Influenza virus polymerase uses a capped primer, derived by 'cap-snatching' from host pre-messenger RNA, to transcribe its RNA genome into mRNA and a stuttering mechanism to generate the poly(A) tail. By contrast, genome replication is unprimed and generates exact full-length copies of the template. Here we use crystal structures of bat influenza A and human influenza B polymerases (FluA and FluB), bound to the viral RNA promoter, to give mechanistic insight into these distinct processes. In the FluA structure, a loop analogous to the priming loop of flavivirus polymerases suggests that influenza could initiate unprimed template replication by a similar mechanism. Comparing the FluA and FluB structures suggests that cap-snatching involves in situ rotation of the PB2 cap-binding domain to direct the capped primer first towards the endonuclease and then into the polymerase active site. The polymerase probably undergoes considerable conformational changes to convert the observed pre-initiation state into the active initiation and elongation states.

Dengue fever, a neglected emerging disease for which no vaccine or antiviral agents exist at present, is caused by dengue virus, a member of the Flavivirus genus, which includes several important human pathogens, such as yellow fever and West Nile viruses. The NS5 protein from dengue virus is bifunctional and contains 900 amino acids. The S-adenosyl methionine transferase activity resides within its N-terminal domain, and residues 270 to 900 form the RNA-dependent RNA polymerase (RdRp) catalytic domain. Viral replication begins with the synthesis of minus-strand RNA from the dengue virus positive-strand RNA genome, which is subsequently used as a template for synthesizing additional plus-strand RNA genomes. This essential function for the production of new viral particles is catalyzed by the NS5 RdRp. Here we present a high-throughput in vitro assay partly recapitulating this activity and the crystallographic structure of an enzymatically active fragment of the dengue virus RdRp refined at 1.85-Å resolution. The NS5 nuclear localization sequences, previously thought to fold into a separate domain, form an integral part of the polymerase subdomains. The structure also reveals the presence of two zinc ion binding motifs. In the absence of a template strand, a chain-terminating nucleoside analogue binds to the priming loop site. These results should inform and accelerate the structure-based design of antiviral compounds against dengue virus.Flaviviridae are enveloped viruses with positive-strand RNA genomes that have been grouped into three genera, Hepacivirus, Pestivirus, and Flavivirus (11,59). Several members of the Flavivirus genus, e.g., dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus, and West Nile virus (WNV), are medically important arthropod-borne pathogens afflicting humans. DENV infects 50 to 100 million people each year, with ϳ500,000 patients developing the more severe disease dengue hemorrhagic fever, leading to hospitalizations and resulting in approximately 20,000 deaths, mainly in children (24,26,27,29). Based on serological studies, DENVs are further classified into four distinct serotypes, DENV 1 to 4, whose respective genomes share ϳ60% sequence identity, with ϳ90% sequence identity within a serotype (7, 26). The DENV RNA genome spans about 10.7 kb and contains a type I methyl guanosine cap structure at its 5Ј end but is devoid of a polyadenylate tail. The genomic RNA is translated into a single polyprotein (58), which is cleaved into three structural (C-prM-E) and seven nonstructural (NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5) proteins by both the viral and cellular proteases (28). The viral serine protease is within the N-terminal region of NS3, and recent structural studies show that part of its catalytic site is formed by the viral cofactor NS2B upon substrate binding (18). The C-terminal region of NS3 forms the RNA helicase domain, which is thought to either separate a double-stranded RNA template into individual strands or disrupt secon...

Macro domains constitute a protein module family found associated with specific histones and proteins involved in chromatin metabolism. In addition, a small number of animal RNA viruses, such as corona-and toroviruses, alphaviruses, and hepatitis E virus, encode macro domains for which, however, structural and functional information is extremely limited. Here, we characterized the macro domains from hepatitis E virus, Semliki Forest virus, and severe acute respiratory syndrome coronavirus (SARS-CoV). The crystal structure of the SARS-CoV macro domain was determined at 1.8-Å resolution in complex with ADP-ribose. Information derived from structural, mutational, and sequence analyses suggests a close phylogenetic and, most probably, functional relationship between viral and cellular macro domain homologs. The data revealed that viral macro domains have relatively poor ADP-ribose 1؆-phosphohydrolase activities (which were previously proposed to be their biologically relevant function) but bind efficiently free and poly(ADP-ribose) polymerase 1-bound poly-(ADP-ribose) in vitro. Collectively, these results suggest to further evaluate the role of viral macro domains in host response to viral infection.

Macro domains (also called"

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.

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 ...

SummarySegmented negative-strand RNA virus (sNSV) polymerases transcribe and replicate the viral RNA (vRNA) within a ribonucleoprotein particle (RNP). We present cryo-EM and X-ray structures of, respectively, apo- and vRNA bound La Crosse orthobunyavirus (LACV) polymerase that give atomic-resolution insight into how such RNPs perform RNA synthesis. The complementary 3′ and 5′ vRNA extremities are sequence specifically bound in separate sites on the polymerase. The 5′ end binds as a stem-loop, allosterically structuring functionally important polymerase active site loops. Identification of distinct template and product exit tunnels allows proposal of a detailed model for template-directed replication with minimal disruption to the circularised RNP. The similar overall architecture and vRNA binding of monomeric LACV to heterotrimeric influenza polymerase, despite high sequence divergence, suggests that all sNSV polymerases have a common evolutionary origin and mechanism of RNA synthesis. These results will aid development of replication inhibitors of diverse, serious human pathogenic viruses.

SummaryRIG-I and MDA5 sense virus-derived short 5′ppp blunt-ended or long dsRNA, respectively, causing interferon production. Non-signaling LGP2 appears to positively and negatively regulate MDA5 and RIG-I signaling, respectively. Co-crystal structures of chicken (ch) LGP2 with dsRNA display a fully or semi-closed conformation depending on the presence or absence of nucleotide. LGP2 caps blunt, 3′ or 5′ overhang dsRNA ends with 1 bp longer overall footprint than RIG-I. Structures of 1:1 and 2:1 complexes of chMDA5 with short dsRNA reveal head-to-head packing rather than the polar head-to-tail orientation described for long filaments. chLGP2 and chMDA5 make filaments with a similar axial repeat, although less co-operatively for chLGP2. Overall, LGP2 resembles a chimera combining a MDA5-like helicase domain and RIG-I like CTD supporting both stem and end binding. Functionally, RNA binding is required for LGP2-mediated enhancement of MDA5 activation. We propose that LGP2 end-binding may promote nucleation of MDA5 oligomerization on dsRNA.