Crystal Structures of Flavivirus NS5 Guanylyltransferase Reveal a GMP-Arginine Adduct.

The positive-sense flavivirus RNA genome bears a cap 1 structure essential for RNA stability and viral protein translation, and the formation of cap 1 requires the virally encoded nonstructural protein NS5 harboring guanylyltransferase (GTase), cap guanine N7 methyltransferase (N7 MTase), and 5'-nucleotide ribose 2'-O MTase activities in its single-domain MTase module. Despite numerous MTase-containing structures reported, the structural evidence for a critical GMP-enzyme intermediate formation and RNA repositioning when transitioning among different reactions is missing. Here, we report 10 high-resolution MTase crystal structures of Omsk hemorrhagic fever virus (OHFV), a representative high-consequence tick-borne flavivirus, capturing previously unidentified GMP-arginine adduct structures and a rarely observed capped RNA conformation. These structures help us thread capping events in the canonical model with a structure-based hypothesis involving the flipping of the 5' nucleotide, while the observation of an m7GMP-arginine adduct is compatible with an alternate capping model that decouples the N7 and 2'-O methylation steps. IMPORTANCE The methyltransferase (MTase) domain of flavivirus NS5 is unique in harboring guanylyltransferase (GTase), N7 MTase, and 2'-O MTase activities, playing a central role in viral RNA capping. However, the detailed mechanisms of the multistep capping process remain elusive. Here, we report 10 crystal structures of a flavivirus MTase to help understand the guanylyl transfer from GTP to the GTase itself and the transition between guanylyl transfer and methylation steps. In particular, a previously unobserved GMP-arginine covalent intermediate was captured multiple times in MTase crystal soaking trials with GTP present in the soaking solution, supporting its role in bridging the guanylyl transfer from GTP to the GTase and subsequent transfer to the 5'-diphosphate RNA.

An induced-fit de novo initiation mechanism suggested by a pestivirus RNA-dependent RNA polymerase.

Viral RNA-dependent RNA polymerases (RdRPs) play central roles in the genome replication and transcription processes of RNA viruses. RdRPs initiate RNA synthesis either in primer-dependent or de novo mechanism, with the latter often assisted by a 'priming element' (PE) within the RdRP thumb domain. However, RdRP PEs exhibit high-level structural diversity, making it difficult to reconcile their conserved function in de novo initiation. Here we determined a 3.1-Å crystal structure of the Flaviviridae classical swine fever virus (CSFV) RdRP with a relative complete PE. Structure-based mutagenesis in combination with enzymology data further highlights the importance of a glycine residue (G671) and the participation of residues 665-680 in RdRP initiation. When compared with other representative Flaviviridae RdRPs, CSFV RdRP PE is structurally distinct but consistent in terminal initiation preference. Taken together, our work suggests that a conformational change in CSFV RdRP PE is necessary to fulfill de novo initiation, and similar 'induced-fit' mechanisms may be commonly taken by PE-containing de novo viral RdRPs.

A Structure-Function Diversity Survey of the RNA-Dependent RNA Polymerases From the Positive-Strand RNA Viruses.

The RNA-dependent RNA polymerases (RdRPs) encoded by the RNA viruses are a unique class of nucleic acid polymerases. Each viral RdRP contains a 500-600 residue catalytic module with palm, fingers, and thumb domains forming an encircled human right hand architecture. Seven polymerase catalytic motifs are located in the RdRP palm and fingers domains, comprising the most conserved parts of the RdRP and are responsible for the RNA-only specificity in catalysis. Functional regions are often found fused to the RdRP catalytic module, resulting in a high level of diversity in RdRP global structure and regulatory mechanism. In this review, we surveyed all 46 RdRP-sequence available virus families of the positive-strand RNA viruses listed in the 2018b collection of the International Committee on Virus Taxonomy (ICTV) and chose a total of 49 RdRPs as representatives. By locating hallmark residues in RdRP catalytic motifs and by referencing structural and functional information in the literature, we were able to estimate the N- and C-terminal boundaries of the catalytic module in these RdRPs, which in turn serve as reference points to predict additional functional regions beyond the catalytic module. Interestingly, a large number of virus families may have additional regions fused to the RdRP N-terminus, while only a few of them have such regions on the C-terminal side of the RdRP. The current knowledge on these additional regions, either in three-dimensional (3D) structure or in function, is quite limited. In the five RdRP-structure available virus families in the positive-strand RNA viruses, only the Flaviviridae family has the 3D structural information resolved for such regions. Hence, future efforts to solve full-length RdRP structures containing these regions and to dissect the functional contribution of them are necessary to improve the overall understanding of the RdRP proteins as an evolutionarily integrated group, and our analyses here may serve as a guideline for selecting representative RdRP systems in these studies.

A dsRNA virus with filamentous viral particles.

Viruses with double-stranded RNA genomes form isometric particles or are capsidless. Here we report a double-stranded RNA virus, Colletotrichum camelliae filamentous virus 1 (CcFV-1) isolated from a fungal pathogen, that forms filamentous particles. CcFV-1 has eight genomic double-stranded RNAs, ranging from 990 to 2444 bp, encoding 10 putative open reading frames, of which open reading frame 1 encodes an RNA-dependent RNA polymerase and open reading frame 4 a capsid protein. When inoculated, the naked CcFV-1 double-stranded RNAs are infectious and induce the accumulation of the filamentous particles in vivo. CcFV-1 is phylogenetically related to Aspergillus fumigatus tetramycovirus-1 and Beauveria bassiana polymycovirus-1, but differs in morphology and in the number of genomic components. CcFV-1 might be an intermediate virus related to truly capsidated viruses, or might represent a distinct encapsidating strategy. In terms of genome and particle architecture, our findings are a significant addition to the knowledge of the virosphere diversity.Viruses with double-stranded RNA (dsRNA) genomes form typically isometric particles or are capsid-less. Here, the authors identify a mycovirus with an eight-segmented dsRNA genome that forms exceptionally long filamentous particles and could represent an evolutionary link between ssRNA and dsRNA viruses.