[How do 2'-O-methylations within Human Immunodeficiency Virus type 1 (HIV-1) genome regulate its replication?] / Effets de la 2'-O-méthylation de l'ARN génomique du VIH-1 sur la réplication virale.

The genomic RNA of HIV-1 is modified by epitranscriptomic modifications, including 2'-O-methylations, which are found on 17 internal positions. These methylations are added by the cellular methyltransferase FTSJ3, and have pro-viral effects, since they shield the viral genome from the detection by the innate immune sensor MDA5. In turn, the production of interferons by infected cells is reduced, limiting the expression of interferon-stimulated genes (ISGs) with antiviral activities. Moreover, 2'-O-methylations protect the HIV-1 genome from its degradation by ISG20, an interferon-induced exonuclease. Conversely, these methylations also exhibit antiviral effects, as they impede reverse-transcription in vitro or in quiescent cells, which are known to contain low nucleotide concentrations. Altogether, these observations suggest a balance between the proviral effect of 2'-O-methylations, related to the protection of the viral genome from detection by MDA5 and degradation by ISG20, and the antiviral effect, associated with the negative impact of 2'-O-methylations on the viral replication. These findings pave the way for further optimization of therapeutic RNA, by selective methylation of specific nucleotides.

Title: Effets de la 2'-O-méthylation de l'ARN génomique du VIH-1 sur la réplication virale. Abstract: Les ARN du virus de l'immunodéficience humaine sont décorés par des marques épitranscriptomiques, dont des 2'-O-méthylations internes. Ces marques ajoutées par une enzyme cellulaire, FTSJ3, sont des marqueurs du « soi ¼. Elles ont des effets proviraux en protégeant l'ARN viral de la détection par le senseur de l'immunité innée MDA5, et en limitant sa dégradation par l'exonucléase cellulaire ISG20, induite par l'interféron. Ces méthylations ont également un effet antiviral, dans la mesure où elles perturbent la rétrotranscription du génome ARN du virus, in vitro et dans des cellules quiescentes. Un équilibre subtil existe donc entre les effets proviraux et antiviraux des 2'-O-méthylations, assurant ainsi une réplication optimale du virus. Ces découvertes ouvrent des perspectives d'optimisation des ARN thérapeutiques à effet antiviral, par la méthylation sélective de certains nucléotides.

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.

Tick-borne flavivirus NS5 antagonizes interferon signaling by inhibiting the catalytic activity of TYK2.

The mechanisms utilized by different flaviviruses to evade antiviral functions of interferons are varied and incompletely understood. Using virological approaches, biochemical assays, and mass spectrometry analyses, we report here that the NS5 protein of tick-borne encephalitis virus (TBEV) and Louping Ill virus (LIV), two related tick-borne flaviviruses, antagonize JAK-STAT signaling through interactions with the tyrosine kinase 2 (TYK2). Co-immunoprecipitation (co-IP) experiments, yeast gap-repair assays, computational protein-protein docking and functional studies identify a stretch of 10 residues of the RNA dependent RNA polymerase domain of tick-borne flavivirus NS5, but not mosquito-borne NS5, that is critical for interactions with the TYK2 kinase domain. Additional co-IP assays performed with several TYK2 orthologs reveal that the interaction is conserved across mammalian species. In vitro kinase assays show that TBEV and LIV NS5 reduce the catalytic activity of TYK2. Our results thus illustrate a novel mechanism by which viruses suppress the interferon response.

Interplay of RNA 2'-O-methylations with viral replication.

Viral RNAs (vRNAs) are decorated by post-transcriptional modifications, including methylation of nucleotides. Methylations regulate biological functions linked to the sequence, structure, and protein interactome of RNA. Several RNA viruses were found to harbor 2'-O-methylations, affecting the ribose moiety of RNA. This mark was initially shown to target the first and second nucleotides of the 5'-end cap structure of mRNA. More recently, nucleotides within vRNA were also reported to carry 2'-O-methylations. The consequences of such methylations are still puzzling since they were associated with both proviral and antiviral effects. Here, we focus on the mechanisms governing vRNA 2'-O-methylation and we explore the possible roles of this epitranscriptomic modification for viral replication.

Properties of rabies virus phosphoprotein and nucleoprotein biocondensates formed in vitro and in cellulo.

Rabies virus (RABV) transcription and replication take place within viral factories having liquid properties, called Negri bodies (NBs), that are formed by liquid-liquid phase separation (LLPS). The co-expression of RABV nucleoprotein (N) and phosphoprotein (P) in mammalian cells is sufficient to induce the formation of cytoplasmic biocondensates having properties that are like those of NBs. This cellular minimal system was previously used to identify P domains that are essential for biocondensates formation. Here, we constructed fluorescent versions of N and analyzed by FRAP their dynamics inside the biocondensates formed in this minimal system as well as in NBs of RABV-infected cells using FRAP. The behavior of N appears to be different of P as there was no fluorescence recovery of N proteins after photobleaching. We also identified arginine residues as well as two exposed loops of N involved in condensates formation. Corresponding N mutants exhibited distinct phenotypes in infected cells ranging from co-localization with NBs to exclusion from them associated with a dominant-negative effect on infection. We also demonstrated that in vitro, in crowded environments, purified P as well as purified N0-P complex (in which N is RNA-free) form liquid condensates. We identified P domains required for LLPS in this acellular system. P condensates were shown to associate with liposomes, concentrate RNA, and undergo a liquid-gel transition upon ageing. Conversely, N0-P droplets were disrupted upon incubation with RNA. Taken together, our data emphasize the central role of P in NBs formation and reveal some physicochemical features of P and N0-P droplets relevant for explaining NBs properties such as their envelopment by cellular membranes at late stages of infection and nucleocapsids ejections from the viral factories.

Structure-function analysis of the nsp14 N7-guanine methyltransferase reveals an essential role in Betacoronavirus replication.

As coronaviruses (CoVs) replicate in the host cell cytoplasm, they rely on their own capping machinery to ensure the efficient translation of their messenger RNAs (mRNAs), protect them from degradation by cellular 5' exoribonucleases (ExoNs), and escape innate immune sensing. The CoV nonstructural protein 14 (nsp14) is a bifunctional replicase subunit harboring an N-terminal 3'-to-5' ExoN domain and a C-terminal (N7-guanine)-methyltransferase (N7-MTase) domain that is presumably involved in viral mRNA capping. Here, we aimed to integrate structural, biochemical, and virological data to assess the importance of conserved N7-MTase residues for nsp14's enzymatic activities and virus viability. We revisited the crystal structure of severe acute respiratory syndrome (SARS)-CoV nsp14 to perform an in silico comparative analysis between betacoronaviruses. We identified several residues likely involved in the formation of the N7-MTase catalytic pocket, which presents a fold distinct from the Rossmann fold observed in most known MTases. Next, for SARS-CoV and Middle East respiratory syndrome CoV, site-directed mutagenesis of selected residues was used to assess their importance for in vitro enzymatic activity. Most of the engineered mutations abolished N7-MTase activity, while not affecting nsp14-ExoN activity. Upon reverse engineering of these mutations into different betacoronavirus genomes, we identified two substitutions (R310A and F426A in SARS-CoV nsp14) abrogating virus viability and one mutation (H424A) yielding a crippled phenotype across all viruses tested. Our results identify the N7-MTase as a critical enzyme for betacoronavirus replication and define key residues of its catalytic pocket that can be targeted to design inhibitors with a potential pan-coronaviral activity spectrum.

The methyltransferase domain of the Respiratory Syncytial Virus L protein catalyzes cap N7 and 2'-O-methylation.

Respiratory syncytial virus (RSV) is a negative sense single-stranded RNA virus and one of the main causes of severe lower respiratory tract infections in infants and young children. RSV RNA replication/transcription and capping are ensured by the viral Large (L) protein. The L protein contains a polymerase domain associated with a polyribonucleotidyl transferase domain in its N-terminus, and a methyltransferase (MTase) domain followed by the C-terminal domain (CTD) enriched in basic amino acids at its C-terminus. The MTase-CTD of Mononegavirales forms a clamp to accommodate RNA that is subsequently methylated on the cap structure and depending on the virus, on internal positions. These enzymatic activities are essential for efficient viral mRNA translation into proteins, and to prevent the recognition of uncapped viral RNA by innate immunity sensors. In this work, we demonstrated that the MTase-CTD of RSV, as well as the full-length L protein in complex with phosphoprotein (P), catalyzes the N7- and 2'-O-methylation of the cap structure of a short RNA sequence that corresponds to the 5' end of viral mRNA. Using different experimental systems, we showed that the RSV MTase-CTD methylates the cap structure with a preference for N7-methylation as first reaction. However, we did not observe cap-independent internal methylation, as recently evidenced for the Ebola virus MTase. We also found that at µM concentrations, sinefungin, a S-adenosylmethionine analogue, inhibits the MTase activity of the RSV L protein and of the MTase-CTD domain. Altogether, these results suggest that the RSV MTase domain specifically recognizes viral RNA decorated by a cap structure and catalyzes its methylation, which is required for translation and innate immune system subversion.