Internal RNA 2'-O-methylation on the HIV-1 genome impairs reverse transcription.

Viral RNA genomes are modified by epitranscriptomic marks, including 2'-O-methylation that is added by cellular or viral methyltransferases. 2'-O-Methylation modulates RNA structure, function and discrimination between self- and non-self-RNA by innate immune sensors such as RIG-I-like receptors. This is illustrated by human immunodeficiency virus type-1 (HIV-1) that decorates its RNA genome through hijacking the cellular FTSJ3 2'-O-methyltransferase, thereby limiting immune sensing and interferon production. However, the impact of such an RNA modification during viral genome replication is poorly understood. Here we show by performing endogenous reverse transcription on methylated or hypomethylated HIV-1 particles, that 2'-O-methylation negatively affects HIV-1 reverse transcriptase activity. Biochemical assays confirm that RNA 2'-O-methylation impedes reverse transcriptase activity, especially at low dNTP concentrations reflecting those in quiescent cells, by reducing nucleotide incorporation efficiency and impairing translocation. Mutagenesis highlights K70 as a critical amino acid for the reverse transcriptase to bypass 2'-O-methylation. Hence, the observed antiviral effect due to viral RNA 2'-O-methylation antagonizes the FTSJ3-mediated proviral effects, suggesting the fine-tuning of RNA methylation during viral replication.

Alarmin S100A9 restricts retroviral infection by limiting reverse transcription in human dendritic cells.

Dendritic cells (DC) subsets, like Langerhans cells (LC), are immune cells involved in pathogen sensing. They express specific antimicrobial cellular factors that are able to restrict infection and limit further pathogen transmission. Here, we identify the alarmin S100A9 as a novel intracellular antiretroviral factor expressed in human monocyte-derived and skin-derived LC. The intracellular expression of S100A9 is decreased upon LC maturation and inversely correlates with enhanced susceptibility to HIV-1 infection of LC. Furthermore, silencing of S100A9 in primary human LC relieves HIV-1 restriction while ectopic expression of S100A9 in various cell lines promotes intrinsic resistance to both HIV-1 and MLV infection by acting on reverse transcription. Mechanistically, the intracellular expression of S100A9 alters viral capsid uncoating and reverse transcription. S100A9 also shows potent inhibitory effect against HIV-1 and MMLV reverse transcriptase (RTase) activity in vitro in a divalent cation-dependent manner. Our findings uncover an unexpected intracellular function of the human alarmin S100A9 in regulating antiretroviral immunity in Langerhans cells.

Skin tropism during Usutu virus and West Nile virus infection: an amplifying and immunological role.

Usutu virus (USUV) and West Nile virus (WNV) are closely related emerging arboviruses belonging to the Flavivirus genus and posing global public health concerns. Although human infection by these viruses is mainly asymptomatic, both have been associated with neurological disorders such as encephalitis and meningoencephalitis. Since USUV and WNV are transmitted through the bite of an infected mosquito, the skin represents the initial site of virus inoculation and provides the first line of host defense. Although some data on the early stages of WNV skin infection are available, very little is known about USUV. Herein, USUV-skin resident cell interactions were characterized. Using primary human keratinocytes and fibroblasts, an early replication of USUV during the first 24 hours was shown in both skin cells. In human skin explants, a high viral tropism for keratinocytes was observed. USUV infection of these models induced type I and III interferon responses associated with upregulated expression of various interferon-stimulated genes as well as pro-inflammatory cytokine and chemokine genes. Among the four USUV lineages studied, the Europe 2 strain replicated more efficiently in skin cells and induced a higher innate immune response. In vivo, USUV and WNV disseminated quickly from the inoculation site to distal cutaneous tissues. In addition, viral replication and persistence in skin cells were associated with an antiviral response. Taken together, these results provide a better understanding of the pathophysiology of the early steps of USUV infection and suggest that the skin constitutes a major amplifying organ for USUV and WNV infection.IMPORTANCEUsutu virus (USUV) and West Nile virus (WNV) are closely related emerging Flaviviruses transmitted through the bite of an infected mosquito. Since they are directly inoculated within the upper skin layers, the interactions between the virus and skin cells are critical in the pathophysiology of USUV and WNV infection. Here, during the early steps of infection, we showed that USUV can efficiently infect two human resident skin cell types at the inoculation site: the epidermal keratinocytes and the dermal fibroblasts, leading to the induction of an antiviral innate immune response. Moreover, following cutaneous inoculation, we demonstrated that both viruses can rapidly spread, replicate, and persist in all distal cutaneous tissues in mice, a phenomenon associated with a generalized skin inflammatory response. These results highlight the key amplifying and immunological role of the skin during USUV and WNV infection.

Internal RNA 2'O-methylation in the HIV-1 genome counteracts ISG20 nuclease-mediated antiviral effect.

RNA 2'O-methylation is a 'self' epitranscriptomic modification allowing discrimination between host and pathogen. Indeed, human immunodeficiency virus 1 (HIV-1) induces 2'O-methylation of its genome by recruiting the cellular FTSJ3 methyltransferase, thereby impairing detection by RIG-like receptors. Here, we show that RNA 2'O-methylations interfere with the antiviral activity of interferon-stimulated gene 20-kDa protein (ISG20). Biochemical experiments showed that ISG20-mediated degradation of 2'O-methylated RNA pauses two nucleotides upstream of and at the methylated residue. Structure-function analysis indicated that this inhibition is due to steric clash between ISG20 R53 and D90 residues and the 2'O-methylated nucleotide. We confirmed that hypomethylated HIV-1 genomes produced in FTSJ3-KO cells were more prone to in vitro degradation by ISG20 than those produced in cells expressing FTSJ3. Finally, we found that reverse-transcription of hypomethylated HIV-1 was impaired in T cells by interferon-induced ISG20, demonstrating the direct antagonist effect of 2'O-methylation on ISG20-mediated antiviral activity.

Despite highly effective antiretroviral therapies, the human immunodeficiency virus (HIV-1) remains a major public health threat. Its pathogenesis depends on its ability to establish a persistent infection in cells of the immune system. Our study highlights a new insight into how HIV-1 evades early restriction by the immune system. We showed that 2'O-methylation marks found inside HIV-1 RNA promote viral evasion from the antiviral action of the interferon-stimulated gene 20-kDa protein (ISG20), an innate immune restriction factor with a nuclease activity. By disrupting the level of 2'O-methylation of the HIV-1 genome, we demonstrated that ISG20 impairs the reverse transcription process of hypomethylated viruses, as a result of viral RNA decay.

Measuring the subcellular compartmentalization of viral infections by protein complementation assay.

The recent emergence and reemergence of viruses in the human population has highlighted the need to develop broader panels of therapeutic molecules. High-throughput screening assays opening access to untargeted steps of the viral replication cycle will provide powerful leverage to identify innovative antiviral molecules. We report here the development of an innovative protein complementation assay, termed αCentauri, to measure viral translocation between subcellular compartments. As a proof of concept, the Centauri fragment was either tethered to the nuclear pore complex or sequestered in the nucleus, while the complementary α fragment (<16 amino acids) was attached to the integrase proteins of infectious HIV-1. The translocation of viral ribonucleoproteins from the cytoplasm to the nuclear envelope or to the nucleoplasm efficiently reconstituted superfolder green fluorescent protein or NanoLuc αCentauri reporters. These fluorescence- or bioluminescence-based assays offer a robust readout of specific steps of viral infection in a multiwell format that is compatible for high-throughput screening and is validated by a short hairpin RNA-based prototype screen.

Modulation of innate immune signaling by a Coxiella burnetii eukaryotic-like effector protein.

The Q fever agent Coxiella burnetii uses a defect in organelle trafficking/intracellular multiplication (Dot/Icm) type 4b secretion system (T4SS) to silence the host innate immune response during infection. By investigating C. burnetii effector proteins containing eukaryotic-like domains, here we identify NopA (nucleolar protein A), which displays four regulator of chromosome condensation (RCC) repeats, homologous to those found in the eukaryotic Ras-related nuclear protein (Ran) guanine nucleotide exchange factor (GEF) RCC1. Accordingly, NopA is found associated with the chromatin nuclear fraction of cells and uses the RCC-like domain to interact with Ran. Interestingly, NopA triggers an accumulation of Ran-GTP, which accumulates at nucleoli of transfected or infected cells, thus perturbing the nuclear import of transcription factors of the innate immune signaling pathway. Accordingly, qRT-PCR analysis on a panel of cytokines shows that cells exposed to the C. burnetii nopA::Tn or a Dot/Icm-defective dotA::Tn mutant strain present a functional innate immune response, as opposed to cells exposed to wild-type C. burnetii or the corresponding nopA complemented strain. Thus, NopA is an important regulator of the innate immune response allowing Coxiella to behave as a stealth pathogen.

SARS-CoV-2 triggers an MDA-5-dependent interferon response which is unable to control replication in lung epithelial cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of coronavirus disease 19 (COVID-19), which ranges from mild respiratory symptoms to acute respiratory distress syndrome, and death in the most severe cases. Immune dysregulation with altered innate cytokine responses is thought to contribute to disease severity. Here, we characterized in depth host cell responses against SARS-CoV-2 in primary human airway epithelia (HAE) and immortalized cell lines. Our results demonstrate that primary HAE and model cells elicit a robust induction of type I and III interferons (IFNs). Importantly, we show for the first time that melanoma differentiation associated gene (MDA)-5 is the main sensor of SARS-CoV-2 in lung cells. IFN exposure strongly inhibited viral replication and de novo production of infectious virions. However, despite high levels of IFNs produced in response to SARS-CoV-2 infection, the IFN response was unable to control viral replication in lung cells, contrary to what was previously reported in intestinal epithelial cells. Altogether, these results highlight the complex and ambiguous interplay between viral replication and the timing of IFN responses.IMPORTANCE Mammalian cells express sensors able to detect specific features of pathogens and induce the interferon response, which is one of the first line of defenses against viruses and help controlling viral replication. The mechanisms and impact of SARS-CoV-2 sensing in lung epithelial cells remained to be deciphered. In this study, we report that despite a high production of type I and III interferons specifically induced by MDA-5-mediated sensing of SARS-CoV-2, primary and immortalized lung epithelial cells are unable to control viral replication. However, exogenous interferons potently inhibited replication, if provided early upon viral exposure. A better understanding of the ambiguous interplay between the interferon response and SARS-CoV-2 replication is essential to guide future therapeutical interventions.

Interplay between SARS-CoV-2 and the type I interferon response.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for the current COVID-19 pandemic. An unbalanced immune response, characterized by a weak production of type I interferons (IFN-Is) and an exacerbated release of proinflammatory cytokines, contributes to the severe forms of the disease. SARS-CoV-2 is genetically related to SARS-CoV and Middle East respiratory syndrome-related coronavirus (MERS-CoV), which caused outbreaks in 2003 and 2013, respectively. Although IFN treatment gave some encouraging results against SARS-CoV and MERS-CoV in animal models, its potential as a therapeutic against COVID-19 awaits validation. Here, we describe our current knowledge of the complex interplay between SARS-CoV-2 infection and the IFN system, highlighting some of the gaps that need to be filled for a better understanding of the underlying molecular mechanisms. In addition to the conserved IFN evasion strategies that are likely shared with SARS-CoV and MERS-CoV, novel counteraction mechanisms are being discovered in SARS-CoV-2-infected cells. Since the last coronavirus epidemic, we have made considerable progress in understanding the IFN-I response, including its spatiotemporal regulation and the prominent role of plasmacytoid dendritic cells (pDCs), which are the main IFN-I-producing cells. While awaiting the results of the many clinical trials that are evaluating the efficacy of IFN-I alone or in combination with antiviral molecules, we discuss the potential benefits of a well-timed IFN-I treatment and propose strategies to boost pDC-mediated IFN responses during the early stages of viral infection.

Original Chemical Series of Pyrimidine Biosynthesis Inhibitors That Boost the Antiviral Interferon Response.

De novo pyrimidine biosynthesis is a key metabolic pathway involved in multiple biosynthetic processes. Here, we identified an original series of 3-(1H-indol-3-yl)-2,3-dihydro-4H-furo[3,2-c]chromen-4-one derivatives as a new class of pyrimidine biosynthesis inhibitors formed by two edge-fused polycyclic moieties. We show that identified compounds exhibit broad-spectrum antiviral activity and immunostimulatory properties, in line with recent reports linking de novo pyrimidine biosynthesis with innate defense mechanisms against viruses. Most importantly, we establish that pyrimidine deprivation can amplify the production of both type I and type III interferons by cells stimulated with retinoic acid-inducible gene 1 (RIG-I) ligands. Altogether, our results further expand the current panel of pyrimidine biosynthesis inhibitors and illustrate how the production of antiviral interferons is tightly coupled to this metabolic pathway. Functional and structural similarities between this new chemical series and dicoumarol, which was reported before to inhibit pyrimidine biosynthesis at the dihydroorotate dehydrogenase (DHODH) step, are discussed.

MxA Mediates SUMO-Induced Resistance to Vesicular Stomatitis Virus.

UNLABELLED: Multiple cellular pathways are regulated by small ubiquitin-like modifier (SUMO) modification, including ubiquitin-mediated proteolysis, signal transduction, innate immunity, and antiviral defense. In the study described in this report, we investigated the effects of SUMO on the replication of two members of the Rhabdoviridae family, vesicular stomatitis virus (VSV) and rabies virus (RABV). We show that stable expression of SUMO in human cells confers resistance to VSV infection in an interferon-independent manner. We demonstrate that SUMO expression did not alter VSV entry but blocked primary mRNA synthesis, leading to a reduction of viral protein synthesis and viral production, thus protecting cells from VSV-induced cell lysis. MxA is known to inhibit VSV primary transcription. Interestingly, we found that the MxA protein was highly stabilized in SUMO-expressing cells. Furthermore, extracts from cells stably expressing SUMO exhibited an increase in MxA oligomers, suggesting that SUMO plays a role in protecting MxA from degradation, thus providing a stable intracellular pool of MxA available to combat invading viruses. Importantly, MxA depletion in SUMO-expressing cells abrogated the anti-VSV effect of SUMO. Furthermore, SUMO expression resulted in interferon-regulatory factor 3 (IRF3) SUMOylation, subsequently decreasing RABV-induced IRF3 phosphorylation and interferon synthesis. As expected, this rendered SUMO-expressing cells more sensitive to RABV infection, even though MxA was stabilized in SUMO-expressing cells, since its expression did not confer resistance to RABV. Our findings demonstrate opposing effects of SUMO expression on two viruses of the same family, intrinsically inhibiting VSV infection through MxA stabilization while enhancing RABV infection by decreasing IFN induction. IMPORTANCE: We report that SUMO expression reduces interferon synthesis upon RABV or VSV infection. Therefore, SUMO renders cells more sensitive to RABV but unexpectedly renders cells resistant to VSV by blocking primary mRNA synthesis. Unlike the interferon-mediated innate immune response, intrinsic antiviral resistance is mediated by constitutively expressed restriction factors. Among the various anti-VSV restriction factors, only MxA is known to inhibit VSV primary transcription, and we show here that its expression does not alter RABV infection. Interestingly, MxA depletion abolished the inhibition of VSV by SUMO, demonstrating that MxA mediates SUMO-induced intrinsic VSV resistance. Furthermore, MxA oligomerization is known to be critical for its protein stability, and we show that higher levels of oligomers were formed in cells expressing SUMO than in wild-type cells, suggesting that SUMO may play a role in protecting MxA from degradation, providing a stable intracellular pool of MxA able to protect cells from viral infection.

PML/TRIM19-Dependent Inhibition of Retroviral Reverse-Transcription by Daxx.

PML (Promyelocytic Leukemia protein), also known as TRIM19, belongs to the family of tripartite motif (TRIM) proteins. PML is mainly expressed in the nucleus, where it forms dynamic structures known as PML nuclear bodies that recruit many other proteins, such as Sp100 and Daxx. While the role of PML/TRIM19 in antiviral defense is well documented, its effect on HIV-1 infection remains unclear. Here we show that infection by HIV-1 and other retroviruses triggers the formation of PML cytoplasmic bodies, as early as 30 minutes post-infection. Quantification of the number and size of PML cytoplasmic bodies revealed that they last approximately 8 h, with a peak at 2 h post-infection. PML re-localization is blocked by reverse-transcription inhibitors and is not observed following infection with unrelated viruses, suggesting it is specifically triggered by retroviral reverse-transcription. Furthermore, we show that PML interferes with an early step of retroviral infection since PML knockdown dramatically increases reverse-transcription efficiency. We demonstrate that PML does not inhibit directly retroviral infection but acts through the stabilization of one of its well-characterized partners, Daxx. In the presence of PML, cytoplasmic Daxx is found in the vicinity of incoming HIV-1 capsids and inhibits reverse-transcription. Interestingly, Daxx not only interferes with exogenous retroviral infections but can also inhibit retrotransposition of endogenous retroviruses, thus identifying Daxx as a broad cellular inhibitor of reverse-transcription. Altogether, these findings unravel a novel antiviral function for PML and PML nuclear body-associated protein Daxx.

Small Ubiquitin-like Modifier Alters IFN Response.

IFNs orchestrate immune defense through induction of hundreds of genes. Small ubiquitin-like modifier (SUMO) is involved in various cellular functions, but little is known about its role in IFN responses. Prior work identified STAT1 SUMOylation as an important mode of regulation of IFN-γ signaling. In this study, we investigated the roles of SUMO in IFN signaling, gene expression, protein stability, and IFN-induced biological responses. We first show that SUMO overexpression leads to STAT1 SUMOylation and to a decrease in IFN-induced STAT1 phosphorylation. Interestingly, IFNs exert a negative retrocontrol on their own signaling by enhancing STAT1 SUMOylation. Furthermore, we show that expression of each SUMO paralog inhibits IFN-γ-induced transcription without affecting that of IFN-α. Further, we focused on IFN-induced gene products associated to promyelocytic leukemia (PML) nuclear bodies, and we show that neither IFN-α nor IFN-γ could increase PML and Sp100 protein expression because they enhanced their SUMO3 conjugation and subsequent proteasomal degradation. Because it is known that SUMO3 is important for the recruitment of RING finger protein 4, a poly-SUMO-dependent E3 ubiquitin ligase, and that PML acts as a positive regulator of IFN-induced STAT1 phosphorylation, we went on to show that RING finger protein 4 depletion stabilizes PML and is correlated with a positive regulation of IFN signaling. Importantly, inhibition of IFN signaling by SUMO is associated with a reduction of IFN-induced apoptosis, cell growth inhibition, antiviral defense, and chemotaxis. Conversely, inhibition of SUMOylation results in higher IFN-γ-induced STAT1 phosphorylation and biological responses. Altogether, our results uncover a new role for SUMO in the modulation of IFN response.

Implication of PMLIV in both intrinsic and innate immunity.

PML/TRIM19, the organizer of nuclear bodies (NBs), has been implicated in the antiviral response to diverse RNA and DNA viruses. Several PML isoforms generated from a single PML gene by alternative splicing, share the same N-terminal region containing the RBCC/tripartite motif but differ in their C-terminal sequences. Recent studies of all the PML isoforms reveal the specific functions of each. The knockout of PML renders mice more sensitive to vesicular stomatitis virus (VSV). Here we report that among PML isoforms (PMLI to PMLVIIb), only PMLIII and PMLIV confer resistance to VSV. Unlike PMLIII, whose anti-VSV activity is IFN-independent, PMLIV can act at two stages: it confers viral resistance directly in an IFN-independent manner and also specifically enhances IFN-ß production via a higher activation of IRF3, thus protecting yet uninfected cells from oncoming infection. PMLIV SUMOylation is required for both activities. This demonstrates for the first time that PMLIV is implicated in innate immune response through enhanced IFN-ß synthesis. Depletion of IRF3 further demonstrates the dual activity of PMLIV, since it abrogated PMLIV-induced IFN synthesis but not PMLIV-induced inhibition of viral proteins. Mechanistically, PMLIV enhances IFN-ß synthesis by regulating the cellular distribution of Pin1 (peptidyl-prolyl cis/trans isomerase), inducing its recruitment to PML NBs where both proteins colocalize. The interaction of SUMOylated PMLIV with endogenous Pin1 and its recruitment within PML NBs prevents the degradation of activated IRF3, and thus potentiates IRF3-dependent production of IFN-ß. Whereas the intrinsic antiviral activity of PMLIV is specific to VSV, its effect on IFN-ß synthesis is much broader, since it affects a key actor of innate immune pathways. Our results show that, in addition to its intrinsic anti-VSV activity, PMLIV positively regulates IFN-ß synthesis in response to different inducers, thus adding PML/TRIM19 to the growing list of TRIM proteins implicated in both intrinsic and innate immunity.

MxA interacts with and is modified by the SUMOylation machinery.

Mx proteins are evolutionarily conserved dynamin-like large GTPases involved in viral resistance triggered by types I and III interferons. The human MxA is a cytoplasmic protein that confers resistance to a large number of viruses. The MxA protein is also known to self-assembly into high molecular weight homo-oligomers. Using a yeast two-hybrid screen, we identified 27 MxA binding partners, some of which are related to the SUMOylation machinery. The interaction of MxA with Small-Ubiquitin MOdifier 1 (SUMO1) and Ubiquitin conjugating enzyme 9 (Ubc9) was confirmed by co-immunoprecipitation and co-localization by confocal microscopy. We identified one SUMO conjugation site at lysine 48 and two putative SUMO interacting motifs (SIMa and SIMb). We showed that MxA interacts with the EIL loop of SUMO1 in a SIM-independent manner via its CID-GED domain. The yeast two-hybrid mapping also revealed that Ubc9 binds to the MxA GTPase domain. Mutation in the putative SIMa and SIMb, which are located in the GTPase binding domain, reduced MxA antiviral activity. In addition, we showed that MxA can be conjugated to SUMO2 or SUMO3 at lysine 48 and that the SUMOylation-deficient mutant of MxA (MxAK48R) retained its capacity to oligomerize and to inhibit Vesicular Stomatitis Virus (VSV) and Influenza A Virus replication, suggesting that MxA SUMOylation is not essential for its antiviral activity.

Daxx, a broad-spectrum viral restriction factor.

Daxx is an ubiquitous protein that was first identified as a Fas-interacting protein. Although cytoplasmic Daxx is involved in apoptosis, it is predominantly a nuclear protein. In the nucleus, Daxx is mainly located within PML nuclear bodies (PML NBs), nuclear structures involved in many cellular processes. The main function of Daxx in the nucleus is transcriptional repression through interactions with multiple factors involved in transcription. Daxx, like other PML NB resident proteins, such as PML itself, but also Sp100 and ATRX, is involved in antiviral defense. In concordance with this, many viruses target Daxx to replicate. Daxx-induced viral restriction has been particularly studied in the case of herpesviruses and adenoviruses, but recent findings suggest that Daxx also has a major role in the replication of retroviruses, including HIV-1. In this review, we summarize and discuss the different antiviral activities of Daxx and detail the strategies that viruses have evolved to escape.

TRIM5α is a SUMO substrate.

BACKGROUND: The TRIM5α restriction factor interferes with retroviral infections by inhibiting an early step of viral replication. TRIM5α activity was recently proposed to be regulated by the SUMO machinery and one SUMO consensus conjugation site as well as three putative SUMO interacting motifs (SIMs) were identified within TRIM5α sequence. Whereas mutation of the SIM sequences was found to abolish TRIM5α antiviral activity, mutation of the consensus SUMO conjugation site did not affect its restriction capacity, although this putative site has never been shown to be actually a SUMO substrate. FINDINGS: Here we further demonstrate that TRIM5α relies on the SUMO machinery to promote restriction, since SUMO1 overexpression enhances TRIM5α-mediated retroviral inhibition whereas knockdown of SUMO1 or E2 SUMO conjugating enzyme Ubc9 prevents restriction. Furthermore, we show for the first time that TRIM5α is SUMOylated both in vitro and in cellulo and that Lysine 10 is the main SUMOylation site. Mutation of the consensus SUMO conjugation motif in position 10 abrogated SUMOylation at this position, but did not disrupt TRIM5α antiviral activity. CONCLUSIONS: Altogether, our results confirm that the SUMO machinery is involved in TRIM5α-mediated retroviral restriction, and demonstrate that TRIM5α is a SUMO 1 and SUMO 2 substrate. The inability to abrogate TRIM5α antiviral activity by mutating its main SUMO conjugation motif supports the notion that non-covalent interaction with SUMO or SUMOylated proteins rather than TRIM5α direct SUMOylation is required.