ARTICLE https://doi.org/10.1038/s42003-023-04841-y OPEN Schlafen 12 restricts HIV-1 latency reversal by a codon-usage dependent post-transcriptional block in CD4+ T cells Mie Kobayashi-Ishihara 1,16,17,18 ✉, Katarína Frazão Smutná1,17, Florencia E. Alonso1, Jordi Argilaguet1,2,3, Anna Esteve-Codina 4, Kerstin Geiger1, Meritxell Genescà 5, Judith Grau-Expósito5, Clara Duran-Castells6, Selina Rogenmoser1, René Böttcher7, Jennifer Jungfleisch7, Baldomero Oliva 8, Javier P. Martinez1, Manqing Li 9, Michael David 9,10, Makoto Yamagishi 11, Marta Ruiz-Riol6,12, Christian Brander 6,13,14, Yasuko Tsunetsugu-Yokota 15, Maria J. Buzon 5, Juana Díez 7,18 ✉ & Andreas Meyerhans 1,14,18 ✉ 1234567890():,; Latency is a major barrier towards virus elimination in HIV-1-infected individuals. Yet, the mechanisms that contribute to the maintenance of HIV-1 latency are incompletely under- stood. Here we describe the Schlafen 12 protein (SLFN12) as an HIV-1 restriction factor that establishes a post-transcriptional block in HIV-1-infected cells and thereby inhibits HIV-1 replication and virus reactivation from latently infected cells. The inhibitory activity is dependent on the HIV-1 codon usage and on the SLFN12 RNase active sites. Within HIV-1- infected individuals, SLFN12 expression in PBMCs correlated with HIV-1 plasma viral loads and proviral loads suggesting a link with the general activation of the immune system. Using an RNA FISH-Flow HIV-1 reactivation assay, we demonstrate that SLFN12 expression is enriched in infected cells positive for HIV-1 transcripts but negative for HIV-1 proteins. Thus, codon-usage dependent translation inhibition of HIV-1 proteins participates in HIV-1 latency and can restrict the amount of virus release after latency reversal. 1 Infection Biology Group, Department of Medicine and Life Sciences, Universitat Pompeu Fabra, Barcelona, Spain. 2 Unitat mixta d’Investigació IRTA-UAB en Sanitat Animal. Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, Spain. 3 IRTA. Programa de Sanitat Animal. Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, Spain. 4 Centro Nacional de Análisis Genómico (CNAG-CRG), Center for Genomic Regulation (CRG), Barcelona Institute of Science and Technology, Barcelona, Spain. 5 Infectious Disease Department, Hospital Universitari Vall d´Hebrón, Institut de Recerca (VHIR), Universitat Autònoma de Barcelona, Barcelona, Spain. 6 IrsiCaixa AIDS Research Institute, Hospital Germans Trias i Pujol, Universitat Autonoma de Barcelona, Badalona, Spain. 7 Molecular Virology Group, Department of Medicine and Life Sciences, Universitat Pompeu Fabra, Barcelona, Spain. 8 Structural Bioinformatics Group, Department of Medicine and Life Sciences, Universitat Pompeu Fabra, Barcelona, Spain. 9 Section of Molecular Biology, Division of Biological Sciences, University of California San Diego, La Jolla, CA, USA. 10 Moores Cancer Center, University of California San Diego, La Jolla, CA, USA. 11 Graduate School of Frontier Sciences, The University of Tokyo, Tokyo, Japan. 12 CIBER de Enfermedades Infecciosas, Madrid, Spain. 13 Universitat de Vic-Universitat Central de Catalunya (UVic-UCC), Vic, Spain. 14 Institució de Recerca i Estudis Avançats (ICREA), Barcelona, Spain. 15 Department of Medical Technology, School of Human Sciences, Tokyo University of Technology, Tokyo, Japan. 16Present address: Department of Molecular Biology, Keio University School of Medicine, Tokyo, Japan. 17These authors contributed equally: Mie Kobayashi-Ishihara, Katarína Frazão Smutná. 18These authors jointly supervised this work: Mie Kobayashi-Ishihara, Juana Díez, Andreas Meyerhans. ✉email: mieki@keio.jp; juana.diez@upf.edu; andreas.meyerhans@upf.edu COMMUNICATIONS BIOLOGY | (2023)6:487 | https://doi.org/10.1038/s42003-023-04841-y | www.nature.com/commsbio 1 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-023-04841-y T he main obstacle in curing an established HIV-1 infection four main groups, referred to as clusters I to IV by k-means is the long-lived reservoir of latently infected CD4+ T clustering17 (Fig. 1c and Supplementary Data 2). Genes of cluster cells1. These cells are treatment-resistant and therefore I (n = 384) are upregulated in both HSP and HSP + TCR con- enable the persistence of HIV-1 proviruses despite combination ditions compared to either TCR or TCR + TCR conditions. Since antiretroviral therapy (cART) and antiviral immune responses. our previous work demonstrated that HIV-1 proviruses in HSP Once cART is interrupted, HIV-1 rapidly rebounds from the viral condition, but not TCR conditions, were refractory to TCR reservoir even after years-long treatment periods1. During treat- activation (HSP + TCR condition)16, cluster I is expected to ment, the reservoir is maintained by T cell expansion that can be contain candidate factors that contribute to HIV-1 restriction. activated by (i) antigen-driven proliferation, (ii) integration-site- GO analysis of the cluster I genes showed significant enrichment driven proliferation, and (iii) homeostatic proliferation (Reviewed of the terms immune response, cell activation and immune system in refs. 2,3). Consequently, targeting the mechanisms governing process (Fig. 1d). Ten of these genes were reported to be linked to the expansion of infected T cells represents a potential treatment HIV-1 inhibition according to the NIH HIV interactome strategy in HIV-1 cure attempts. database18–20 (Supplementary Data 3). For example, CD63 has Antigen-driven proliferation triggered by T cell receptor (TCR) been shown to repress HIV-1 infection21–24 and HAVCR2 inhi- signaling is a strong physiological inducer of CD4+ T cell expan- bits viral budding25 (Fig. 1e). sion. While this also reactivates latent HIV-1 and thus, can be To further narrow down candidate restriction factors, we the source of viral rebound4,5, sequential waves of polyclonal selected from the cluster I genes those that are (i) differentially T cell stimulation in the presence of cART may result in viral expressed either in HSP vs TCR, or in HSP + TCR vs TCR + TCR reservoir reduction according to the “rinse and replace” strategy6. (FDR < 5%, Supplementary Data 4), and that are (ii) members of a Latently infected T cells may also expand clonally when virus gene family that contains a known restriction factor (RF). The latter integration takes place within cancer-associated genes that affect criterion was used because RF-containing gene families often have cell proliferation7–10. Given the observed enrichment for such members with partially redundant functions to safeguard an integration events over non-cancer-associated genes, this expan- organism against pathogenic threats26,27. A family member list was sion mechanism seems common10. Nonetheless, latency reversal therefore created that includes known restriction factors28. It is depends on position effects i.e., the exact location of the pro- given in Supplementary Data 4. From the total of 1,501 family virus within the chromosome11 and thus, the mere expansion of members, 58 genes fulfill the above criteria as candidate RFs for infected T cell clones does not necessarily guarantee provirus HIV-1 latency control (Fig. 1f). Among these 58 candidates, 44 transcriptional activity. Finally, infected CD4+ T cells may gene products were attributed to clusters of differentiation (CD) expand through homeostatic proliferation (HSP) driven by molecules like the restriction factor CD317 (BST2 = Tetherin) interleukins IL-7 and IL-15. Unlike antigen-driven prolifera- (Fig. 1g and Supplementary Data 5). However, since our previous tion, HSP allows the expansion of HIV-1-infected CD4+ T cells work pointed to a post-transcriptional restriction of HIV-1 in HSP without activating HIV-1 expression12,13. While these condi- condition cultured CD4+ T cells16, we paid particular attention to tions strongly activate STAT5 signaling14,15, the mechanisms members of three gene families that could potentially be involved in for HIV-1 containment are unknown. Our previous work using post-transcriptional and/or translational events (Fig. 1g). Protein HIV-1-infected primary CD4+ T cells maintained under HSP phosphatase 1 regulatory subunits (PPP1Rs) are a gene family culture conditions suggested a post-transcriptional block as a whose members interact with and regulate serine/threonine cause of the containment16. Here we demonstrate that SLFN12, phosphatases. One of these is EIF2AK2 (also known as Protein a member of a conserved family of proteins with antiviral kinase R) that senses viral RNAs and enhances the integrated stress activities, participates in the maintenance of HIV-1 latency and response (ISR)29–31, which enables the shutdown of global protein may enable to replenish the HIV-1 reservoir pool during synthesis32. Four other family members are PPP1R9A, BCL2L2, homeostatic proliferation in vivo. AATK, and SPRED1 (Supplementary Fig. 1). PPP1R9A is predicted to interact with F-actin and inhibits protein phosphatase 1-ɑ activity33. BCL2L2 encodes a BCL2-like protein which is usually Results involved in apoptosis and thus could contribute to cell expansion SLFN12 is differentially expressed in homeostatic proliferating under HSP conditions34. AATK is a serine/threonine-protein primary CD4+ T cells and is a candidate for post- kinase, which is likely involved in neuronal differentiation35. transcriptional blockage of HIV-1. To decipher the mechan- SPRED1 activates MAP-kinase signaling36. Although these pro- isms that contribute to the HIV-1 refractory state in homeostatic teins have various functions, they all have the potential to proliferating CD4+ T cells, we analyzed differentially expressed deactivate translation factors by modifying their phosphorylation genes in primary CD4+ T cells that were cultured either under status in response to a viral infection. In addition, the candidate HSP conditions or after TCR-stimulation. For this, naive CD4+ genes UACA and ANKRD50 are members of the Ankyrin repeat T cells were purified from peripheral blood mononuclear cells family to which RNASEL (RNase L) also belongs (Supplementary (PBMCs) of three HIV-1 uninfected blood donors (purity > 90%) Fig. 1). RNase L can cleave viral RNAs to induce inflammatory and cultured in four different ways schematically shown in responses37,38. The Ankyrin domain of RNase L is critical to Fig. 1a, A–D; cultures with IL-7 and IL-15 (HSP-cultured CD4+ forming functional dimers and sensing viral RNAs39. While the T cells) after stimulation via the TCR at day 12 (B; HSP + TCR) function of ANKRD50 has not been well characterized, UACA is a or not stimulated (A; HSP), and cultures with IL-2 after anti- known repressor of NF-κB transcription40,41 and these proteins CD3/CD28 activation (TCR-cultured CD4+ T cells) with a sec- may together participate in viral RNA sensing and innate immune ond TCR activation at day 12 (D; TCR + TCR) or without (C; activation within HSP-cultured CD4+ T cells. Finally, within TCR). Around half of the cells maintained their naive phenotype cluster I there was SLFN12, a member of the Schlafen protein under HSP culture conditions whereas the majority of cells family (SLFN). This protein has been identified in two high- became memory cells under TCR conditions (Fig. 1b). On day 13, throughput screenings for interferon-induced antiviral and anti- RNA was isolated from the cultured cells and transcriptomes retroviral factors42,43. It has moderate activity against vesicular analyzed by RNA-seq (Supplementary Data 1). Genes were stomatitis virus (VSV)43 and a mouse gammaherpes virus (MHV- ranked by their standard deviation across the four culture con- 68)43 as well as activity against several retroviruses including ditions. The top 2000 variably expressed genes were classified into HIV-142. Its mechanism of antiviral activity has not yet been 2 COMMUNICATIONS BIOLOGY | (2023)6:487 | https://doi.org/10.1038/s42003-023-04841-y | www.nature.com/commsbio COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-023-04841-y ARTICLE studied. For several of the other SLFN proteins, effective antiviral the statistical signal observed in our in vitro stimulation, we functions have been reported (recently reviewed by Kim et al.44). hypothesized that SLFN12 might be another member of the SLFN11 was shown to be a restriction factor (RF) repressing HIV-1 SLFN family with an anti-HIV-1 activity that might act at a post- protein translation45. Other human SLFN proteins namely SLFN5, transcriptional level. SLFN13 and SLFN14 are known to attenuate the production of To first confirm SLFN12 gene expression in CD4+ T cells under several viruses including influenza virus, retroviruses, and HSP and TCR culture conditions, CD4+ T cells from five flaviviruses46–49. Based on these descriptions of SLFN genes and additional healthy blood donors were cultured under the respective COMMUNICATIONS BIOLOGY | (2023)6:487 | https://doi.org/10.1038/s42003-023-04841-y | www.nature.com/commsbio 3 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-023-04841-y Fig. 1 Differential RNA-seq analysis of cultured naive CD4+ T cells to identify candidate HIV-1 restriction factors. a Scheme of naïve CD4+ T cell cultures. Naïve CD4+ T cells from 3 different HIV-1-negative blood donors were maintained for 13 days under 4 different conditions: IL-7+IL-15 alone (A- HSP) or with anti-CD3/CD28 activation at day 12 (B-HSP+ TCR); IL-2 after anti-CD3/CD28 activation alone (C-TCR) or with a second activation by anti- CD3/CD28 at day 12 (D-TCR+ TCR). On day 13, total RNA was isolated and processed for transcriptome analysis to identify candidate HIV-1 restriction factors. b Phenotypes of CD4+ T cells maintained under HSP or TCR condition. A representative result of one of the three blood donors utilized in a is shown. Isolated naive CD4+ T cell purity was above 91%, and 96.5% for the donor shown in the upper panel. After 12-day cultivation, a naïve T cell phenotype is partially maintained in HSP conditions (CD27+/CD45RO−, 45.1%; on average >30%), while TCR conditions increased cells with central memory phenotype (CD27+/CD45RO+, 35.1%; on average >35%). TN, naïve T cells; TCM, central memory T cells; TEM, effector memory T cells; TTM, terminally differentiated memory T cells. c Heatmap of the top 2,000 variable genes clustered by k-means analysis (red: upregulated, blue: downregulated). Four main clusters were identified: Cluster I, genes upregulated in both HSP and HSP + TCR conditions compared to either TCR or TCR + TCR conditions (n = 384). Cluster II, genes upregulated in HSP, HSP + TCR and TCR conditions (n = 677). Cluster III, genes with a trend to be upregulated in TCR condition (n = 205). Cluster IV, genes upregulated by TCR stimulation at day12 (n = 734). These differences in expression are visualized on the right panel for clarity. d Top 5 gene ontology terms of the cluster I genes. e Expression patterns of two known inhibitors of HIV-1 replication/function in the cluster I according to the NIH HIV interaction database18–20. Mean counts per million reads (CPM) and the standard error of the mean (SEM; n = 3) are shown. f Venn diagram shows overlap among (i) cluster I genes, (ii) differentially expressed genes in HSP vs TCR and in HSP + TCR vs TCR + TCR conditions (DEGs), and (iii) members of gene families that contain a known restriction factor (RF gene family members). g Candidate restriction factors identified in f and their corresponding gene families. The asterisks show gene families that could potentially be involved in post- transcriptional and/or translational events. h Expression pattern of SLFN11 and SLFN12 from 3 blood donors. Plots represent mean CPM ± SEM. conditions (Fig. 1a) and SLFN12 mRNAs were quantified by qPCR SLFN12 restricts HIV-1 production by selectively inhibiting (Supplementary Fig. 2). The expression pattern was consistent virus protein translation. HEK 293T cells do not express SLFN11 with that of the RNA-seq analysis (Fig. 1h) and differed from the or SLFN12 (Fig. 2a). To test how SLFN12 influences HIV-1 repli- other SLFN family mRNAs that we analyzed in comparison cation, we co-transfected HEK 293T cells with pmCherry-SLFN11 (Supplementary Figs. 2 and 3). Indeed, these other SLFNs did not (positive control), pmCherry-SLFN12 or empty pmCherry vector show a trend towards a decrease from HSP + TCR to TCR culture (mock) together with the HIV-1 pNL-E vector, an HIV-1 provirus conditions and thus were considered non-consistent with a cluster I clone encoding EGFP between the env and nef coding region50 expression pattern (Fig. 1c). (Fig. 3a). Expression system of SLFN proteins was confirmed by Western blots (Fig. 3b). 48 h post-transfection, supernatants and SLFN12 inhibits HIV-1 reactivation in a post-transcriptional cell lysates were collected for further analyses. SLFN12 affected process. To establish a suitable in vitro model of viral reactivation HIV-1 production and strongly diminished HIV-1 titres as well as and SLFN12 modulation, we evaluated different cell lines for SLFN Gag-p24 protein in supernatants in a dose-dependent manner gene expression profiles (T cell lines Jurkat, A3.01, ACH2 and (Fig. 3c, d). This decrease was unlikely due to inhibitory effects on embryonic kidney cells HEK 293T; Fig. 2a). SLFN5 was expressed viral RNA processing or export (Fig. 3e, f, Supplementary Fig. 4). in all cell lines and was the only SLFN family member expressed in Western blot analysis with specific antibodies showed down- HEK 293T cells. SLFN14 expression was not found in any of the regulation of Gag-p24 and the Nef accessory protein (Fig. 3g). tested cells. SLFN11 and SLFN12 were expressed in Jurkat, However, we did not observe any change in cellular GAPDH or A3.01 cells and in HIV-1 latently infected ACH2 cells. Importantly, enhanced green fluorescent protein (EGFP) derived from the pNL- ACH2 cells had a similar expression pattern of SLFN11 and E vector. Thus, SLFN12, as SLFN11, inhibit HIV-1 production by SLFN12 as primary CD4+ T cells under HSP culture conditions. selectively reducing translation of at least some viral proteins. Therefore, given that HSP has been suggested to maintain the HIV- 1 latent reservoir and that the ACH2 cell line is an established SLFN12 affects the translational machinery to stall ribosomes model for HIV-1 latency, we used ACH2 cells to study the effect of on HIV-1 mRNA. To determine how SLFN12 may influence the SLFN12 on HIV-1 reactivation. SLFN11 has been shown to restrict process of translation of viral proteins, we co-transfected HEK HIV-1 at a translational level45 and was used as a positive control 293T cells with SLFN vectors and pNL-E, and performed a poly- for the subsequent experiments. some profiling analysis (Fig. 4a). UV absorbance profiles showed To knockdown SLFN12 expression in ACH2 cells, we first no significant differences in total cellular RNA distribution among generated retroviral vectors expressing specific shRNAs against the three transfected cells (Fig. 4b). Given similar monosome to SLFN12 (shSLFN12#1/ shSLFN12#2), SLFN11 (shSLFN11) or polysome ratios (Fig. 4c), SLFN12, as well as SLFN11, seems not to expressing a scrambled shRNA (shSc) as control. ACH2 affect global mRNA translation. Next, we analyzed the distribution cells were then transduced with these vectors and HIV-1 was of GAPDH mRNAs, whose expression levels are not affected by reactivated by treatment with the HDAC inhibitor SAHA SLFN12 or SLFN11 expression (Fig. 3g), and HIV-1-gag mRNAs (also known as Vorinostat) as shown schematically in Fig. 2b. within monosome and polysome fractions. GAPDH mRNAs were Cell lysates and supernatants (SN) were harvested at 48 and mainly distributed in light polysomes even in the presence of the 72 h (h) post-reactivation, respectively, and analyzed. The SLFN proteins (Fig. 4d, fractions 8–12, 13–17, and 18–23). How- mRNAs of SLFN12 or SLFN11 were roughly 60% suppressed ever, upon SLFN12 or SLFN11 expression, HIV-1-gag mRNAs by the specific shRNAs (Fig. 2c). After treatment with SAHA or were shifted from light polysomes towards heavy, elongating the vehicle control DMSO, virus-containing supernatants from polysomes (Fig. 4e). This increase of ribosomes per HIV-1 gag ACH2 cells were titrated using TZM-bl cells. Knockdown of mRNA, together with the observed inhibition of HIV-1-p24 SLFN12 (SLFN11) expression resulted in at least 5.7-fold (2.8- expression levels (Fig. 3g), is concordant with slow or stuck ribo- fold) increase in infectious HIV-1 production (Fig. 2d). More- somes i.e., slowing down translation elongation. over, the translation efficiency of HIV-1 Gag-Pr55 significantly increased (Fig. 2e, p < 0.05), indicating that SLFN12, as SLFN11, control HIV-1 reactivation from latently infected T cells by Translational block by SLFN12 is codon-usage-dependent. The repressing the translation efficiency of Gag-Pr55. translation elongation rate is severely affected by codon optimality 4 COMMUNICATIONS BIOLOGY | (2023)6:487 | https://doi.org/10.1038/s42003-023-04841-y | www.nature.com/commsbio COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-023-04841-y ARTICLE SLFN5 10-3 SLFN11 Relative RNA level SLFN12 10-4 SLFN13 10-5 10-6 3T at 01 2 T T H 4+ rk 4+ 3. 29 C D Ju A A D EK C C R H SP TC H Fig. 2 SLFN12 affects HIV-1 latency reversal from ACH2 cells. a Relative transcript levels of the human SLFN family members 5, 11, 12 and 13 in different cell lines (mean values of two repeated measurements) as well as in naive CD4+ T cells under HSP or TCR culture conditions. Naïve CD4+ T cells were isolated from five independent HIV-1-negative blood donors and cultivated for 13 days with IL-7 + IL-15 (HSP condition) or with anti-CD3/CD28 antibodies + IL-2 (TCR condition) as in Fig. 1a. Transcripts were quantified by RT-qPCR with specific primer pairs and normalized to cellular 18S rRNA. Dot plots of the individual values and p-values were provided in Supplementary Fig. 2. b Flow chart of SLFNs knockdown and HIV-1 reactivation. HIV-1 latently infected ACH2 cells were transduced with retroviral vectors expressing specific shRNAs against SLFN11 (shSLFN11) or SLFN12 (shSLFN12#1/shSLFN12#2), or with a scrambled shRNA (shSc) as a negative control. The cells were then treated with SAHA to reactivate HIV-1. DMSO at a concentration that equals the activation mix served as the vehicle control. Cell lysates and supernatants (SN) were harvested at 48- and 72-h post-reactivation, respectively, and analyzed. c Relative RNA levels of SLFN11 (left panel) and SLFN12 (right panel) in the knockdown ACH2 cells (n = 4 biological replicates; mean ± SD). *** represents p < 0.01 by Student’s t test. d SAHA-induced HIV-1 reactivation from ACH2 cells after SLFN knockdown. Fold HIV-1 reactivation was determined by titration of HIV-1-containing supernatants on TZM-bl cells and normalized to the basal HIV-1 level in DMSO-treated samples. Given is the mean ± SD for three independent samples. e Knockdown of SLFN11 and SLFN12 in ACH2 cells increases translation efficiency of HIV-1 Gag-Pr55. Plots represent fold change (FC) translation efficiency of Gag-Pr55 in SAHA-treated samples to DMSO-treated samples. Pr55 translation efficiency was calculated as a ratio of cellular Pr55 protein levels to gag RNA levels. The cellular Pr55 protein and HIV-1 gag RNA levels were quantified by Western blot and RT-qPCR, respectively. The mean FC efficiency of the control knockdown cells (shSc) was set to 1. Results show the mean ± SD of three independent experiments. Ratio paired t-test was used to calculate statical significance (*p < 0.05, **p < 0.005). of transcripts and the availability of cognate transfer RNAs vector expressing either HIV-1-gag wt (pGag-wt) or a codon- (tRNAs)51,52. SLFN11 has been shown to inhibit HIV-1 translation optimized HIV-1-gag (pGag-opt). Protein and mRNA levels were in a codon-usage-dependent manner45. To test whether also then analyzed 48 h later (Fig. 5b). Compared to mock, SLFN12 affects HIV-1 translation elongation in a codon-usage- SLFN12 strongly diminished translation of Gag-p24 protein tran- dependent manner, we first compared viral codon frequencies to scribed from HIV-1-gag wt (Fig. 5c), however, protein expression RefSeq-based human coding sequences using the Codon Adapta- in cells transfected with pGag-opt remained unchanged (Fig. 5d). tion Index (CAI), a general metric to analyze codon usage bias53. Since the RNA levels of HIV-1-gag wt and HIV-1-gag opt were not As previously described54, HIV-1 sequences harbor less optimal significantly altered by SLFN12, HIV-1 inhibition by SLFN12 codons (lower CAI values) compared to most human transcripts (Fig. 3) may indeed occur at a post-transcriptional step. including GAPDH (Fig. 5a). Next, we compared the effect of To identify which codons may de-optimize the HIV-1 codon SLFN12 on Gag-p24 protein production from wild-type (wt) and usage, we compared the relative synonymous codon usage codon-optimized HIV-1-gag transcripts. For this, HEK 293T cells (RSCU)55 amongst HIV-1, HIV-2, Chikungunya virus (CHIKV) were co-transfected with the SLFN12 expression vector plus a as an example of an unrelated virus, and humans (H.sapiens). COMMUNICATIONS BIOLOGY | (2023)6:487 | https://doi.org/10.1038/s42003-023-04841-y | www.nature.com/commsbio 5 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-023-04841-y Fig. 3 SLFN12 inhibits HIV-1 at the level of translation. a Experimental outline. HEK 293T cells were co-transfected with an expression plasmid encoding mCherry-fused SLFN11 or SLFN12, or mCherry alone, and the HIV-1 vector pNL-E. At 48 h post-transfection, supernatants (SN) and cell lysates were collected and processed for further analyses as indicated. b Expression of recombinant SLFN proteins in the transfected HEK 293T cells was studied by Western blots with an anti-mCherry antibody. c SLFN12 expression strongly diminishes HIV-1 production from transfected HEK 293T cells. Supernatants from the co-transfected cells were titrated by the TZM-bl assay (upper panel; n = 3 for each SLFN11 (denoted as rectangles) or SLFN12 (triangles) independent transfection; mean ± SD; *p < 0.05 by Student’s t test) and analyzed by Western blot (lower panel; a representative example of three independent experiments is shown). d Dose-dependent inhibition of HIV-1 production by SLFN12. HEK 293T cells were co-transfected with pNL-E and increasing amounts of pmCherry-SLFN12 (0, 0.2, 0.4, 0.8 or 1.6 µg). The empty mCherry plasmid was added to maintain constant DNA amounts for all transfections. SNs were harvested 48 h after transfection and titrated by TZM-bl assay (n = 3 biological replicates; mean ± SD; *p < 0.05 by Student’s t test). SLFN12 expression did not significantly affect the levels of HIV-1 gag RNA (e) or total HIV-1 RNA (f). RNA levels were quantified by RT-qPCR and normalized to 18S rRNA levels (n = 4 biological replicates; mean ± SD). There were no significant differences amongst the indicated samples (Student’s t test). g Western blot from cell lysates of co-transfected HEK 293T cells with indicated antibodies. An arrow and asterisk highlight the bands of Nef and a nonspecific protein, respectively. Representative results of three independent experiments are shown. As previously reported56,57, HIV-1 and -2 utilize many A-ending known to degrade tRNAs49,61–63. Comparison of sequences and codons that are rare in humans and CHIKV (Fig. 5e and conformations of SLFN11, 12 and 13 showed a 34.8% similarity Supplementary Data 6). Among A-ending codons, both HIV-1 between SLFN12 and SLFN11 or SLFN13, and a 75.8% similarity and HIV-2 use Leu-UUA very frequently instead of Leu-CUG between SLFN11 and SLFN13. Protein backbone structures were that is common in humans. To test whether Leu codon swapping similar between the 3 proteins except that SLFN12 had a short within EGFP would render its expression sensitive to SLFN12 C-terminal domain (Fig. 6a). Based on these structural and func- inhibition, HEK 293T cells were co-transfected with the SLFN12 tional features, we hypothesized that SLFN12 may inhibit HIV-1 expression vector plus synonymous EGFP constructs differing in protein translation by cleaving the rare Leu-UUA tRNA and reduce their codon usage. EGFP expression was analyzed by flow its cellular concentration. cytometry 48 h later (Fig. 5f). SLFN12 inhibited only Leu-UUA Leucine tRNAs are categorized as type II tRNAs that have an swapped EGFP. Altogether our results demonstrate that the expanded variable loop compared to type I tRNAs64. We, therefore, attenuation of HIV-1 protein production by SLFN12 is codon- simulated docking between the SLFN12 dimer and human type II usage dependent. tRNA structure available in the Uniplot database (Fig. 6b). A type II tRNA potentially fits into a space formed by the SLFN12 dimer. SLFN12-mediated HIV-1 suppression depends on a putative Furthermore, putative catalytic sites, which are composed of the tRNase cleavage domain. To better understand the mechanism by negatively charged glutamic acids E200 and E205 in SLFN12, which SLFN12 may interfere with HIV-1 protein translation, we mapped closely to the docked tRNA. These putative RNase active concentrated on the structural features of SLFN protein family sites were conserved as E209 and E214 in SLFN1163. Thus, SLFN12 members. The structure of SLFN12 was recently resolved by cryo- may exert its codon-specific HIV-1 inhibitory effect through the EM and suggested an RNase activity58,59. This was subsequently two putative cleavage sites E200 and E205. To investigate this confirmed biochemically demonstrating a selective cleavage activity hypothesis, we generated expression vectors of SLFN11 and 12 of SLFN12 for Leu-UUA tRNA60. SLFN11 and SLFN13 are also in which the respective active site glutamic acids were converted 6 COMMUNICATIONS BIOLOGY | (2023)6:487 | https://doi.org/10.1038/s42003-023-04841-y | www.nature.com/commsbio COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-023-04841-y ARTICLE Fig. 4 SLFN12 slows down translation elongation. a Outline of polysome profiling. HEK 293T cells were co-transfected with expression plasmids encoding mCherry-fused SLFN11 or SLFN12, or mCherry alone, and the HIV-1 vector pNL-E. At 48 h post-transfection, cells were lysed and subjected to a 10–50% sucrose gradient centrifugation. The gradient was fractionated, and samples were processed for specific RT-qPCR shown in d and e. b UV absorbance profiles of lysed transfected HEK 293T cells after sucrose gradient fractionation (representative result of three independent samples). c SLFN12 expression does not influence the global ratio between monosomes and polysomes (mean ± SD; n = 3 biological replicates). d, e SLFN12 expression shifts the HIV-1 RNA distribution towards heavy polysomes. Distribution of GAPDH RNA (d) or HIV-1 gag RNA (e) in different fractions of HEK 293T cells expressing SLFN11 or SLFN12. Pooled fractions represent free proteins (mRNPs fraction; 1–7), single ribosomal subunits (40S + 60S; 8–12), 80S monosome (13–17), light- (18–23) and heavy -polysomes (24–29). The total amount of the RNAs in all fractions was set to 100% (mean ± SD; n = 2 biological replicates). to alanine. HEK 293T cells were then co-transfected with the Involvement of SLFN12 in HIV-1 post-transcriptional individual SLFN constructs and pNL-E, and the antiviral activity restriction within infected individuals. To study SLFN12 was tested by TZM-bl assays and Western blot (Fig. 6c). All active- expression and its putative role in the control of HIV-1 latency in site mutants of SLFN11 and SLFN12 reduced SLFNs anti-HIV-1 HIV-1-infected individuals, we first analyzed SLFN12 expression in activity (Fig. 6d) and increased levels of Gag-Pr55 and its PBMCs from HIV-1 high (High, n = 16) and low viremic proteolyzed product, p24 (Fig. 6e). Likewise, these mutants lost (Low, n = 30) individuals, viremic controllers (VC, n = 11), and their codon-dependent inhibition of Leu-UUA swapped-EGFP elite controllers (EC, n = 12). SLFN12 mRNA expression levels per expression as shown by the increase of mean fluorescence CD4 count showed significant differences among each group, and a intensities of HEK 293T cells co-transfected with Leu-UUA tendency to increase with disease progression (Fig. 7a). The swapped-EGFP plus the individual SLFN mutants (Fig. 6f). SLFN12 mRNA level was also positively correlated with viral RNA Together these results demonstrate that the putative tRNA cleavage and DNA level, respectively, indicating that SLFN12 is expressed in active sites E200 and E205 of SLFN12 mediate the post- response to viral load (Fig. 7b, c). To further test whether SLFN12 transcriptional, codon-dependent blockage of HIV-1. may be involved in post-transcriptional HIV-1 latency control To test whether SLFN12 degrades tRNAs, we quantified tRNAs in vivo, patient’s PBMCs were treated with or without LRAs in SLFN-transfected cells by RNA gel electrophoresis. Visual (Romidepsin and Ingenol), stained with anti-Gag-p24 and anti- inspection of the ethidium bromide-stained polyacrylamide gel SLFN12 antibodies plus anti-HIV-1-RNA probes, and analyzed by showed a slight decrease in type II tRNA abundance (Supple- FISH-Flow assays as previously described65,66. The gating strategy mentary Fig. 5a). Likewise, type I and II tRNA quantification by is shown in Fig. 7d. SLFN12 expressions in p24 positive(+), image J normalized to 5.8S or 5S rRNA levels showed a decrease HIV-1-RNA+/ p24 negative(−), and both negative populations in type II tRNA by SLFN12, although with less activity than the were characterized (Fig. 7e, f). SLFN12-expressing cells were positive control SLFN11 (Supplementary Fig. 5b, c). enriched in the population of HIV-1-RNA+/ p24 – cells, which COMMUNICATIONS BIOLOGY | (2023)6:487 | https://doi.org/10.1038/s42003-023-04841-y | www.nature.com/commsbio 7 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-023-04841-y is consistent with a role in post-transcriptional HIV-1 latency Schlafen proteins, is an HIV-1 restriction factor that inhibits HIV-1 control. translation in a codon-usage dependent manner and participates in virus containment in homeostatically proliferating CD4+ T cells. Discussion The mechanism of action of SLFN12 was similar to that of the The factors and mechanisms that maintain HIV-1 latency and thus previously described SLFN11, despite a limited sequence homology hamper antiviral cure strategies are incompletely understood. Here between the two Schlafen family members. As the two genes we demonstrate that SLFN12, a member of the conserved family of showed differences in their transcription patterns, their antiviral 8 COMMUNICATIONS BIOLOGY | (2023)6:487 | https://doi.org/10.1038/s42003-023-04841-y | www.nature.com/commsbio COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-023-04841-y ARTICLE Fig. 5 SLFN12-mediated inhibition of HIV-1 translation is codon-usage dependent. a Histogram of codon adaptation indices (CAIs) for the human RefSeq transcript hg38. CAIs of HIV-1 sequences, GAPDH and EGFP are highlighted for comparison. The mean CAI of human transcripts was 0.77. Gag-opt, codon- optimized gag sequence as in plasmid pGag-opt. b Experimental outline. HEK 293T cells were co-transfected with expression plasmids encoding mCherry- fused SLFN11 or SLFN12, or mCherry alone, and an HIV-1 vector expressing Gag-p24 with either wild-type (pGag-wt) or optimized codon usage (pGag-opt). At 48 h post-transfection, transfected cells were lysed and analyzed by Western blot and RT-qPCR. c, d Upper panel: Western blot detection of p24 from HEK 293T cells expressing wild-type Gag (Gag-wt, c) or codon-optimized Gag (Gag-opt, d). Lower panel: Relative HIV-1-gag RNA levels from indicated expression vectors (n = 3 biological replicates; mean ± SD). There were no significant differences between the RNA levels amongst the indicated samples (Student’s t test). e Heatmap of relative synonymous codon usage of human (H. sapiens), HIV-1, HIV-2 and Chikungunya virus (CHIKV) transcripts. Codons Leu-UUA (Leu(TTA) in the graph) and Leu-CUG (Leu(CTG)) are highlighted with arrows. f SLFN11 and SLFN12 specifically inhibit Leu-UUA codon- swapped EGFP expression (n = 3 biological replicates; mean ± SD). The upper panel shows the outline of the experiment. Wild-type EGFP expression vector (WT) or Leu codon-swapped vectors (Leu-CUG, Leu-CUC, Leu-UUA, Leu-CUU, Leu-UUG, and Leu-CUA) were individually transfected together with mCherry (MOCK), mCherry-fused-SLFN11 or 12 expression vector into HEK 293T cells. After 48 h, relative mean fluorescence intensities (MFI) were measured by flow cytometry. The fold change MFI values are shown; the MFI of mock transfection was set to 1. The asterisks represent statistical significance (*p < 0.02, by one sample t-test). functional redundancy may indicate a way to ensure virus control SLFN1349, and suggested for SLFN12 here as well (Fig. 6a–f), an under different cell growth conditions. effect in the nucleus where the tRNA is generated and in the Homeostatic proliferation is a physiological process that restores cytoplasm where the tRNA is used for mRNA-directed transla- the peripheral T cell pool after lymphopenia. It requires the cyto- tion would be most effective. However, many unknowns with kines IL-7 and IL-15, and HLA-restricted T cell receptor triggering respect to the regulation of the RNase activities of SLFN11 and of low affinity67. Importantly, these conditions allow HIV-1- SLFN12 as well as their possible individual and combined effects infected T cells to expand without virus production12,13. Our on the tRNA pool under different cellular growth conditions previous observations with latently infected primary CD4+ T cells remain and require further studies. under HSP culture conditions16, and that of Mohammadi et al. Our data from a cohort of HIV-1-infected individuals showed using another primary CD4+ T cell model of HIV-1 latency68, a positive correlation between SLFN12 expression and HIV-1 suggested that virus production from provirus-containing cells can viral load (Fig. 7a–c), suggesting a link with the general activation be restricted by a post-transcriptional rather than a transcriptional of the immune system. Indeed, SLFN family gene expression is block. Our work here now suggests that SLFN12 is an important considered interferon-inducible42,43,73,74. Furthermore, our component of this post-transcriptional block. It may enable HIV-1- transcriptome-based identification of SLFN12 was derived from infected cells to remain undetectable to adaptive immune responses the screening of homeostatic proliferating primary CD4+ T cells and antiviral therapies because the underlying effector mechanisms ex vivo (Fig. 1). This mode of proliferation requires the cytokines require viral protein expression for exerting their antiviral function. IL-7 and IL-15 whose expression in vivo is directly linked to Consequently, blocking SLFN11 and SLFN12 function and thus, reduced CD4+ T cell counts, and high HIV-1 loads75–77. Inter- increasing viral protein production would help expose HIV-1 estingly, SLFN12 was just one amongst several candidate HIV-1 infected cells more effectively to the immune system and antiviral restriction factors induced under these conditions (Fig. 1c–g), drugs. This should lead to increased HIV-1-infected cell elimina- thus suggesting that CD4+ T cells during homeostatic pro- tion by the immune system and the reduction of the latent reservoir liferation expand highly protected. of infected cells in vivo. Studies in this direction are clearly worth To finally evaluate whether SLFN12 restricts HIV-1 protein pursuing. expression at a post-transcriptional level ex vivo, we combined Functionally, SLFN gene members have multiple and divergent FISH-Flow assays to detect intracellular HIV-1 RNA with anti- biological roles in host defense45–49, cell differentiation69,70 and Gag-p24 and anti-SLFN12 antibody staining and analyzed PBMCs cancer drug sensitivity58,61,71,72. SLFNs 5, 11, 13 and 14 have of several infected individuals. A trend towards the enrichment of been described as virus restriction factors showing activities SLFN12 expression within HIV-1-RNA+/p24– cells was observed against diverse RNA and DNA viruses while SLFN12 until now in these patients. The same was true when considering the mean was considered a candidate antiviral protein based solely on the fluorescence intensities of SLFN12-expressing cells. This supports result of high-throughput screens for interferon-induced antiviral our in vitro-experiments with HIV-1-infected cell lines and sug- factors42–44. Our present work here extends these studies and gests that SLFN12 also restricts the generation of infectious HIV-1 demonstrates the mechanism by which SLFN12 inhibits HIV-1 in people living with HIV (PLWH). However, the frequency of and restricts latency reversal in a codon usage-dependent manner. HIV-1 RNA+ and/or p24+ CD4+ T cells within the PBMCs of Interestingly, SLFN11 and 12 showed comparable anti-HIV-1 HIV-1-infected individuals was low (0.01∼0.1%), and the current properties that in both cases were abrogated by active site FISH-flow analysis is technically limited in that it cannot dis- mutations in the proteins´ RNase domains. However, the type II criminate whether infected cells containing complete and compe- tRNA decrease by SLFN12 was less than that by SLFN11 (Sup- tent HIV-1 proviruses from those with defectives forms. Therefore, plementary Fig. 5). This observations is in line with recent find- our observations should be interpreted with caution. ings of a specific cleavage of the low abundant Leu-UUA tRNA by In conclusion, SLFN12 now adds to the growing number of SLFN1260 and the broader cleavage activity of SLFN1161,63. In restriction factors that a host utilizes to downregulate HIV-1 addition, while the C-terminal helicase domain of SLFN11 seems production. Importantly, by blocking virus protein production at required for its anti-HIV-1 activity62, SLFN12 lacks this region a translational level, SLFN11 and 12 also help HIV-1 and infected and thus exerts its antiviral activity without it. Furthermore, cells to escape from antiviral therapy and avoid immune- SLFN11 also encodes a nuclear localization signal while SLFN12 mediated destruction, facilitating the virus to reside within its does not. Thus, the two SLFN proteins might exert their effect host organism. This makes SLFN11 and 12 interesting therapeutic from both the nuclear as well as the cytoplasmic compartment of targets in strategies aiming to eliminate HIV-1 persistence. An the cell. Supposing that the antiviral mechanism is mediated by experimental examination of this interesting hypothesis clearly the tRNA cleavage activity as demonstrated for SLFN1161–63 and deserves further consideration. COMMUNICATIONS BIOLOGY | (2023)6:487 | https://doi.org/10.1038/s42003-023-04841-y | www.nature.com/commsbio 9 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-023-04841-y Fig. 6 SLFN12-mediated HIV-1 suppression depends on a putative tRNase cleavage domain. a Conformational similarity of human SLFN11 (beige), SLFN12 (cyan) and SLFN13 (magenta). Monomer structures retrieved from the cryo-EM structures of SLFN12 (7LRE) and SLFN11 (7ZEL), and from AlphaFold predicted model of SLFN13 (downloaded from Uniprot, code Q68D06) were superimposed and compared in ribbon plates obtained with Chimera88. SLFN12 has a short C-terminal domain. b Structural models of SLFN12 dimer/tRNA complexes. The cryo-EM structure of SLFN12 dimer (7LRE) docked with the structure of type II tRNA (selenocysteine tRNA, 3HL2) and relaxed, adding the relaxed structure of type I tRNAPhe (5AXM) for comparison, are shown as surface representations (chain A of SLFN12 in light blue, chain B of SLNF12 in cyan, type II tRNA in yellow and type I tRNA in green). In the upper right is shown in ribbon plates a comparison of the structures of SLFN11 (7ZEL) and the relaxed model of SLFN12 docked with type I (green) and type II (yellow) tRNAs, highlighting in van der Waals spheres the side-chain atoms of E200 and E205 of SLFN12, and E209 and E214 of SLFN11, of both chains. In the bottom are shown the surface of the relaxed structure of SLFN12, colored by Coulombic potential the positively (red) and negatively (blue) charged areas, plus a ribbon plate of the structures of type I and type II tRNAs, with arrows pointing to the positions of E200 and E205 in both chains and a detailed view of the active site in chain A. The positively charged surface of SLFN12 was responsible of the binding of the tRNA helping to locate the tRNA in a cleft close to the active site formed by glutamates E200 and E205. c Experimental outline. mCherry-fused SLFN expression vectors or their respective active site mutants E209A and E214A of SLFN11, and E200A and E205A of SLFN12 were individually co-transfected with HIV-1 pNL-E vector into HEK 293T cells. 48 h post-transfection, cells and supernatants were harvested and processed for further analyses as indicated. The following results are from three independent experiments (d, e). d Mutants of the tRNase cleavage sites of SLFN12 lose their anti-HIV-1 activity (mean ± SD). Asterisks indicate significant differences compared to MOCK-transfected cells calculated by Student’s t test. The lower panel shows expression levels of the SLFN proteins in a Western blot using an anti-mCherry antibody. e Western blot showing intracellular HIV-1 Gag Pr55 and p24 levels in SLFN-expressing cells. EGFP and GAPDH were used as controls. f The Leu-UUA codon swapped vector was transfected with indicated SLFN mutant expression vectors into HEK 293T cells. After 48 h, relative mean fluorescence intensity (MFI) was measured by flow cytometry. The fold change MFI values are shown; the MFI of mock transfection was set to 1 (n = 2 biological replicates; mean ± SD). 10 COMMUNICATIONS BIOLOGY | (2023)6:487 | https://doi.org/10.1038/s42003-023-04841-y | www.nature.com/commsbio COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-023-04841-y ARTICLE Fig. 7 Involvement of SLFN12 gene in HIV-1 post-transcriptional restriction in patients. a Expression level of SLFN12 in PBMCs from HIV-1-infected individuals without ART. SLFN12 RNA levels were quantified using PBMCs from HIV-1 high viremic- (High, n = 16) and low viremic- (Low, n = 30) patients, and viremic- (VC, n = 11) and elite-controllers (EC, n = 12). SLFN12 levels were normalized by TBP RNA levels and CD4+ T cell counts per µl. Each point represents the values of a single individual. p-values were calculated by Mann–Whitney test. b Correlation between SLFN12 expression levels and viral RNA loads. Each point represents the values of a single individual. The correlation was calculated by Spearman’s rank test. c Correlation between SLFN12 expression levels and HIV-1 provirus loads. Each point represents the values of a single individual. Spearman’s rank test was applied to analyze the correlation. d–f SLFN12 expressing CD4+ T cells are enriched in HIV-1 RNA+/p24- cells. Gating strategy to measure SLFN12 expression in CD4+ T cells from patients on suppressive ART (d). Gating strategy consisted of selecting lymphocytic cells (“Lymphocytes” on the plots) by FSC- and SSC-scatter, followed by a double doublet exclusion (“Singlets1” and “Singlets2”), dead cells exclusion (“Live cells”) and finally an HIV-1-RNA+ and p24+ gate from where SLFN12+ cells were identified. The proportion of SLFN12 expressing cells (e) or relative MFI of SLFN12 (f) in p24-/HIV-1 RNA−, p24-/HIV-1 RNA+, or p24+ cells. Blue circles, patient samples treated with Romidepsin plus Ingenol (RMD + ING in d) ex vivo (n = 3); White circles, untreated patient samples (R10 in d, n = 3). Limitation of the study. The physiological conditions under committees in the Hospital Universitari Vall d’Hebron (PR(AG)270/2015), and which SLFN11 and/or SLFN12 restrict HIV-1 expression and IrsiCaixa (CEIC: EO-12-042 and PI-18-183) in Barcelona, Spain. Experiments with HIV-1-infected cells were conducted at biosafety level 3 (BSL3) facilities at the suppress latency reversal in vivo are not completely defined. This Universitat Pompeu Fabra, Hospital Universitari Vall d’Hebron and IrsiCaixa. includes infected cell types e.g. CD4+ T cells and macrophages, location of function e.g. nuclear or cytoplasmic, and the activa- Cell lines and PBMCs. ACH2 cells (NIH AIDS Reagent Program) were cultured in tion of the RNase activity of the SLFNs. RPMI medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Sigma Aldrich) and 1% of penicillin/streptomycin mix (P/S, Gibco). HEK 293T cells (ATCC) and TZM-bl cells (NIH AIDS Reagent Program) were main- Methods tained in DMEM medium (Gibco) supplemented with 10% heat-inactivated FBS and Ethics statement. PBMCs from healthy donors and HIV-1 infected patients were 1% P/S. All cell lines were incubated at 37 °C in presence of 5% CO2. PBMC were obtained with a written consent and by a protocol which was approved by the ethic isolated by Histopaque-1077 density centrifugation. The separation and culture of COMMUNICATIONS BIOLOGY | (2023)6:487 | https://doi.org/10.1038/s42003-023-04841-y | www.nature.com/commsbio 11 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-023-04841-y naive CD4+ T cells were conducted according to our previous study16. The culture expression vectors were produced previously61. The primers and shRNA sequences conditions used in this study were as follows: HSP condition – cell cultured with IL- utilized here are listed in Supplementary Data 7. 15 and −7 (10 μg/ml, PeproTech); HSP + TCR condition – cells cultured with IL-15 and −7 and activated by anti-CD3 (5 μg/ml, BD Biosciences) and CD28 (1 μg/ml, BD Biosciences) on day 12; TCR condition – cells activated with anti-CD3/CD28 Transfection and transduction. Transduction and transfection were done antibodies on day 0 and cultured with IL-2 (2 ng/ml, PeproTech); TCR + TCR according to our previous study84. To prepare recombinant retroviruses carrying condition – cells activated with anti-CD3/CD28 on day 0, cultured with IL-2 and the shRNAs, HEK 293T cells were seeded in a 6-well plate at a density of 6 × 105 second time activated with anti-CD3/CD28 at day 12. cells/well and co-transfected with pSIN-siU6 – shSLFN11/ shSLFN12/ shSc (2 μg) along with plasmids pGP (1 μg, Takara) and pPE ampho (1 μg, Takara) using 12 μl of Lipofectamine 2000 (ThermoFisher) per well. At 48 h after transfection, the RNA-sequencing and bioinformatic analysis. Total RNA from cultured cells was supernatant was harvested and added on to ACH2 cells (1 ml of supernatant/105 isolated according to the manufacturer’s instructions using Qiagen RNeasy Micro cells). Transduction was performed by spinoculation (1200 × g, 25 °C, 2 h). Pelleted kit (Qiagen) and submitted to the Genomics Unit of Centre for Genomic Reg- cells were resuspended in 500 μl of RPMI with 10% FBS and placed into 24-well ulation (CRG, PRBB, Spain) for sequencing. Quality and concentration of RNA plates. Selection of transduced cells was done by adding G418 (1 mg/ml, Invivo- were determined by an Agilent Bioanalyzer. Sequencing libraries were generated by Gen) into the cell at 48 h after transduction. a Ribo-Zero kit (Illumina). cDNA was synthesized and tagged by addition of For the expression studies in HEK 293T cells, the cells were seeded in 24-well barcoded Truseq adapters. Libraries were quantified using the KAPA Library plate at a density of 2 × 105, and after 24 h co-transfected with construct Quantification Kit (KapaBiosystems) prior to amplification with Illumina’s cBot. pmCherry-SLFN11/ pmCherry-SLFN12 along with vector HIV-1-pNL-E, pGag-wt, Four libraries were pooled and sequenced (single strand, 50nts) on an Illumina pGag-opt, or codon-swapped EGFP using Lipofectamine 2000 (2.5 μl/well). At 3- HiSeq2000 sequencer to obtain 50–60 million reads per sample. RNA-seq reads 4 h post transfection, fresh medium was replaced, and the cells were kept at 37 °C were mapped against the Homo sapiens reference genome (GRCh37.p13) with the with 5% CO2 for 24 or 48 h. GEMtools RNA-seq pipeline (http://gemtools.github.io/docs/rna_pipeline.html). Genes were quantified with the same pipeline using the Gencode version 19 as an annotation. Normalization was performed with the edgeR TMM method78. The SAHA treatment of ACH cells. ACH2 cells with blocked expression of SLFN11 k-means clustering was performed by iDEP. 94 interface17 and the heatmap was and SLFN12 (as described above) were seeded in a 24-well plate and treated with generated by ggplot2 R package. Gene ontology (GO) enrichment analysis was DMSO (0.01%, Sigma-Aldrich) or SAHA (Vorinostat, Sigma-Aldrich) at 0.5 μM. performed with DAVID (http://david.ncifcrf.gov/). Differentially expression ana- The cells were lysed for RNA or protein isolation at 48 h after treatment. Super- lysis was performed with the ‘robust’ version of the edgeR R package79. Genes with natants were harvested at 72 h after treatment to test for virus titer by TZM- FDR < 5% were considered differentially expressed. The list of human gene families bl assay. was obtained from the HUGO gene nomenclature committee (https://www. genenames.org/). The list of HIV-interacting genes and restriction factors were TZM-bl assay. Virus titers in supernatants from transfected HEK 293T cells and obtained from the NIH HIV interaction database18–20 and a previous report28, HIV-1-reactivated ACH2 cells were determined by the TZM-bl assay as described respectively. previously85. Briefly, 11 serial dilutions of supernatants were prepared and added into fresh TZM-bl cells (104 cells/well) in 96-well flat-bottom culture plates RNA isolation and quantitative PCR. Total RNA was extracted from cells and (Greiner Bio-One). After 72 h incubation at 37 °C, 5% CO2 the luciferase activity treated with DNase I according to the manufacturer’s instructions using Qiagen was measured on Centro LB 960 Microplate Luminometer (Berthold Technologies) RNeasy Mini Kit (Qiagen). 100–1000 ng RNA was reverse-transcribed into using Britelite PlusTM (PerkinElmer) according to the manufacturer’s protocol. cDNA in a total volume of 20 μl using SuperScript IV Reverse Transcriptase Based on the luciferase levels, the TCID50 was calculated. (ThermoFisher). 2 μl of cDNA was used for quantitative PCR (qPCR) in a 10 μl reaction using SYBR master mix (ThermoFisher). Each reaction was performed Western blot and antibodies. The cells were lysed with 1× passive lysis buffer in triplicates in 348-well plates in a QuantStudio 12K flex (ThermoFisher). (Promega), snap-frozen in liquid nitrogen and kept at −80 °C overnight. The next Relative RNA levels were calculated after normalization to 18S rRNA unless day, cell debris was removed by centrifugation at 15,000 RPM for 5 min and specified otherwise. Primers used this study are described in Supplementary supernatants were mixed with 2× Laemmli buffer, heated at 97 °C for 5 min, Data 7. transferred to the nitrocellulose membrane and immunoblot using specific anti- For SLFN12 expression quantification in HIV-1 participants, total RNA samples bodies was performed. Detection was done using secondary antibodies conjugated from available PBMC dry-pellets were obtained using Qiagen RNeasy Mini Kit with horseradish peroxidase (HRP). Protein bands were developed on Medical (Qiagen) following manufacturer´s recommendations. cDNA was obtained using X-Ray Blue Films (AGFA) using PierceTM ECL Plus Western Blotting Substrate or SuperScript IV Reverse Transcriptase (ThermoFisher) and TaqMan gene SuperSignal WestFemto Maximum Sensitivity Substrate (ThermoFisher) and expression assay (Applied Biosystems) was used for detection of SLFN12 quantified in Image-J software. The antibodies used in this study are listed in (Hs00430118_m1) and TBP (Hs99999910_m1). Gene amplification was performed Supplementary Data 8. on an Applied Biosystems 7500 Fast Real-Time PCR System thermocycler, and the relative expression was calculated as 2 − ΔCT (where CT is the median threshold cycle from 3 replicates). Flow cytometry. CD4+ T cells after HSP or TCR culture were incubated with Quantification of integrated HIV-1-proviral DNA was performed from PBMC LIVE/DEAD® Fixable Aqua (ThermoFisher) and human Fc blocker (BD by droplet digital PCR (ddPCR) as described80. Briefly, two different primer/probe Bioscience). Then the cells were washed and stained with anti-CD4, CD8, CD27, sets annealing to the 5′ long terminal repeat (LTR) and gag regions, respectively, and CD45RO- antibodies (Supplementary Data 8) on ice for 20 min. Transfected were used to circumvent sequence mismatch in the patient proviruses, and the HEK 293T cells were suspended with PBS containing 1% FBS and 1 μg/mL 4’,6- RPP30 housekeeping gene was quantified in parallel to normalize sample input. diamidino-2-phenylindole (DAPI) before data acquisition. Fluorescence was Raw ddPCR data were analyzed using the QX100™ droplet reader and QuantaSoft measured on a BD LSRFortessa (Becton, Dickinson) and analyzed with FlowJo v10 v.1.6 software (Bio-Rad). (Becton, Dickinson). Plasmids. SLFN11/12 sequences were acquired from the plasmids clone MGS: Polysome profiling. HEK 293T were co-transfected with pmCherry/ pmCherry- 59997 (SLFN11) and clone MGS: 45076 (SLFN12) provided by Dharmacon and SLFN11/ pmCherry-SLFN12 (12 μg) and HIV-1-pNL-E (12 μg) using Lipo- cloned into mCherry expression vector, pmCherry-N’ (Clontech) between NheI fectamine 2000 (48 μl) in a T-150 culture dish (ThermoFisher). In order to and HindIII restriction sites. The plasmids generated were named pmCherry- freeze elongation ribosomes, 48 h after transfection cells were treated with 10 ml SLFN11 and pmCherry-SLFN12, respectively. pNL-E, the HIV-1 proviral clone of DMEM containing cycloheximide (CHX, 100 μg/ml) during 2 min at 37 °C pNL4-381-derived EGFP-expressing plasmid, was generated previously50. The and washed with 10 ml of PBS containing CHX (100 μg/ml) using a vacuum plasmid expressing HIV-1 wild-type gag sequence in pEF-BOS_bsr backbone was system. Cells were lysed with 700 μl of lysis buffer (10 mM Tris-HCl (pH = 7.4), produced previously (pGag-wt)82. To generate HIV-1 codon-optimized gag 10 mM MgCl2, 100 mM NaCl, 1% Triton X100, 2 mM DTT, 100 ug/ml CHX), expressing plasmid (pGag-opt), the codon-optimized gag sequence was obtained scraped and immediately frozen in liquid nitrogen and stored at −80 °C. Cell from the plasmid p96ZM651gag-opt (Drs. Y. Li, F. Gao, and B.H. Hahn through lysates were thawed at 25 °C and centrifuged at 12,000 × g, 5 min, 4 °C and the the National Institutes of Health (NIH) AIDS Reagent Program, Reference# 8675) supernatants were transferred to new tubes. After quantification, aliquots of 8 and ligated between BamHI and NotI sites of pEF-BOS_bsr. Retrovirus vectors UA260 were made and stored at −80 °C. Linear gradients of 10–50% sucrose carrying shRNA sequences were constructed by inserting annealed oligonucleotides were prepared in polysome buffer (20 mM Tris-HCl (pH = 7.4), 10 mM MgCl2, into a pSIN-siU6 vector (Takara) between BamHI and ClaI sites. As a negative 100 mM NH4Cl). The Gradient Master (Biocomp) was used to prepare the control pSIN-siU6 vector expressing shRNA scramble sequences was used83. The gradients in polyallomer tubes (Beckman Coulter). One aliquot of 8 UA260 was constructs expressing mutant SLFNs (SLFN11 E209A, SLFN11 E214A, SLFN12 loaded on each gradient and centrifuged in Beckman SW41 rotor at 35 000 E200A and SLFN12 E205A) were generated using Q5 Site-Directed Mutagenesis RPM, 3 h, 4 °C. Gradients were fractionated with fraction collector Model 2128 Kit (NEB). The mutagenesis was performed according to the substitution protocol (BioRad). These fractions were used for phenol:chloroform RNA extraction and using primers designed with the NEBaseChanger (NEB). Codon-swapped EGFP analyzed by RT-qPCR. 12 COMMUNICATIONS BIOLOGY | (2023)6:487 | https://doi.org/10.1038/s42003-023-04841-y | www.nature.com/commsbio COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-023-04841-y ARTICLE Structural comparison. The dimer structures of human SLFN11 and SLFN12 were RNA gel electrophoresis. Transfected HEK 293T cells were collected and sub- downloaded from PDB86 (codes 7ZEL and 7LRE, respectively). A model of the jected to total RNA extraction through phenol-chloroform extraction as described monomeric structure of SLFN13 was downloaded from Uniprot (model predicted above. 2 µg of RNA were resolved in a 10% TBE-Urea polyacrylamide gel. Bands of by AlphaFold87, AF-Q68D06-F1). The model of the dimer structure of SLFN13 was tRNA and 5.8S rRNA were visualized by ethidium bromide staining and quantified constructed by superimposition on the structure of SLFN11 with the program by Image J. Matchmaker using Chimera software (version 1. 13. 1)88. The model of the complex of SLFN12 with yeast tRNAPhe Type I was obtained by docking the Reporting summary. Further information on research design is available in the Nature structure of the dimer of SLNF12 with the tRNA structure (code 5AXM) according Portfolio Reporting Summary linked to this article. to Metzner et al.63 using SLFN11 as template. The model of the complex of SLFN12 with human selenocystein tRNA Type II was obtained by docking it with the tRNA structure (code 3HL2) according to Yang et al.49 using SLFN13 as template. The structures of the complexes were relaxed and optimized with Rosetta89 and ribbon Data availability plates and surfaces, colored by Coulombic potential, were calculated and plotted The RNA-seq data from this study are available from the NCBI Expression Omnibus with Chimera (version 1. 13. 1)88. GEO; GSE221332. Gating strategy of flow cytometry performed in Figs. 1b, 5f and 6f are shown in Supplementary Figs. 6 and 7. Uncropped version of Western blot images are Calculating and visualizing gene-wise CAI and RSCU. For all hg38 RefSeq genes, available from Supplementary Figs. 8 to 11. The numerical source data are provided in we used the seqinr (v.3.3-6) and ggplot2 (3.1.0) R-packages and codon weights Supplementary Data 9–15. All other data are available from the corresponding authors obtained from CAIcal (http://genomes.urv.es/CAIcal/CU_human_nature). We on reasonable request. used Codon Usage Table database (https://hive.biochemistry.gwu.edu/cuts/about) for calculation of RSCU55. Received: 9 January 2023; Accepted: 13 April 2023; FISH-flow. PBMC from ART-treated HIV-1-infected patients were obtained from the HIV Unit of the Hospital Universitari Vall d’Hebron (Barcelona, Spain) and CD4+ T cells were isolated by negative selection using magnetic beads (MagniSort Human CD4+ T Cell Enrichment; eBioscience). 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Nucleic Acids Res. 42, Correspondence and requests for materials should be addressed to Mie Kobayashi- W478–W484 (2014). Ishihara, Juana Díez or Andreas Meyerhans. 89. Conway, P., Tyka, M. D., DiMaio, F., Konerding, D. E. & Baker, D. Relaxation of backbone bond geometry improves protein energy landscape modeling. Peer review information Communications Biology thanks Heidi Greulich and the other Protein Sci. 23, 47–55 (2014). anonymous reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Zhijuan Qiu and Karli Montague-Cardoso. Peer reviewer reports are available. Acknowledgements We thank Drs Yingying Li, Feng Gao and Beatrice H. Hahn for providing codon- Reprints and permission information is available at http://www.nature.com/reprints optimized HIV-1 Gag expression vector, Drs James Hoxie and Susan Zolla-Pazner for supplying anti-Nef and -p24 antibodies, respectively through the NIH AIDS reagent Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in program. We also thank Dr Song Gao for providing SLFN13-tRNA structure informa- published maps and institutional affiliations. tion, and Dr Maria-Eugenia Gas Lopez and Dr Ester Gea-Mallorquí for advise. This work was supported by following grants: M.K.I., JSPS Oversea Research Fellowship and Takeda Science Foundation; A.E.C., PT17/0009/0019 (ISCIII/MINECO and FEDER); M.J.B., Open Access This article is licensed under a Creative Commons RTI2018-101082-B-I00 and PID2021-123321OB-I00 [MINECO/FEDER]), and the Attribution 4.0 International License, which permits use, sharing, Miguel Servet program by ISCIII (CP17/00179 and CPII22/00005); C.B., M.R.R., C.D.C., adaptation, distribution and reproduction in any medium or format, as long as you give European Union’s Horizon 2020 research and innovation program under grant agree- appropriate credit to the original author(s) and the source, provide a link to the Creative ment 681137-EAVI2020 and NIH grant P01-AI131568; J.D., the Spanish Ministry of Commons license, and indicate if changes were made. The images or other third party Science and Innovation (PID2019106959RB-I00/AEI/10.13039/501100011033); A.M., material in this article are included in the article’s Creative Commons license, unless the Spanish Ministry of Science and Innovation (PID2019-106323RB-I00 AEI//10.13039/ indicated otherwise in a credit line to the material. If material is not included in the 501100011033) and the institutional “María de Maeztu” Programme for Units of Excellence in R&D (CEX2018-000792-M). article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ Author contributions licenses/by/4.0/. M.K.I., J.P.M., Y.T.Y., J.A., J.D. and A.M. designed the research study. M.K.I., K.F.S., J.P.M., Y.T.Y., F.E.A., K.G., M.G., J.G.E., C.D.C., and S.R. performed the experiments. J.J., M.L., M.D., and M.Y. provided essential tools. A.E.C., R.B., B.O., M.R.R., and M.J.B. © The Author(s) 2023 COMMUNICATIONS BIOLOGY | (2023)6:487 | https://doi.org/10.1038/s42003-023-04841-y | www.nature.com/commsbio 15