The Signal Peptide of a Simple Retrovirus Envelope Functions as a Posttranscriptional Regulator of Viral Gene Expression

ABSTRACT Retroviruses use different strategies to regulate transcription and translation and exploit the cellular machinery involved in these processes. This study shows that the signal peptide of the envelope glycoprotein (Env) of Jaagsiekte sheep retrovirus (JSRV) plays a major role in posttranscriptional viral gene expression. Expression of the JSRV Env in trans increases viral particle production by mechanisms dependent on (i) its leader sequence, (ii) an intact signal peptide cleavage site, (iii) a cis-acting RNA-responsive element located in the viral genome, (iv) Crm1, and (v) B23. The signal peptide of the JSRV Env (JSE-SP) is 80 amino acid residues in length and contains putative nuclear localization and export signals, in addition to an arginine-rich RNA binding motif. JSE-SP localizes both in the endoplasmic reticulum and in the nucleus, where it colocalizes with nucleolar markers. JSE-SP is a multifunctional protein, as it moderately enhances nuclear export of unspliced viral mRNA and considerably increases viral particle release by favoring a posttranslational step of the replication cycle.

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Nuclear Export Signal Masking Regulates HIV-1 Rev Trafficking and Viral RNA Nuclear Export

Journal of Virology ◽

10.1128/jvi.02107-16 ◽

2016 ◽

Vol 91 (3) ◽

Cited By ~ 17

Author(s):

Ryan T. Behrens ◽

Mounavya Aligeti ◽

Ginger M. Pocock ◽

Christina A. Higgins ◽

Nathan M. Sherer

Keyword(s):

Gene Expression ◽

Nuclear Export ◽

Rna Binding ◽

Nuclear Export Signal ◽

Viral Gene Expression ◽

Viral Gene ◽

Virion Production ◽

Export Signal ◽

Hiv 1 ◽

Rev Protein

ABSTRACT HIV-1's Rev protein forms a homo-oligomeric adaptor complex linking viral RNAs to the cellular CRM1/Ran-GTP nuclear export machinery through the activity of Rev's prototypical leucine-rich nuclear export signal (NES). In this study, we used a functional fluorescently tagged Rev fusion protein as a platform to study the effects of modulating Rev NES identity, number, position, or strength on Rev subcellular trafficking, viral RNA nuclear export, and infectious virion production. We found that Rev activity was remarkably tolerant of diverse NES sequences, including supraphysiological NES (SNES) peptides that otherwise arrest CRM1 transport complexes at nuclear pores. Rev's ability to tolerate a SNES was both position and multimerization dependent, an observation consistent with a model wherein Rev self-association acts to transiently mask the NES peptide(s), thereby biasing Rev's trafficking into the nucleus. Combined imaging and functional assays also indicated that NES masking underpins Rev's well-known tendency to accumulate at the nucleolus, as well as Rev's capacity to activate optimal levels of late viral gene expression. We propose that Rev multimerization and NES masking regulates Rev's trafficking to and retention within the nucleus even prior to RNA binding. IMPORTANCE HIV-1 infects more than 34 million people worldwide causing >1 million deaths per year. Infectious virion production is activated by the essential viral Rev protein that mediates nuclear export of intron-bearing late-stage viral mRNAs. Rev's shuttling into and out of the nucleus is regulated by the antagonistic activities of both a peptide-encoded N-terminal nuclear localization signal and C-terminal nuclear export signal (NES). How Rev and related viral proteins balance strong import and export activities in order to achieve optimal levels of viral gene expression is incompletely understood. We provide evidence that multimerization provides a mechanism by which Rev transiently masks its NES peptide, thereby biasing its trafficking to and retention within the nucleus. Targeted pharmacological disruption of Rev-Rev interactions should perturb multiple Rev activities, both Rev-RNA binding and Rev's trafficking to the nucleus in the first place.

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Mapping of Functional Regions in the Amino-Terminal Portion of the Herpes Simplex Virus ICP27 Regulatory Protein: Importance of the Leucine-Rich Nuclear Export Signal and RGG Box RNA-Binding Domain

Journal of Virology ◽

10.1128/jvi.76.23.11866-11879.2002 ◽

2002 ◽

Vol 76 (23) ◽

pp. 11866-11879 ◽

Cited By ~ 45

Author(s):

Joy Lengyel ◽

Chandra Guy ◽

Vivian Leong ◽

Sarah Borge ◽

Stephen A. Rice

Keyword(s):

Gene Expression ◽

Nuclear Export ◽

Rna Binding ◽

Regulatory Protein ◽

Nuclear Export Signal ◽

Viral Gene Expression ◽

Vero Cells ◽

Viral Gene ◽

Functional Regions ◽

Hsv 1

ABSTRACT Infected-cell protein 27 (ICP27) is an essential herpes simplex virus type 1 (HSV-1) regulatory protein that activates a subset of viral delayed-early and late genes, at least in part through posttranscriptional mechanisms. Previous studies have shown that the amino (N)-terminal half of the protein contains important functional regions, including a leucine-rich nuclear export signal (NES). However, to date, the phenotype of an HSV-1 ICP27 NES mutant has not been reported. In this study, we engineered and characterized dLeu, an HSV-1 deletion mutant that specifically lacks ICP27's NES (amino acids 6 to 19). The phenotype of dLeu was analyzed alongside those of eight other ICP27 N-terminal deletion mutants. We found that in Vero cells, dLeu displays modest defects in viral gene expression and an approximately 100-fold reduction in the production of viral progeny. Unlike wild-type (WT) ICP27, which exhibits a cytoplasmic distribution in addition to its predominant nuclear localization, dLeu ICP27 is highly restricted to the cell nucleus. This strongly suggests that the N-terminal leucine-rich sequence functions as an NES during viral infection. Our analysis of dLeu and the other mutants has enabled us to genetically define the regions in the N-terminal 200 residues of ICP27 which are required for efficient viral growth in Vero cells. Only two regions appear to be important: (i) the leucine-rich NES and (ii) the RGG box RNA-binding domain, encoded by residues 139 to 153. A virus lacking the RGG box-encoding sequence, d4-5, has a phenotype similar to that of dLeu in that it displays modest defects in viral gene expression and grows poorly. Interestingly, deletion of both the NES and RGG box, as well as the sequences in between, is lethal. The resulting virus, d1-5, displays severe defects in viral gene expression and DNA synthesis and is unable to produce significant amounts of infectious progeny. Therefore, the N-terminal portion of ICP27 contains at least two functional domains which collectively are absolutely essential for viral infection.

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Protein-RNA interactome analysis reveals wide association of KSHV ORF57 with host non-coding RNAs and polysomes

Journal of Virology ◽

10.1128/jvi.01782-21 ◽

2021 ◽

Author(s):

Beatriz Alvarado-Hernandez ◽

Yanping Ma ◽

Nishi R. Sharma ◽

Vladimir Majerciak ◽

Alexei Lobanov ◽

...

Keyword(s):

Gene Expression ◽

Rna Splicing ◽

Rna Binding ◽

Viral Gene Expression ◽

Viral Gene ◽

28S Rrna ◽

Protein Coding ◽

Infected Cells ◽

Non Coding Rnas ◽

Rna Interaction

Kaposi’s sarcoma-associated herpesvirus (KSHV) ORF57 is an RNA-binding post-transcriptional regulator. We recently applied an affinity-purified anti-ORF57 antibody to conduct ORF57-CLIP (Cross-linking Immunoprecipitation) in combination with RNA-sequencing (CLIP-seq) and analyzed the genome-wide host RNA transcripts in association with ORF57 in BCBL-1 cells with lytic KSHV infection. Mapping of the CLIPed RNA reads to the human genome (GRCh37) revealed that most of the ORF57-associated RNA reads were from rRNAs. The remaining RNA reads mapped to several classes of host non-coding and protein-coding mRNAs. We found ORF57 binds and regulates expression of a subset of host lncRNAs, including LINC00324, LINC00355, and LINC00839 which are involved in cell growth. ORF57 binds snoRNAs responsible for 18S and 28S rRNA modifications, but does not interact with fibrillarin and NOP58. We validated ORF57 interactions with 67 snoRNAs by ORF57-RNA immunoprecipitation (RIP)-snoRNA-array assays. Most of the identified ORF57 rRNA binding sites (BS) overlap with the sites binding snoRNAs. We confirmed ORF57-snoRA71B RNA interaction in BCBL-1 cells by ORF57-RIP and Northern blot analyses using a 32 P-labeled oligo probe from the 18S rRNA region complementary to snoRA71B. Using RNA oligos from the rRNA regions that ORF57 binds for oligo pulldown-Western blot assays, we selectively verified ORF57 interactions with 5.8S and 18S rRNAs. Polysome profiling revealed that ORF57 associates with both monosomes and polysomes and its association with polysomes increases PABPC1 binding to, but prevent Ago2 from polysomes. Our data indicate a functional correlation with ORF57 binding and suppression of Ago2 activities for ORF57 promotion of gene expression. Significance As an RNA-binding protein, KSHV ORF57 regulates RNA splicing, stability, and translation and inhibits host innate immunity by blocking the formation of RNA granules in virus infected cells. In this report, ORF57 was found to interact many host non-coding RNAs, including lncRNAs, snoRNAs and ribosomal RNAs to carry out additional unknown functions. ORF57 binds a group of lncRNAs via the identified RNA motifs by ORF57 CLIP-seq to regulate their expression. ORF57 associates with snoRNAs independently of fibrillarin and NOP58 proteins, and with ribosomal RNA in the regions that commonly bind snoRNAs. Knockdown of fibrillarin expression decreases the expression of snoRNAs and CDK4, but not affect viral gene expression. More importantly, we found that ORF57 binds translationally active polysomes and enhances PABPC-1 but prevents Ago2 association with polysomes. Data provide a compelling evidence on how ORF57 in KSHV infected cells might regulate protein synthesis by blocking Ago2’s hostile activities on translation.

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Influenza Virus Usurps an Interferon-Induced Translational Program to Promote Viral Replication

Proceedings ◽

10.3390/proceedings2020050134 ◽

2020 ◽

Vol 50 (1) ◽

pp. 134

Author(s):

Mitchell P. Ledwith ◽

Vy Tran ◽

Thiprampai Thamamongood ◽

Christina A. Higgins ◽

Shashank Tripathi ◽

...

Keyword(s):

Gene Expression ◽

Influenza Virus ◽

Viral Replication ◽

Rna Binding ◽

Viral Gene Expression ◽

Viral Gene ◽

Translational Efficiency ◽

A Genome ◽

Cellular Mrnas ◽

Stimulation Of

Hosts mount prudently tuned responses to viral infection in an attempt to block nearly every step of the replication cycle. Viruses must adapt to replicate in this hostile antiviral cellular state. Interferon stimulation or pathogen challenge robustly induces expression of IFIT (interferon-induced proteins with tetratricopeptide repeats) proteins. IFITs are a family of proteins that bind RNA and play antiviral roles during infection. Thus, we were surprised to identify the IFIT family as top candidate proviral host factors for influenza A virus (IAV) in a genome-wide CRISPR–Cas9 knockout screen. We validated the proviral activity of IFIT2 by showing that IFIT2-deficient cells support lower levels of IAV replication and exhibit defects in viral gene expression. The molecular functions of IFIT2, let alone how they are used by influenza virus, are unknown. Using CLIP-seq, we showed that IFIT2 binds directly to viral and cellular mRNAs in AU-rich regions largely in the 3’UTR, with a preference for a subset of interferon-stimulated mRNAs. IFIT2 also associates with actively translating ribosomes in infected cells to facilitate the translation of viral messages. IFIT2-responsive elements from an IAV mRNA were sufficient to confer translational enhancement to exogenous transcripts in cis. Conversely, mutation of these elements or the use of an IFIT2 RNA-binding mutant ablated stimulation of viral gene expression. Together, these data link the RNA-binding capability of IFIT2 to changes in translational efficiency of target viral mRNAs and the stimulation of viral replication. They establish a model for the normal function of IFIT2 as an antiviral protein affecting the post-transcriptional fate of cellular mRNAs and explain how influenza virus repurposes IFIT2 to support viral replication. Our work highlights a new node for the regulation of translation during interferon responses and highlights how canonical antiviral responses may be repurposed to support viral replication.

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Widespread remodeling of the m6A RNA-modification landscape by a viral regulator of RNA processing and export

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2104805118 ◽

2021 ◽

Vol 118 (30) ◽

pp. e2104805118

Author(s):

Kalanghad Puthankalam Srinivas ◽

Daniel P. Depledge ◽

Jonathan S. Abebe ◽

Stephen A. Rice ◽

Ian Mohr ◽

...

Keyword(s):

Gene Expression ◽

Messenger Rna ◽

Rna Binding ◽

Viral Gene Expression ◽

Rna Modification ◽

Viral Gene ◽

Viral Mrnas ◽

Simplex Virus ◽

Processing Stability ◽

Hsv 1

N6-methyladenosine (m6A) is the most abundant internal messenger RNA (mRNA) modification, contributing to the processing, stability, and function of methylated RNAs. Methylation occurs in the nucleus during pre-mRNA synthesis and requires a core methyltransferase complex consisting of METTL3, METTL14, and WTAP. During herpes simplex virus (HSV-1) infection, cellular gene expression is profoundly suppressed, allowing the virus to monopolize the host transcription and translation apparatus and antagonize antiviral responses. The extent to which HSV-1 uses or manipulates the m6A pathway is not known. Here, we show that, in primary fibroblasts, HSV-1 orchestrates a striking redistribution of the nuclear m6A machinery that progresses through the infection cycle. METTL3 and METTL14 are dispersed into the cytoplasm, whereas WTAP remains nuclear. Other regulatory subunits of the methyltransferase complex, along with the nuclear m6A-modified RNA binding protein YTHDC1 and nuclear demethylase ALKBH5, are similarly redistributed. These changes require ICP27, a viral regulator of host mRNA processing that mediates the nucleocytoplasmic export of viral late mRNAs. Viral gene expression is initially reduced by small interfering RNA (siRNA)-mediated inactivation of the m6A methyltransferase but becomes less impacted as the infection advances. Redistribution of the nuclear m6A machinery is accompanied by a wide-scale reduction in the installation of m6A and other RNA modifications on both host and viral mRNAs. These results reveal a far-reaching mechanism by which HSV-1 subverts host gene expression to favor viral replication.

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Pat1 proteins: regulating mRNAs from birth to death?

BioMolecular Concepts ◽

10.1515/bmc-2012-0005 ◽

2012 ◽

Vol 3 (4) ◽

pp. 295-306

Author(s):

Nancy Standart ◽

Aline Marnef

Keyword(s):

Gene Expression ◽

Mammalian Cells ◽

Mrna Decay ◽

Rna Binding ◽

Rna Binding Proteins ◽

Viral Gene Expression ◽

Model Systems ◽

Viral Gene ◽

Nuclear Speckles ◽

Mrna Targets

AbstractThe Pat1 protein family has been the subject of several recent extensive investigations of diverse model systems ranging from yeast, flies and worms to man, using a variety of experimental approaches. Although some contradictions remain, the emerging consensus view is that these RNA-binding proteins act in mRNA decay by physically linking deadenylation with decapping and by regulating gene expression as translational repressors. These multiple functions are present in the single invertebrate Pat1 proteins, whereas, in vertebrates, one Pat1 variant represses translation in early development, while a somatic version synthesised in embrogenesis and in adults acts in mRNA decay. At steady state, Pat1 proteins are found enriched in cytoplasmic P(rocessing)-bodies, and related mRNP complexes and granules. Evidence recently obtained from mammalian tissue culture cells shows that Pat1 shuttles in and out of the nucleus, where it localises to nuclear speckles, PML bodies and nucleolar caps, which suggests RNA-related nuclear functions. Less well understood, Pat1 proteins may play additional roles in miRNA silencing and/or biogenesis, as well in the regulation of viral gene expression. Due to the relatively low level of sequence conservation between Pat1 proteins from different species and lacking any discernable motifs, determining their functional domains has proved difficult, as is obtaining a simple unified view of the location of the binding sites of their interacting proteins in all examined species. Questions that remain to be addressed include the following: 1) What are their roles in the nucleus? 2) What is the link, if one exists, between their cytoplasmic and nuclear roles? 3) Do they have specific mRNA targets? 4) Which signalling pathways regulate their P-body localisation in mammalian cells, which may affect quiescent cell survival?

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The HIV 5′ Gag Region Displays a Specific Nucleotide Bias Regulating Viral Splicing and Infectivity

Viruses ◽

10.3390/v13060997 ◽

2021 ◽

Vol 13 (6) ◽

pp. 997

Author(s):

Bastian Grewe ◽

Carolin Vogt ◽

Theresa Horstkötter ◽

Bettina Tippler ◽

Han Xiao ◽

...

Keyword(s):

Gene Expression ◽

Alternative Splicing ◽

Nuclear Export ◽

Rna Binding ◽

Gc Content ◽

Splice Donor Site ◽

Viral Gene ◽

Nucleotide Bias ◽

Rna Sequence ◽

Hiv Rna

Alternative splicing and the expression of intron-containing mRNAs is one hallmark of HIV gene expression. To facilitate the otherwise hampered nuclear export of non-fully processed mRNAs, HIV encodes the Rev protein, which recognizes its intronic response element and fuels the HIV RNAs into the CRM-1-dependent nuclear protein export pathway. Both alternative splicing and Rev-dependency are regulated by the primary HIV RNA sequence. Here, we show that these processes are extremely sensitive to sequence alterations in the 5’coding region of the HIV genomic RNA. Increasing the GC content by insertion of either GFP or silent mutations activates a cryptic splice donor site in gag, entirely deregulates the viral splicing pattern, and lowers infectivity. Interestingly, an adaptation of the inserted GFP sequence toward an HIV-like nucleotide bias reversed these phenotypes completely. Of note, the adaptation yielded completely different primary sequences although encoding the same amino acids. Thus, the phenotypes solely depend on the nucleotide composition of the two GFP versions. This is a strong indication of an HIV-specific mRNP code in the 5′ gag region wherein the primary RNA sequence bias creates motifs for RNA-binding proteins and controls the fate of the HIV-RNA in terms of viral gene expression and infectivity.

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Influenza A Virus NS1 Protein Promotes Efficient Nuclear Export of Unspliced Viral M1 mRNA

Journal of Virology ◽

10.1128/jvi.00528-17 ◽

2017 ◽

Vol 91 (15) ◽

Cited By ~ 15

Author(s):

Carina F. Pereira ◽

Eliot K. C. Read ◽

Helen M. Wise ◽

Maria J. Amorim ◽

Paul Digard

Keyword(s):

Influenza A Virus ◽

Nuclear Export ◽

Influenza A ◽

Matrix Protein ◽

Rna Binding ◽

Viral Gene Expression ◽

Mutant Virus ◽

Viral Gene ◽

Ns1 Protein ◽

Viral Mrnas

ABSTRACT Influenza A virus mRNAs are transcribed by the viral RNA-dependent RNA polymerase in the cell nucleus before being exported to the cytoplasm for translation. Segment 7 produces two major transcripts: an unspliced mRNA that encodes the M1 matrix protein and a spliced transcript that encodes the M2 ion channel. Export of both mRNAs is dependent on the cellular NXF1/TAP pathway, but it is unclear how they are recruited to the export machinery or how the intron-containing but unspliced M1 mRNA bypasses the normal quality-control checkpoints. Using fluorescent in situ hybridization to monitor segment 7 mRNA localization, we found that cytoplasmic accumulation of unspliced M1 mRNA was inefficient in the absence of NS1, both in the context of segment 7 RNPs reconstituted by plasmid transfection and in mutant virus-infected cells. This effect was independent of any major effect on steady-state levels of segment 7 mRNA or splicing but corresponded to a ∼5-fold reduction in the accumulation of M1. A similar defect in intronless hemagglutinin (HA) mRNA nuclear export was seen with an NS1 mutant virus. Efficient export of M1 mRNA required both an intact NS1 RNA-binding domain and effector domain. Furthermore, while wild-type NS1 interacted with cellular NXF1 and also increased the interaction of segment 7 mRNA with NXF1, mutant NS1 polypeptides unable to promote mRNA export did neither. Thus, we propose that NS1 facilitates late viral gene expression by acting as an adaptor between viral mRNAs and the cellular nuclear export machinery to promote their nuclear export. IMPORTANCE Influenza A virus is a major pathogen of a wide variety of mammalian and avian species that threatens public health and food security. A fuller understanding of the virus life cycle is important to aid control strategies. The virus has a small genome that encodes relatively few proteins that are often multifunctional. Here, we characterize a new function for the NS1 protein, showing that, as well as previously identified roles in antagonizing the innate immune defenses of the cell and directly upregulating translation of viral mRNAs, it also promotes the nuclear export of the viral late gene mRNAs by acting as an adaptor between the viral mRNAs and the cellular mRNA nuclear export machinery.

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Influenza Virus NS1 Protein-RNA Interactome Reveals Intron Targeting

Journal of Virology ◽

10.1128/jvi.01634-18 ◽

2018 ◽

Vol 92 (24) ◽

Cited By ~ 10

Author(s):

Liang Zhang ◽

Juan Wang ◽

Raquel Muñoz-Moreno ◽

Min Kim ◽

Ramanavelan Sakthivel ◽

...

Keyword(s):

Gene Expression ◽

Influenza Virus ◽

Virus Infection ◽

Influenza A ◽

Rna Binding ◽

Influenza Virus Infection ◽

Viral Gene Expression ◽

Viral Gene ◽

Ns1 Protein ◽

Cellular Processes

ABSTRACTThe NS1 protein of influenza A virus is a multifunctional virulence factor that inhibits cellular processes to facilitate viral gene expression. While NS1 is known to interact with RNA and proteins to execute these functions, the cellular RNAs that physically interact with NS1 have not been systematically identified. Here we reveal a NS1 protein-RNA interactome and show that NS1 primarily binds intronic sequences. Among this subset of pre-mRNAs is the RIG-I pre-mRNA, which encodes the main cytoplasmic antiviral sensor of influenza virus infection. This suggested that NS1 interferes with the antiviral response at a posttranscriptional level by virtue of its RNA binding properties. Indeed, we show that NS1 is necessary in the context of viral infection and sufficient upon transfection to decrease the rate of RIG-I intron removal. This NS1 function requires a functional RNA binding domain and is independent of the NS1 interaction with the cleavage and polyadenylation specificity factor CPSF30. NS1 has been previously shown to abrogate RIG-I-mediated antiviral immunity by inhibiting its protein function. Our data further suggest that NS1 also posttranscriptionally alters RIG-I pre-mRNA processing by binding to the RIG-I pre-mRNA.IMPORTANCEA key virulence factor of influenza A virus is the NS1 protein, which inhibits various cellular processes to facilitate viral gene expression. The NS1 protein is localized in the nucleus and in the cytoplasm during infection. In the nucleus, NS1 has functions related to inhibition of gene expression that involve protein-protein and protein-RNA interactions. While several studies have elucidated the protein interactome of NS1, we still lack a clear and systematic understanding of the NS1-RNA interactome. Here we reveal a nuclear NS1-RNA interactome and show that NS1 primarily binds intronic sequences within a subset of pre-mRNAs, including the RIG-I pre-mRNA that encodes the main cytoplasmic antiviral sensor of influenza virus infection. Our data here further suggest that NS1 is necessary and sufficient to impair intron processing of the RIG-I pre-mRNA. These findings support a posttranscriptional role for NS1 in the inhibition of RIG-I expression.

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Hepatitis B virus Core protein nuclear interactome identifies SRSF10 as a host RNA-binding protein restricting HBV RNA production

10.1101/2020.05.04.076646 ◽

2020 ◽

Author(s):

Hélène Chabrolles ◽

Héloïse Auclair ◽

Serena Vegna ◽

Thomas Lahlali ◽

Caroline Pons ◽

...

Keyword(s):

Gene Expression ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Rna Binding ◽

Rna Binding Proteins ◽

Core Protein ◽

Viral Gene Expression ◽

Viral Gene ◽

B Virus ◽

Regulatory Functions

AbstractDespite the existence of a preventive vaccine, chronic infection with Hepatitis B virus (HBV) affects more than 250 million people and represents a major global cause of hepatocellular carcinoma (HCC) worldwide. Current clinical treatments, in most of cases, do not eliminate viral genome that persists as a DNA episome in the nucleus of hepatocytes and constitutes a stable template for the continuous expression of viral genes. Several studies suggest that, among viral factors, the HBV core protein (HBc), well-known for its structural role in the cytoplasm, could have critical regulatory functions in the nucleus of infected hepatocytes. To elucidate these functions, we performed a proteomic analysis of HBc-interacting host-factors in the nucleus of differentiated human hepatocytes. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs), which are involved in various aspects of mRNA metabolism. Among them, we focused our studies on SRSF10, a RBP that was previously shown to regulate alternative splicing in a phosphorylation-dependent manner and to control stress and DNA damage responses, as well as viral replication. Functional studies combining SRSF10 knockdown and a pharmacological inhibitor of SRSF10 phosphorylation (1C8) showed that SRSF10 behaves as a restriction factor that regulates HBV RNAs levels and that its dephosphorylated form is likely responsible for the anti-viral effect. Surprisingly, neither SRSF10 knock-down nor 1C8 treatment modified the splicing of HBV RNAs but rather modulated the level of nascent HBV RNA. Altogether, our work suggests that in the nucleus of infected cells HBc interacts with multiple RBPs that regulate viral RNA metabolism. Our identification of SRSF10 as a new anti-HBV restriction factor offers new perspectives for the development of new host-targeted antiviral strategies.Author SummaryChronic infection with Hepatitis B virus (HBV) affects more than 250 millions of people world-wide and is a major global cause of liver cancer. Current treatments lead to a significant reduction of viremia in patients. However, viral clearance is rarely obtained and the persistence of the HBV genome in the hepatocyte’s nucleus generates a stable source of viral RNAs and subsequently proteins which play important roles in immune escape mechanisms and liver disease progression. Therapies aiming at efficiently and durably eliminating viral gene expression are still required. In this study, we identified the nuclear partners of the HBV Core protein (HBc) to understand how this structural protein, responsible for capsid assembly in the cytoplasm, could also regulate viral gene expression. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs). One of these RBPs, SRSF10, was demonstrated to restrict HBV RNA levels and a drug, able to alter its phosphorylation, behaved as an antiviral compound capable of reducing viral gene expression. Altogether, this study sheds new light novel regulatory functions of HBc and provides information relevant for the development of antiviral strategies aiming at preventing viral gene expression.

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