A specific domain within the 3' untranslated region of Usutu virus confers resistance to the exonuclease ISG20.

Usutu virus (USUV) and West Nile virus (WNV) are two closely related emerging mosquito-borne flaviviruses. Their natural hosts are wild birds, but they can also cause severe neurological disorders in humans. Both viruses are efficiently suppressed by type I interferon (IFN), which interferes with viral replication, dissemination, pathogenesis and transmission. Here, we show that the replication of USUV and WNV are inhibited through a common set of IFN-induced genes (ISGs), with the notable exception of ISG20, which USUV is resistant to. Strikingly, USUV was the only virus among all the other tested mosquito-borne flaviviruses that demonstrated resistance to the 3'-5' exonuclease activity of ISG20. Our findings highlight that the intrinsic resistance of the USUV genome, irrespective of the presence of cellular or viral proteins or protective post-transcriptional modifications, relies on a unique sequence present in its 3' untranslated region. Importantly, this genomic region alone can confer ISG20 resistance to a susceptible flavivirus, without compromising its infectivity, suggesting that it could be acquired by other flaviviruses. This study provides new insights into the strategy employed by emerging flaviviruses to overcome host defense mechanisms.

[When 2'-O-methylation throws a wrench in HIV-1 reverse transcriptase]. / La 2'-O-méthylation : un grain de sable dans les rouages de la transcriptase inverse du VIH-1.

HIV-1 polymerase, commonly known as HIV reverse transcriptase (RT), catalyzes the critical reaction of reverse transcription by synthesizing a double-stranded DNA copy of the viral genomic RNA. During the replication cycle, this synthesized DNA is integrated into the host genome. This entire process is essential for viral replication and is targeted by several antiviral drugs. Numerous studies in biochemistry and structural biology have led to a good understanding of HIV-1 RT functions. However, the discovery of epitranscriptomic marks, such as 2'-O-methylations, on the HIV-1 RNA genome raise the questions about RT's ability to copy RNAs decorated with these biochemical modifications. This review focuses on the importance of RT in the viral cycle, its structure and function and the impact of 2'-O-methylations on its activity and replication regulation, particularly in quiescent cells.

JNJ-7184, a respiratory syncytial virus inhibitor targeting the connector domain of the viral polymerase.

Respiratory syncytial virus (RSV) can cause pulmonary complications in infants, elderly and immunocompromised patients. While two vaccines and two prophylactic monoclonal antibodies are now available, treatment options are still needed. JNJ-7184 is a non-nucleoside inhibitor of the RSV-Large (L) polymerase, displaying potent inhibition of both RSV-A and -B strains. Resistance selection and hydrogen-deuterium exchange experiments suggest JNJ-7184 binds RSV-L in the connector domain. JNJ-7184 prevents RSV replication and transcription by inhibiting initiation or early elongation. JNJ-7184 is effective in air-liquid interface cultures and therapeutically in neonatal lambs, acting to drastically reverse the appearance of lung pathology.

[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.

N-Arylsulfonamide-based adenosine analogues to target RNA cap N7-methyltransferase nsp14 of SARS-CoV-2.

RNA cap methylations have been shown to be crucial for the life cycle, replication, and infection of ssRNA viruses, as well as for evading the host's innate immune system. Viral methyltransferases (MTases) therefore represent an attractive target for the development of compounds as tools and inhibitors. In coronaviruses, N7-methyltransferase function is localized in nsp14, which has become an increasingly important therapeutic target with the COVID-19 pandemic. In recent years, we have been developing SAH-derived bisubstrates with adenosine and an N-arylsulfonamide moiety targeting both SAM and RNA binding sites in nsp14. We report here the synthesis of 31 SAH analogues with the N-arylsulfonamide attached to the 5'-position of adenosine via different linkers such as N-ethylthioether, N-ethylsulfone, N-ethylamino or N-methyltriazole. The compounds were obtained efficiently by amine sulfonylation or click chemistry. Their ability to inhibit SARS-CoV-2 N7-MTase was evaluated and the best inhibitors showed a submicromolar inhibitory activity against N7-MTase nsp14.

Identification of Novel Non-Nucleoside Inhibitors of Zika Virus NS5 Protein Targeting MTase Activity.

Zika virus (ZIKV) is a positive-sense single-stranded virus member of the Flaviviridae family. Among other arboviruses, ZIKV can cause neurological disorders such as Guillain Barré syndrome, and it can have congenital neurological manifestations and affect fertility. ZIKV nonstructural protein 5 (NS5) is essential for viral replication and limiting host immune detection. Herein, we performed virtual screening to identify novel small-molecule inhibitors of the ZIKV NS5 methyltransferase (MTase) domain. Compounds were tested against the MTases of both ZIKV and DENV, demonstrating good inhibitory activities against ZIKV MTase. Extensive molecular dynamic studies conducted on the series led us to identify other derivatives with improved activity against the MTase and limiting ZIKV infection with an increased selectivity index. Preliminary pharmacokinetic parameters have been determined, revealing excellent stability over time. Preliminary in vivo toxicity studies demonstrated that the hit compound 17 is well tolerated after acute administration. Our results provide the basis for further optimization studies on novel non-nucleoside MTase inhibitors.

5'-cap RNA/SAM mimetic conjugates as bisubstrate inhibitors of viral RNA cap 2'-O-methyltransferases.

Viral RNA cap 2'-O-methyltransferases are considered promising therapeutic targets for antiviral treatments, as they play a key role in the formation of viral RNA cap-1 structures to escape the host immune system. A better understanding of how they interact with their natural substrates (RNA and the methyl donor SAM) would enable the rational development of potent inhibitors. However, as few structures of 2'-O-MTases in complex with RNA have been described, little is known about substrate recognition by these MTases. For this, chemical tools mimicking the state in which the cap RNA substrate and SAM cofactor are bound in the enzyme's catalytic pocket may prove useful. In this work, we designed and synthesized over 30 RNA conjugates that contain a short oligoribonucleotide (ORN with 4 or 6 nucleotides) with the first nucleotide 2'-O-attached to an adenosine by linkers of different lengths and containing S or N-heteroatoms, or a 1,2,3-triazole ring. These ORN conjugates bearing or not a cap structure at 5'-extremity mimic the methylation transition state with RNA substrate/SAM complex as bisubstrates of 2'-O-MTases. The ORN conjugates were synthesized either by the incorporation of a dinucleoside phosphoramidite during RNA elongation or by click chemistry performed on solid-phase post-RNA elongation. Their ability to inhibit the activity of the nsp16/nsp10 complex of SARS-CoV-2 and the NS5 protein of dengue and Zika viruses was assessed. Significant submicromolar IC50 values and Kd values in the µM range were found, suggesting a possible interaction of some ORN conjugates with these viral 2'-O-MTases.

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.

Structural and mechanistic insights into the inhibition of respiratory syncytial virus polymerase by a non-nucleoside inhibitor.

The respiratory syncytial virus polymerase complex, consisting of the polymerase (L) and phosphoprotein (P), catalyzes nucleotide polymerization, cap addition, and cap methylation via the RNA dependent RNA polymerase, capping, and Methyltransferase domains on L. Several nucleoside and non-nucleoside inhibitors have been reported to inhibit this polymerase complex, but the structural details of the exact inhibitor-polymerase interactions have been lacking. Here, we report a non-nucleoside inhibitor JNJ-8003 with sub-nanomolar inhibition potency in both antiviral and polymerase assays. Our 2.9 Å resolution cryo-EM structure revealed that JNJ-8003 binds to an induced-fit pocket on the capping domain, with multiple interactions consistent with its tight binding and resistance mutation profile. The minigenome and gel-based de novo RNA synthesis and primer extension assays demonstrated that JNJ-8003 inhibited nucleotide polymerization at the early stages of RNA transcription and replication. Our results support that JNJ-8003 binding modulates a functional interplay between the capping and RdRp domains, and this molecular insight could accelerate the design of broad-spectrum antiviral drugs.

Structure-guided optimization of adenosine mimetics as selective and potent inhibitors of coronavirus nsp14 N7-methyltransferases.

The COVID-19 pandemic reveals the urgent need to develop new therapeutics targeting the SARS-CoV-2 replication machinery. The first antiviral drugs were nucleoside analogues targeting RdRp and protease inhibitors active on nsp5 Mpro. In addition to these common antiviral targets, SARS-CoV-2 codes for the highly conserved protein nsp14 harbouring N7-methyltransferase (MTase) activity. Nsp14 is involved in cap N7-methylation of viral RNA and its inhibition impairs viral RNA translation and immune evasion, making it an attractive new antiviral target. In this work, we followed a structure-guided drug design approach to design bisubstrates mimicking the S-adenosylmethionine methyl donor and RNA cap. We developed adenosine mimetics with an N-arylsulfonamide moiety in the 5'-position, recently described as a guanine mimicking the cap structure in a potent adenosine-derived nsp14 inhibitor. Here, the adenine moiety was replaced by hypoxanthine, N6-methyladenine, or C7-substituted 7-deaza-adenine. 26 novel adenosine mimetics were synthesized, one of which selectively inhibits nsp14 N7-MTase activity with a subnanomolar IC50 (and seven with a single-digit nanomolar IC50). In the most potent inhibitors, adenine was replaced by two different 7-deaza-adenines bearing either a phenyl or a 3-quinoline group at the C7-position via an ethynyl linker. These more complex compounds are barely active on the cognate human N7-MTase and docking experiments reveal that their selectivity of inhibition might result from the positioning of their C7 substitution in a SAM entry tunnel present in the nsp14 structure and absent in the hN7-MTase. These compounds show moderate antiviral activity against SARS-CoV-2 replication in cell culture, suggesting delivery or stability issue.

Design and synthesis of naturally-inspired SARS-CoV-2 inhibitors.

A naturally inspired chemical library of 25 molecules was synthesised guided by 3-D dimensionality and natural product likeness factors to explore a new chemical space. The synthesised chemical library, consisting of fused-bridged dodecahydro-2a,6-epoxyazepino[3,4,5-c,d]indole skeletons, followed lead likeness factors in terms of molecular weight, C-sp3 fraction and Clog P. Screening of the 25 compounds against lung cells infected with SARS-CoV-2 led to the identification of 2 hits. Although the chemical library showed cytotoxicity, the two hits (3b, 9e) showed the highest antiviral activity (EC50 values of 3.7 and 1.4 µM, respectively) with an acceptable cytotoxicity difference. Computational analysis based on docking and molecular dynamics simulations against main protein targets in SARS-CoV-2 (main protease Mpro, nucleocapsid phosphoprotein, non-structural protein nsp10-nsp16 complex and RBD/ACE2 complex) were performed. The computational analysis proposed the possible binding targets to be either Mpro or the nsp10-nsp16 complex. Biological assays were performed to confirm this proposition. A cell-based assay for Mpro protease activity using a reverse-nanoluciferase (Rev-Nluc) reporter confirmed that 3b targets Mpro. These results open the way towards further hit-to-lead optimisations.

AT-752 targets multiple sites and activities on the Dengue virus replication enzyme NS5.

AT-752 is a guanosine analogue prodrug active against dengue virus (DENV). In infected cells, it is metabolized into 2'-methyl-2'-fluoro guanosine 5'-triphosphate (AT-9010) which inhibits RNA synthesis in acting as a RNA chain terminator. Here we show that AT-9010 has several modes of action on DENV full-length NS5. AT-9010 does not inhibit the primer pppApG synthesis step significantly. However, AT-9010 targets two NS5-associated enzyme activities, the RNA 2'-O-MTase and the RNA-dependent RNA polymerase (RdRp) at its RNA elongation step. Crystal structure and RNA methyltransferase (MTase) activities of the DENV 2 MTase domain in complex with AT-9010 at 1.97 Å resolution shows the latter bound to the GTP/RNA-cap binding site, accounting for the observed inhibition of 2'-O but not N7-methylation activity. AT-9010 is discriminated ∼10 to 14-fold against GTP at the NS5 active site of all four DENV1-4 NS5 RdRps, arguing for significant inhibition through viral RNA synthesis termination. In Huh-7 cells, DENV1-4 are equally sensitive to AT-281, the free base of AT-752 (EC50 ≈ 0.50 µM), suggesting broad spectrum antiviral properties of AT-752 against flaviviruses.

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.

Biochemistry of the Respiratory Syncytial Virus L Protein Embedding RNA Polymerase and Capping Activities.

The human respiratory syncytial virus (RSV) is a negative-sense, single-stranded RNA virus. It is the major cause of severe acute lower respiratory tract infection in infants, the elderly population, and immunocompromised individuals. There is still no approved vaccine or antiviral treatment against RSV disease, but new monoclonal prophylactic antibodies are yet to be commercialized, and clinical trials are in progress. Hence, urgent efforts are needed to develop efficient therapeutic treatments. RSV RNA synthesis comprises viral transcription and replication that are catalyzed by the large protein (L) in coordination with the phosphoprotein polymerase cofactor (P), the nucleoprotein (N), and the M2-1 transcription factor. The replication/transcription is orchestrated by the L protein, which contains three conserved enzymatic domains: the RNA-dependent RNA polymerase (RdRp), the polyribonucleotidyl transferase (PRNTase or capping), and the methyltransferase (MTase) domain. These activities are essential for the RSV replicative cycle and are thus considered as attractive targets for the development of therapeutic agents. In this review, we summarize recent findings about RSV L domains structure that highlight how the enzymatic activities of RSV L domains are interconnected, discuss the most relevant and recent antivirals developments that target the replication/transcription complex, and conclude with a perspective on identified knowledge gaps that enable new research directions.

Amino Acid Polymorphisms on the Brazilian Strain of Yellow Fever Virus Methyltransferase Are Related to the Host's Immune Evasion Mediated by Type I Interferon.

Since late 2016, a yellow fever virus (YFV) variant carrying a set of nine amino acid variations has circulated in South America. Three of them were mapped on the methyltransferase (MTase) domain of viral NS5 protein. To assess whether these changes affected viral infectivity, we synthesized YFV carrying the MTase of circulating lineage as well as its isoform with the residues of the previous strains (NS5 K101R, NS5 V138I, and NS5 G173S). We observed a slight difference in viral growth properties and plaque phenotype between the two synthetic YFVs. However, the MTase polymorphisms associated with the Brazilian strain of YFV (2016-2019) confer more susceptibility to the IFN-I. In addition, in vitro MTase assay revealed that the interaction between the YFV MTase and the methyl donor molecule (SAM) is altered in the Brazilian MTase variant. Altogether, the results reported here describe that the MTase carrying the molecular signature of the Brazilian YFV circulating since 2016 might display a slight decrease in its catalytic activity but virtually no effect on viral fitness in the parameters comprised in this study. The most marked influence of these residues stands in the immune escape against the antiviral response mediated by IFN-I.

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.

A second type of N7-guanine RNA cap methyltransferase in an unusual locus of a large RNA virus genome.

The order Nidovirales is a diverse group of (+)RNA viruses, with a common genome organization and conserved set of replicative and editing enzymes. In particular, RNA methyltransferases play a central role in mRNA stability and immune escape. However, their presence and distribution in different Nidovirales families is not homogeneous. In Coronaviridae, the best characterized family, two distinct methytransferases perform methylation of the N7-guanine and 2'-OH of the RNA-cap to generate a cap-1 structure (m7GpppNm). The genes of both of these enzymes are located in the ORF1b genomic region. While 2'-O-MTases can be identified for most other families based on conservation of both sequence motifs and genetic loci, identification of the N7-guanine methyltransferase has proved more challenging. Recently, we identified a putative N7-MTase domain in the ORF1a region (N7-MT-1a) of certain members of the large genome Tobaniviridae family. Here, we demonstrate that this domain indeed harbors N7-specific methyltransferase activity. We present its structure as the first N7-specific Rossmann-fold (RF) MTase identified for (+)RNA viruses, making it remarkably different from that of the known Coronaviridae ORF1b N7-MTase gene. We discuss the evolutionary implications of such an appearance in this unexpected location in the genome, which introduces a split-off in the classification of Tobaniviridae.