contributed equally to this work Viruses represent an attractive system with which to study the molecular basis of mRNA capping and its relation to the RNA transcription machinery. The RNA-dependent RNA polymerase NS5 of¯avi-viruses presents a characteristic motif of S-adenosyl-L-methionine-dependent methyltransferases at its N-terminus, and polymerase motifs at its C-terminus. The crystal structure of an N-terminal fragment of Dengue virus type 2 NS5 is reported at 2.4 A Ê resolution. We show that this NS5 domain includes a typical methyltransferase core and exhibits a (nucleoside-2¢-O-)-methyltransferase activity on capped RNA. The structure of a ternary complex comprising S-adenosyl-L-homocysteine and a guanosine triphosphate (GTP) analogue shows that 54 amino acids N-terminal to the core provide a novel GTP-binding site that selects guanine using a previously unreported mechanism. Binding studies using GTP-and RNA cap-analogues, as well as the spatial arrangement of the methyltransferase active site relative to the GTPbinding site, suggest that the latter is a speci®c capbinding site. As RNA capping is an essential viral function, these results provide a structural basis for the rational design of drugs against the emerging¯avi-viruses. Keywords: crystal structure/¯avivirus/GTP/RNA capping/RNA polymerase IntroductionThe cap is a unique structure found at the 5¢-end of viral and cellular eukaryotic mRNA (Bisaillon and Lemay, 1997;Furuichi and Shatkin, 2000). It is critical for both mRNA stability and binding to the ribosome during translation. mRNA capping is a co-transcriptional modi®cation resulting from a series of three chemical reactions (Shuman, 2001). The 5¢-triphosphate of the mRNA is ®rst converted to a diphosphate by an RNA triphosphatase. The second reaction is a transfer of a guanosine monophosphate (GMP) moiety from GTP to the 5¢-diphosphate RNA by a guanylyltransferase to yield G 5 ¢-ppp 5 ¢-N. In a third reaction utilizing S-adenosyl-L-methionine (AdoMet) as the methyl donor, the transferred guanosine moiety is methylated by a (guanine-N7)-methyltransferase (N7MTase) to yield 7Me G 5 ¢-ppp 5 ¢-N (cap 0 structure). A second methylation reaction catalysed by a (nucleoside-2¢-O-)-methyltransferase (2¢OMTase) occurs on the ®rst nucleotide 3¢ to the triphosphate bridge to yield 7Me G 5 ¢-ppp 5 ¢-N Me (cap 1 structure). Adjacent nucleotides 3¢ to the ®rst one can also be 2¢-O-methylated to various extents. The order of the methyltransfer reactions is variable, and in some viruses GTP is methylated at its N7 position before being transferred to the RNA 5¢-end (Ahola and Kaariainen, 1995;Furuichi and Shatkin, 2000).Few enzymes involved in the RNA capping pathway have been structurally characterized. The crystal structures of two cellular RNA triphosphatases from yeast and mouse have been determined at 2.0 and 1.6 A Ê resolution, respectively. These structures provide a mechanistic insight into the ®rst reaction in the RNA capping pathway catalysed by prototypic enzymes of the metal-dependent and -indep...

SARS-coronavirus (SARS-CoV) genome expression depends on the synthesis of a set of mRNAs, which presumably are capped at their 5′ end and direct the synthesis of all viral proteins in the infected cell. Sixteen viral non-structural proteins (nsp1 to nsp16) constitute an unusually large replicase complex, which includes two methyltransferases putatively involved in viral mRNA cap formation. The S-adenosyl-L-methionine (AdoMet)-dependent (guanine-N7)-methyltransferase (N7-MTase) activity was recently attributed to nsp14, whereas nsp16 has been predicted to be the AdoMet-dependent (nucleoside-2′O)-methyltransferase. Here, we have reconstituted complete SARS-CoV mRNA cap methylation in vitro. We show that mRNA cap methylation requires a third viral protein, nsp10, which acts as an essential trigger to complete RNA cap-1 formation. The obligate sequence of methylation events is initiated by nsp14, which first methylates capped RNA transcripts to generate cap-0 7MeGpppA-RNAs. The latter are then selectively 2′O-methylated by the 2′O-MTase nsp16 in complex with its activator nsp10 to give rise to cap-1 7MeGpppA2′OMe-RNAs. Furthermore, sensitive in vitro inhibition assays of both activities show that aurintricarboxylic acid, active in SARS-CoV infected cells, targets both MTases with IC50 values in the micromolar range, providing a validated basis for anti-coronavirus drug design.

The rapid global emergence of SARS-CoV-2 has been the cause of significant health concern, highlighting the immediate need for antivirals. Viral RNA-dependent RNA polymerases (RdRp) play essential roles in viral RNA synthesis, and thus remains the target of choice for the prophylactic or curative treatment of several viral diseases, due to high sequence and structural conservation. To date, the most promising broad-spectrum class of viral RdRp inhibitors are nucleoside analogues (NAs), with over 25 approved for the treatment of several medically important viral diseases. However, Coronaviruses stand out as a particularly challenging case for NA drug design due to the presence of an exonuclease (ExoN) domain capable of excising incorporated NAs and thus providing resistance to many of these available antivirals. Here we use the available structures of the SARS-CoV RdRp and ExoN proteins, as well as Lassa virus N exonuclease to derive models of catalytically competent SARS-CoV-2 enzymes. We then map a promising NA candidate, GS-441524 (the active metabolite of Remdesivir) to the nucleoside active site of both proteins, identifying the residues important for nucleotide recognition, discrimination, and excision. Interestingly, GS-441524 addresses both enzyme active sites in a manner consistent with significant incorporation, delayed chain termination, and altered excision due to the ribose 1'-CN group, which may account for the increased antiviral effect compared to other available analogues. Additionally, we propose structural and function implications of two previously identified RdRp resistance mutations in relation to resistance against Remdesivir.This study highlights the importance of considering the balance between incorporation and excision properties of NAs between the RdRp and ExoN.

The coronavirus family of positive-strand RNA viruses includes important pathogens of livestock, companion animals, and humans, including the severe acute respiratory syndrome coronavirus that was responsible for a worldwide outbreak in 2003. The unusually complex coronavirus replicase/transcriptase is comprised of 15 or 16 virus-specific subunits that are autoproteolytically derived from two large polyproteins. In line with bioinformatics predictions, we now show that feline coronavirus (FCoV) nonstructural protein 16 (nsp16) possesses an S-adenosyl-L-methionine (AdoMet)-dependent RNA (nucleoside-2O)-methyltransferase (2O-MTase) activity that is capable of cap-1 formation. Purified recombinant FCoV nsp16 selectively binds to short capped RNAs. Remarkably, an N7-methyl guanosine cap ( 7Me GpppAC 3-6 ) is a prerequisite for binding. High-performance liquid chromatography analysis demonstrated that nsp16 mediates methyl transfer from AdoMet to the 2O position of the first transcribed nucleotide, thus converting 7Me GpppAC 3-6 into 7Me GpppA 2OMe C 3-6 . The characterization of 11 nsp16 mutants supported the previous identification of residues K45, D129, K169, and E202 as the putative K-D-K-E catalytic tetrad of the enzyme. Furthermore, residues Y29 and F173 of FCoV nsp16, which may be the functional counterparts of aromatic residues involved in substrate recognition by the vaccinia virus MTase VP39, were found to be essential for both substrate binding and 2O-MTase activity. Finally, the weak inhibition profile of different AdoMet analogues indicates that nsp16 has evolved an atypical AdoMet binding site. Our results suggest that coronavirus mRNA carries a cap-1, onto which 2O methylation follows an order of events in which 2O-methyl transfer must be preceded by guanine N7 methylation, with the latter step being performed by a yet-unknown N7-specific MTase.

Viruses of the family Flaviviridae are important human and animal pathogens. Among them, the Flaviviruses dengue (DENV) and West Nile (WNV) cause regular outbreaks with fatal outcomes. The RNA-dependent RNA polymerase (RdRp) activity of the non-structural protein 5 (NS5) is a key activity for viral RNA replication. In this study, crystal structures of enzymatically active and inactive WNV RdRp domains were determined at 3.0-and 2.35-Å resolution, respectively. The determined structures were shown to be mostly similar to the RdRps of the Flaviviridae members hepatitis C and bovine viral diarrhea virus, although with unique elements characteristic for the WNV RdRp. Using a reverse genetic system, residues involved in putative interactions between the RNA-cap methyltransferase (MTase) and the RdRp domain of Flavivirus NS5 were identified. This allowed us to propose a model for the structure of the full-length WNV NS5 by in silico docking of the WNV MTase domain (modeled from our previously determined structure of the DENV MTase domain) onto the RdRp domain. The Flavivirus RdRp domain structure determined here should facilitate both the design of anti-Flavivirus drugs and structure-function studies of the Flavivirus replication complex in which the multifunctional NS5 protein plays a central role.The Flaviviridae form a large family of single-stranded positive-sense RNA viruses comprising the three genera Hepacivirus, Pestivirus, and Flavivirus. The genus Flavivirus contains more than 80 known arthropod-borne viruses, including major human and animal pathogens such as dengue virus (DENV), 3 yellow fever virus, Japanese encephalitis virus, and West Nile virus (WNV). Both DENV and WNV are considered as emerging pathogens. Dengue fever is one of the most important mosquito-borne viral diseases in the world, with more than 3 billion people at risk in endemic tropical areas (1). Dengue outbreaks are increasingly severe in terms of cases and fatalities in many regions of the world (2). WNV was discovered in the West Nile district in Uganda in 1937 and was subsequently shown to have an extensive worldwide distribution with the exception of the Americas (1). In 1999, WNV was introduced into the Americas in the New York City area and has since spread throughout the mainland United States, southern Canada, and Mexico. WNV epidemics in the United States have resulted in a total of 23,925 cases of human disease and 946 deaths reported to the Centers for Disease Control (CDC) from 1999 to 2006. WNV consists of 2 lineages (I and II). The North American WNV isolates belong to lineage I, which also includes the Australian subtype Kunjin (3). In contrast to other lineage I WNV strains (4), infections with the Kunjin subtype of WNV do not cause fatal disease in humans (5).The Flavivirus positive sense RNA genome contains a single open reading frame encoding a polyprotein that is processed into three structural and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). Signature-sequence analysis suggests that the ...

Flaviviridae are small enveloped viruses hosting a positive-sense single-stranded RNA genome. Besides yellow fever virus, a landmark case in the history of virology, members of the Flavivirus genus, such as West Nile virus and dengue virus, are increasingly gaining attention due to their re-emergence and incidence in different areas of the world. Additional environmental and demographic considerations suggest that novel or known flaviviruses will continue to emerge in the future. Nevertheless, up to few years ago flaviviruses were considered low interest candidates for drug design. At the start of the European Union VIZIER Project, in 2004, just two crystal structures of protein domains from the flaviviral replication machinery were known. Such pioneering studies, however, indicated the flaviviral replication complex as a promising target for the development of antiviral compounds. Here we review structural and functional aspects emerging from the characterization of two main components (NS3 and NS5 proteins) of the flavivirus replication complex. Most of the reviewed results were achieved within the European Union VIZIER Project, and cover topics that span from viral genomics to structural biology and inhibition mechanisms. The ultimate aim of the reported approaches is to shed light on the design and development of antiviral drug leads.

The ongoing Corona Virus Disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has emphasized the urgent need for antiviral therapeutics. The viral RNA-dependent-RNA-polymerase (RdRp) is a promising target with polymerase inhibitors successfully used for the treatment of several viral diseases. We demonstrate here that Favipiravir predominantly exerts an antiviral effect through lethal mutagenesis. The SARS-CoV RdRp complex is at least 10-fold more active than any other viral RdRp known. It possesses both unusually high nucleotide incorporation rates and high-error rates allowing facile insertion of Favipiravir into viral RNA, provoking C-to-U and G-to-A transitions in the already low cytosine content SARS-CoV-2 genome. The coronavirus RdRp complex represents an Achilles heel for SARS-CoV, supporting nucleoside analogues as promising candidates for the treatment of COVID-19.

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