence of the dinucleotide ApA entire HIV-1 genome, as well as alternative conformations and a lower-than-expected proportion of CpG. Reduced CpG at critical splice sites that influence the ratio of transcript frequencies are also observed in many other RNA viruses, and in isoforms. Overall, these results strongly suggest that HIV-1 RNA most vertebrate genomes. The low CpG abundance in vertebrate conformation regulates splice-site use and viral gene expression genomes can be explained by the methylation/deamination (Tomezsko et al., 2020). process that promotes the mutation of CpG to TpG/CpA (Karlin In SIV and HIV-1, the expression of Env depends on the and Mrázek, 1997). In contrast to HIV-1, although many RNA nature of the codons used (Shin et al., 2015). One interesting viruses mimic the CpG suppression of their vertebrate hosts hypothesis is that in different families of persistent viruses, codon (Simmonds et al., 2013), this phenomenon cannot be explained usage is skewed in a distinctive manner to enable temporal by methylation since they lack a DNA intermediate. Thus, regulation of late-expressed structural gene products, which is the explanation for the low CpG abundance in RNA viruses the case for HIV-1 Env. Temporal regulation of the lentiviral remains controversial. Env protein ensures its production late in the lytic cycle of Studies of several RNA viruses, including HIV-1, have revealed these persisting viruses. Notably, expression of these late gene that increases of CpG in the virus genome negatively impact products is typically induced by viral transducers produced their replication capacity (Burns et al., 2009; Atkinson et al., earlier in the viral replication cycle. One example of such a 2014; Tulloch et al., 2014; Gaunt et al., 2016; Antzin-Anduetza transducer is the HIV-1/SIV protein Rev. A nuclear localization et al., 2017; Takata et al., 2017). In HIV-1-infected individuals, the signal encoded in the rev gene enables localization of the Rev in vivo generation of de novo CpG sites carries twice the fitness protein to the nucleus, where it participates in the export of cost of mutations that do not generate CpG sites (Theys et al., unspliced and incompletely spliced mRNAs. In the absence of 2018). These findings raise questions regarding why CpG sites Rev, mRNAs of the late (structural) HIV-1/SIV genes (e.g., Env are suppressed in the HIV-1 genome, and why an increase of this protein) are retained in the nucleus, preventing their translation dinucleotide carries a fitness cost. (Karn and Stoltzfus, 2012). Toll-like receptor 9 (TLR9) recognizes bacterial and viral Env exhibits unusual codon usage that differs from that of DNA that is rich in unmethylated CpG DNA (Figure 4; Pandey the host cell. It was recently demonstrated that Rev induction et al., 2015). TLR9 is highly expressed in plasmacytoid dendritic of Env protein expression is dependent on this biased codon cells. Since the replication of HIV-1 in plasmacytoid dendritic usage (Shin et al., 2015). To determine whether codon usage cells is not fully demonstrated, the role of TLR9 in HIV-1 affected HIV-1 Env protein expression and virus viability, the CpG suppression is also unclear. Recently, Takata and colleagues codons AGG, GAG, CCU, ACU, CUC, and GGG of the HIV-1 generated several CpG-rich HIV-1 variants through the synthetic env gene were substituted with the synonymous codons CGU, random synonymous mutagenesis of different HIV-1 regions GAA, CCG, ACG, UUA, and GGA, respectively (Jordan-Paiz (Takata et al., 2017). As previously described (Atkinson et al., et al., 2020). This approach revealed that synonymous recoding 2014; Gaunt et al., 2016; Antzin-Anduetza et al., 2017), they Frontiers in Microbiology | www.frontiersin.org 6 March 2021 | Volume 12 | Article 606087 Jordan-Paiz et al. HIV Synonymous Genome Recoding FIGURE 4 | Factors of the innate immune system that might affect the nucleotide composition of the HIV-1 genome. SLFN11 sequesters tRNAs in a codon-dependent manner. Toll-like receptor 9 recognizes unmethylated CpGs in DNA, and activates the immune cascade (Pandey et al., 2015). Finally, ZAP recognizes CpG-rich non-self RNAs and induces their degradation. found that variants containing larger numbers of CpG sites had relationship between CpG frequency, ZAP, and virus replication significantly lower replication capacity. By targeting different capacity (Odon et al., 2019). cellular proteins implicated in mRNA degradation, they revealed Since the demonstration that ZAP induces the degradation that knockdown of a zinc-finger antiviral protein (ZAP) restored of viral CpG-rich mRNA, several studies have focused on the normal replication capacity of CpG-rich HIV-1 variants the mechanism underlying this binding and inhibition. As (Figure 4). ZAP is a factor involved in the innate immune previously described, ZAP recruits the RNA processing exosome response, which was first described as an inhibitor of viral RNA (Guo et al., 2007) and this complex degrades CpG-rich viral production (Gao et al., 2002). ZAP specifically binds viral mRNAs mRNAs. However, the regulation of ZAP’s antiviral activity is (Guo et al., 2004) and prevents their cytoplasmic accumulation not completely understood, as other cellular cofactors might by recruiting and promoting degradation of the RNA processing be involved in its activation. The E3 ubiquitin ligase TRIM25 exosome (Guo et al., 2007) – thereby promoting specific loss of reportedly enhances ZAP and is required for its activity (Li et al., cytoplasmic viral mRNA without affecting nuclear mRNA. While 2017; Zheng et al., 2017). ZAP interacts with several components previous studies could not determine how ZAP targets viral of the RNA exosome complex, suggesting that TRIM25 may mRNA (Guo et al., 2004), Takata and colleagues demonstrated not be the only cofactor that mediates ZAP activity. Indeed, that ZAP showed specificity for CpG-rich HIV-1 mRNA exonic it was recently demonstrated that KHNYN, a newly described regions. These authors suggested that RNA virus evolution may cytoplasmic human protein, is also essential for ZAP activity have favored low CpG content to avoid selective inhibition by against foreign CpG-rich mRNAs (Ficarelli et al., 2019b). This ZAP. Examination of another RNA virus has corroborated the study revealed how KHNYN interacted with ZAP, and that Frontiers in Microbiology | www.frontiersin.org 7 March 2021 | Volume 12 | Article 606087 Jordan-Paiz et al. HIV Synonymous Genome Recoding its overexpression dramatically reduced the infectivity capacity These variants were not inhibited by ZAP, but rather through of a synonymous recoded CpG-rich HIV-1 variant. Moreover, other ZAP-independent mechanisms, mainly related to pre- KHNYN inhibition enabled this CpG-rich variant to reach WT mRNA splicing. As described in a previous section, synonymous replication levels. mutations can also alter splicing (Takata et al., 2018). In this case, The N-terminal domain of ZAP includes four CCCH- synonymous CpGs were responsible for aberrant mRNA splicing type zinc-finger motifs that together form the ZAP RNA- and reduced viral fitness. binding domain (RBD), and are responsible for targeting As discussed above, CpG suppression in HIV-1 might be viral mRNA (Chen et al., 2012). A recent paper describes partly explained by the actions of the innate response factor the RBD structure, and how it selectively binds CpG-rich ZAP. However, other mechanisms may also contribute, including mRNA sequences (Meagher et al., 2019). Structural analysis mRNA splicing and mRNA secondary structure. The exact of ZAP revealed a pocket on the protein surface, which only mechanism of ZAP targeting and binding remains to be binds CpG dinucleotides (Meagher et al., 2019). This ZAP elucidated. This phenomenon may be further studied through the structure may explain how ZAP avoids low-CpG host mRNA generation of different HIV-1 variants with increasing amounts of and recognizes foreign mRNAs. Importantly, another recent CpG sites in different regions of the genome, which could also be publication describes the molecular mechanism through which useful for identifying new factors that might be involved in the ZAP only detects CpG sites in a single-stranded form (Luo inhibition of HIV-1 replication. et al., 2020). The four zinc-finger motifs of ZAP form a specific architecture that enables extensive interactions with RNA. They further revealed that an RNA containing several ZAP-binding HIV-1 CODON USAGE AND INNATE sites can be recognized by multiple ZAP molecules, which likely IMMUNE RESPONSE enhances the activation of the exosome complex. The authors hypothesize that a single CpG dinucleotide cannot bind ZAP and HIV-1-biased nucleotide composition can produce activate the exosome complex, but rather must be single-stranded overstimulation of the type I interferon, suggesting that and surrounded by other CpG sequences that can bind several RNA sequences may be discriminated based on their nucleotide ZAP molecules, potentially exerting a synergistic effect. composition (Vabret et al., 2012). Type I interferon is a major Despite recent descriptions of the ZAP structure and antiviral cytokine that contributes to chronic immune system molecular mechanism, there remains some controversy activation and progression to AIDS during HIV infection surrounding how CpG-rich mRNAs are targeted. Investigations (Jacquelin et al., 2009). Pathogenicity is reportedly correlated of the genomes of different primate lentiviruses do not reveal any with divergent nucleotide composition of HIV-1 compared to correlation between the number of CpG sites and the sensitivity host (Vabret et al., 2012), suggesting that virus-host interactions to ZAP. As previously noted, the HIV-1 genome contains a very might be altered by artificially changing the nucleotide frequency low proportion of CpG sites. In contrast, HIV-2 shows less CpG of the HIV-1 genome. With this aim, SIV codon usage was suppression. However, although the CpG content is higher in optimized to be closer to the average nucleotide composition HIV-2 than in HIV-1, no ZAP inhibition is observed in HIV-2 of the SIV macaque host (Vabret et al., 2014). A synthetic (Kmiec et al., 2020). This phenomenon has also been observed SIV optimized with 169 synonymous mutations in gag and pol for another lentivirus, SIVmac, which exhibits a higher CpG exhibited a 100-fold decrease of replicative capacity. Interestingly, content but significantly lower ZAP inhibition compared to this optimized variant also exhibited reduced ability to stimulate HIV-1. This raises the question of why ZAP can inhibit HIV-1, type I interferon in infected human and macaque PBMCs but not other related lentiviruses. (Vabret et al., 2014). No reversion of the introduced mutations It is hypothesized that a region in the genome, termed ZAPsen, was observed after ten serial cell passages, suggesting that this may confer ZAP sensitivity. In HIV-1, this ZAPsen region is variant may be a safe candidate for an attenuated vaccine. Still, described as located in the 5’ region of the env gene (from position further experiments should be performed to confirm the stability 6,239–6,947 of the HIV-1 HXB2 reference genome) (Kmiec et al., of this attenuated variant. Live attenuated SIV vaccines are 2020). And increase of synonymous CpG sites in ZAPsen confers highly protective in the macaque AIDS model (Koff et al., 2006). higher ZAP sensitivity and lower virus replication capacity However, in addition to safety concerns, this optimized SIV (Ficarelli et al., 2019a; Kmiec et al., 2020). However, this is variant raises intriguing questions related to the fact that a type not the only factor that affects replication. ZAP sensitivity is I interferon response is necessary for a vaccine to shape adaptive also increased by the introduction of other ZAPsen mutations immune responses and memory. that alter the RNA secondary structure, without modifying the The Schlafen (SLFN) gene family was first discovered in 1998, number of CpGs. Thus, ZAP may detect the ZAPsen region as regulators of T-cell maturation. The name Schlafen means based on either the number of CpG sites, or changes in the “to sleep” in German, and was chosen based on the observation RNA secondary structure. ZAP shows greater affinity to ZAPsen that enhanced SLFN1 expression resulted in G0 /G1 cell cycle when higher amounts of CpGs are introduced, but other factors arrest (Schwarz et al., 1998). SLFN genes are categorized as may also explain why CpG suppression is observed along the interferon-stimulated genes (ISGs), as their expression is induced whole HIV-1 genome. Accordingly, synonymous mutations that by type I interferon. Some SLFN proteins possess RNA cleavage increase the number of CpGs in other HIV-1 regions also activity, and exhibit antiviral activity against RNA and DNA lead to reduced replication capacity (Ficarelli et al., 2019a). viruses. In particular, SLFN11 potently and selectively inhibits Frontiers in Microbiology | www.frontiersin.org 8 March 2021 | Volume 12 | Article 606087 Jordan-Paiz et al. HIV Synonymous Genome Recoding HIV-1 protein translation and virus production (Li et al., 2012), frequencies has been applied in several RNA virus genomes and is considered an HIV-1 restriction factor. SLFN11 also to generate new attenuated vaccines. Nevertheless, the safety inhibits other retroviruses, including murine leukemia virus and and stability of these attenuated vaccines remain to be feline immunodeficiency virus (Stabell et al., 2016), such that elucidated. Due to safety concerns, it is very difficult to its antiretroviral effect has been classified as host-specific but envision the successful development of an attenuated HIV-1 virus-independent. vaccine. However, recoded HIV-1 variants can be used in gene Interestingly, the inhibitory effect of SLFN11 on viral protein therapy, as a vaccine vector for immunization against diverse expression is intrinsic to the transcripts. SLFN11 acts at the microorganisms, or in immunotherapy to elicit specific innate point of virus protein synthesis by exploiting the unique viral immune responses to treat particular conditions. Importantly, codon bias toward A/U nucleotides, sequestering tRNAs in artificial HIV-1 synonymous recoding has greatly increased our a codon-dependent manner (Figure 4). Accordingly, SLFN11 knowledge regarding its interaction with the host. Specifically, only affects WT HIV-1, and does not recognize synonymously HIV-1 synonymous recoding impacts virus RNA nucleotide recoded viruses in which the HIV-1 structural gag sequences composition and RNA secondary structure which regulate splice- are optimized for human codon usage (i.e., without A/U in the site use and viral gene expression. Together with structural RNA third position) (Li et al., 2012). Again, and similar to the findings features, changes in nucleotide composition also affect HIV-1 obtained with SIV, HIV-1 replication was attenuated by codon susceptibility to endogenous cell innate responses and correlated optimization for host translation. This model is in line with with differences in clinical progression rates, suggesting a findings that SLFN11’s antiviral activity extends to other viruses potential role of virus RNA nucleotide composition in HIV-1 with rare codon bias (e.g., influenza, which also has a high A in vivo pathogenicity. content) but not to adeno-associated virus or herpes simplex virus (Li et al., 2012). Overall, investigations in SIV and HIV-1 have shown that innate immunity restricts sequence landscapes AUTHOR CONTRIBUTIONS by targeting specific sequences and sequence patterns that are primarily found in pathogens (Vabret et al., 2017). AJ-P, SF, and MM discussed and wrote this review. All authors contributed to the article and approved the submitted version. CONCLUSION FUNDING Synonymous rewriting of the HIV-1 genome is helping to elucidate essential genome functions. 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U S A. 110, potential conflict of interest. 9481–9486. doi: 10.1073/pnas.1307473110 Zheng, X., Wang, X., Tu, F., Wang, Q., Fan, Z., and Gao, G. (2017). TRIM25 Is Copyright © 2021 Jordan-Paiz, Franco and Martínez. This is an open-access article Required for the Antiviral Activity of Zinc Finger Antiviral Protein. J. Virol. 91, distributed under the terms of the Creative Commons Attribution License (CC BY). 88–17e. The use, distribution or reproduction in other forums is permitted, provided the Zhou, Z., Dang, Y., Zhou, M., Li, L., Yu, C. H., Fu, J., et al. (2016). Codon usage original author(s) and the copyright owner(s) are credited and that the original is an important determinant of gene expression levels largely through its effects publication in this journal is cited, in accordance with accepted academic practice. No on transcription. Proc. Natl. Acad. Sci. U S A. 113, E6117–E6125. use, distribution or reproduction is permitted which does not comply with these terms. 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