The Structure of the RNA-Dependent RNA Polymerase of a Permutotetravirus Suggests a Link between Primer-Dependent and Primer-Independent Polymerases

Thosea asigna virus (TaV), an insect virus belonging to the Permutatetraviridae family, has a positive-sense single-stranded RNA (ssRNA) genome with two overlapping open reading frames, encoding for the replicase and capsid proteins. The particular TaV replicase includes a structurally unique RNA-dependent RNA polymerase (RdRP) with a sequence permutation in the palm sub-domain, where the active site is anchored. This non-canonical arrangement of the RdRP palm is also found in double-stranded RNA viruses of the Birnaviridae family. Both virus families also share a conserved VPg sequence motif at the polymerase N-terminus which in birnaviruses appears to be used to covalently link a fraction of the replicase molecules to the 5’-end of the genomic segments. Birnavirus VPgs are presumed to be used as primers for replication initiation. Here we have solved the crystal structure of the TaV RdRP, the first non-canonical RdRP of a ssRNA virus, in its apo- form and bound to different substrates. The enzyme arranges as a stable dimer maintained by mutual interactions between the active site cleft of one molecule and the flexible N-terminal tail of the symmetrically related RdRP. The latter, partially mimicking the RNA template backbone, is involved in regulating the polymerization activity. As expected from previous sequence-based bioinformatics predictions, the overall architecture of the TaV enzyme shows important resemblances with birnavirus polymerases. In addition, structural comparisons and biochemical analyses reveal unexpected similarities between the TaV RdRP and those of Flaviviruses. In particular, a long loop protruding from the thumb domain towards the central enzyme cavity appears to act as a platform for de novo initiation of RNA replication. Our findings strongly suggest an unexpected evolutionary relationship between the RdRPs encoded by these distant ssRNA virus groups.     

Flock House Virus RNA Polymerase Initiates RNA Synthesis De Novo and Possesses a Terminal Nucleotidyl Transferase Activity

Flock House virus (FHV) is a positive-stranded RNA virus with a bipartite genome of RNAs, RNA1 and RNA2, and belongs to the family Nodaviridae. As the most extensively studied nodavirus, FHV has become a well-recognized model for studying various aspects of RNA virology, particularly viral RNA replication and antiviral innate immunity. FHV RNA1 encodes protein A, which is an RNA-dependent RNA polymerase (RdRP) and functions as the sole viral replicase protein responsible for RNA replication. Although the RNA replication of FHV has been studied in considerable detail, the mechanism employed by FHV protein A to initiate RNA synthesis has not been determined. In this study, we characterized the RdRP activity of FHV protein A in detail and revealed that it can initiate RNA synthesis via a de novo (primer-independent) mechanism. Moreover, we found that FHV protein A also possesses a terminal nucleotidyl transferase (TNTase) activity, which was able to restore the nucleotide loss at the 3′-end initiation site of RNA template to rescue RNA synthesis initiation in vitro, and may function as a rescue and protection mechanism to protect the 3′ initiation site, and ensure the efficiency and accuracy of viral RNA synthesis. Altogether, our study establishes the de novo initiation mechanism of RdRP and the terminal rescue mechanism of TNTase for FHV protein A, and represents an important advance toward understanding FHV RNA replication.     

Mutational Analysis of Hepatitis C Virus NS5B in the Subgenomic Replicon Cell Culture

The hepatitis C virus (HCV) NS5B is an RNA-dependent RNA polymerase (RdRP), a central catalytic enzyme of HCV RNA replication. We previously identified five novel residues of NS5B in a JK-1 isolate indispensable for RdRP activity in vitro (Qin, W., Yamashita, T., Shirota, Y., Lin, Y., Wei, W., and Murakami, S. (2001) Hepatology 33, 728-737). We addressed the role of these residues in HCV RNA replication using a HCV replicon system derived from an M1LE isolate (Kishine, H., Sugiyama, K., Hijikata, M., Kato, N., Takahashi, H., Noshi, T., Nio, Y., Hosaka, M., Miyanari, Y., and Shimotohno, K. (2002) Biochem. Biophys. Res. Commun. 293, 993-999). The five residues of NS5B in M1LE were found to be critical for HCV replication in vivo and also indispensable for RdRP activity in vitro along with purified bacterial recombinant proteins. We also found a chimeric replicon of JK-1 and M1LE in which only the NS5B sequence derived from JK-1 could not replicate in Huh-7 cells. The residues responsible for the phenomenon were mapped by several chimeric and substituted forms of NS5B M1LE and/or JK-1 isolates in the HCV RNA replicon. Two residues, amino acids 220 and 288, were critical, and two residues, amino acids 213 and 231, were important for efficient HCV replication. Mutant JK-1 NS5B harboring all four residues of M1LE was replication-competent in the chimeric replicon and was as efficient as the original M1LE replicon. By comparing the replication competence in vivo and RdRP activity in vitro with various chimeric and mutated versions of NS5B, the HCV replication ability was found to correlate well with the RdRP activity. However, heat- and dilution-sensitive NS5Bs exhibiting weaker RdRP activity in vitro were found to be replication-incompetent, suggesting that HCV replication requires RdRP activity higher than a certain critical threshold.     

Characterization of a Nodavirus Replicase Revealed a de Novo Initiation Mechanism of RNA Synthesis and Terminal Nucleotidyltransferase Activity

Nodaviruses are a family of positive-stranded RNA viruses with a bipartite genome of RNAs. In nodaviruses, genomic RNA1 encodes protein A, which is recognized as an RNA-dependent RNA polymerase (RdRP) and functions as the sole viral replicase protein responsible for its RNA replication. Although nodaviral RNA replication has been studied in considerable detail, and nodaviruses are well recognized models for investigating viral RNA replication, the mechanism(s) governing the initiation of nodaviral RNA synthesis have not been determined. In this study, we characterized the RdRP activity of Wuhan nodavirus (WhNV) protein A in detail and determined that this nodaviral protein A initiates RNA synthesis via a de novo mechanism, and this RNA synthesis initiation could be independent of other viral or cellular factors. Moreover, we uncovered that WhNV protein A contains a terminal nucleotidyltransferase (TNTase) activity, which is the first time such an activity has been identified in nodaviruses. We subsequently found that the TNTase activity could function in vitro to repair the 3′ initiation site, which may be digested by cellular exonucleases, to ensure the efficiency and accuracy of viral RNA synthesis initiation. Furthermore, we determined the cis-acting elements for RdRP or TNTase activity at the 3′-end of positive or negative strand RNA1. Taken together, our data establish the de novo synthesis initiation mechanism and the TNTase activity of WhNV protein A, and this work represents an important advance toward understanding the mechanism(s) of nodaviral RNA replication.     

RNA-dependent RNA polymerase of hepatitis C virus: Study on inhibition by α,γ-diketo acid derivatives

It is supposed that α,γ-diketo acids (DKAs) inhibit the activity of hepatitis C virus RNA-dependent RNA poly-merase (RdRP HCV) via chelation of catalytic magnesium ions in the active center of the enzyme. However, DKAs display noncompetitive mode of inhibition with respect to NTP substrate, which contradicts the proposed mechanism. We have examined the NTP substrate entry channel and the active site of RdRP HCV for their possible interaction with DKAs. The substitutions R48A, K51A, and R222A greatly facilitated RdRP inhibition by DKAs and simultaneously increased K _m values for UTP substrate. Interestingly, C223A was the only one of a number of substitutions that decreased K _m(UTP) but facilitated the inhibitory action of DKAs. The findings allowed us to model an enzyme-inhibitor complex. According to the proposed model, DKAs introduce an additional Mg²⁺ ion into the active site of the enzyme at a stage of phosphodiester bond formation, which results in displacement of the NTP substrate triphosphate moiety to a catalytically inactive binding mode. This mechanism, in contrast to the currently adopted one, explains the noncompetitive mode of inhibition.     

The Interface between Methyltransferase and Polymerase of NS5 Is Essential for Flavivirus Replication

The flavivirus NS5 harbors both a methyltransferase (MTase) and an RNA-dependent RNA polymerase (RdRP). Both enzyme activities of NS5 are critical for viral replication. Recently, the full-length NS5 crystal structure of Japanese encephalitis virus reveals a conserved MTase-RdRP interface that features two conserved components: a six-residue hydrophobic network and a GTR sequence. Here we showed for the first time that these key interface components are essential for flavivirus replication by various reverse genetics approaches. Interestingly, some replication-impaired variants generated a common compensatory NS5 mutation outside the interface (L322F), providing novel routes to further explore the crosstalk between MTase and RdRP.     

Structure of foot-and-mouth disease virus RNA-dependent RNA polymerase and its complex with a template-primer RNA

Genome replication in picornaviruses is catalyzed by a virally encoded RNA-dependent RNA polymerase, termed 3D. The enzyme performs this operation, together with other viral and probably host proteins, in the cytoplasm of their host cells. The crystal structure of the 3D polymerase of foot-and-mouth disease virus, one of the most important animal pathogens, has been determined unliganded and bound to a template-primer RNA decanucleotide. The enzyme folds in the characteristic fingers, palm and thumb subdomains, with the presence of an NH2-terminal segment that encircles the active site. In the complex, several conserved amino acid side chains bind to the template-primer, likely mediating the initiation of RNA synthesis. The structure provides essential information for studies on RNA replication and the design of antiviral compounds.     

Crystallographic identification of a noncompetitive inhibitor binding site on the hepatitis C virus NS5B RNA polymerase enzyme

The virus-encoded nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is an RNA-dependent RNA polymerase and is absolutely required for replication of the virus. NS5B exhibits significant differences from cellular polymerases and therefore has become an attractive target for anti-HCV therapy. Using a high-throughput screen, we discovered a novel NS5B inhibitor that binds to the enzyme noncompetitively with respect to nucleotide substrates. Here we report the crystal structure of NS5B complexed with this small molecule inhibitor. Unexpectedly, the inhibitor is bound within a narrow cleft on the protein's surface in the "thumb" domain, about 30 A from the enzyme's catalytic center. The interaction between this inhibitor and NS5B occurs without dramatic changes to the structure of the protein, and sequence analysis suggests that the binding site is conserved across known HCV genotypes. Possible mechanisms of inhibition include perturbation of protein dynamics, interference with RNA binding, and disruption of enzyme oligomerization.     

A Compact Viral Processing Proteinase/Ubiquitin Hydrolase from the OTU Family

Turnip yellow mosaic virus (TYMV) - a member of the alphavirus-like supergroup of viruses - serves as a model system for positive-stranded RNA virus membrane-bound replication. TYMV encodes a precursor replication polyprotein that is processed by the endoproteolytic activity of its internal cysteine proteinase domain (PRO). We recently reported that PRO is actually a multifunctional enzyme with a specific ubiquitin hydrolase (DUB) activity that contributes to viral infectivity. Here, we report the crystal structure of the 150-residue PRO. Strikingly, PRO displays no homology to other processing proteinases from positive-stranded RNA viruses, including that of alphaviruses. Instead, the closest structural homologs of PRO are DUBs from the Ovarian tumor (OTU) family. In the crystal, one molecule''s C-terminus inserts into the catalytic cleft of the next, providing a view of the N-terminal product complex in replication polyprotein processing. This allows us to locate the specificity determinants of PRO for its proteinase substrates. In addition to the catalytic cleft, at the exit of which the active site is unusually pared down and solvent-exposed, a key element in molecular recognition by PRO is a lobe N-terminal to the catalytic domain. Docking models and the activities of PRO and PRO mutants in a deubiquitylating assay suggest that this N-terminal lobe is also likely involved in PRO''s DUB function. Our data thus establish that DUBs can evolve to specifically hydrolyze both iso- and endopeptide bonds with different sequences. This is achieved by the use of multiple specificity determinants, as recognition of substrate patches distant from the cleavage sites allows a relaxed specificity of PRO at the sites themselves. Our results thus shed light on how such a compact protein achieves a diversity of key functions in viral genome replication and host-pathogen interaction.     

Nucleolin interacts with the feline calicivirus 3' untranslated region and the protease-polymerase NS6 and NS7 proteins, playing a role in virus replication

Cellular proteins play many important roles during the life cycle of all viruses. Specifically, host cell nucleic acid-binding proteins interact with viral components of positive-stranded RNA viruses and regulate viral translation, as well as RNA replication. Here, we report that nucleolin, a ubiquitous multifunctional nucleolar shuttling phosphoprotein, interacts with the Norwalk virus and feline calicivirus (FCV) genomic 3' untranslated regions (UTRs). Nucleolin can also form a complex in vitro with recombinant Norwalk virus NS6 and -7 (NS6/7) and can be copurified with the analogous protein from feline calicivirus (p76 or NS6/7) from infected feline kidney cells. Nucleolin RNA levels or protein were not modified during FCV infection; however, as a consequence of the infection, nucleolin was seen to relocalize from the nucleoli to the nucleoplasm, as well as to the perinuclear area where it colocalizes with the feline calicivirus NS6/7 protein. In addition, antibodies to nucleolin were able to precipitate viral RNA from feline calicivirus-infected cells, indicating a direct or indirect association of nucleolin with the viral RNA during virus replication. Small interfering RNA (siRNA)-mediated knockdown of nucleolin resulted in a reduction of the cytopathic effect and virus yield in CrFK cells. Taken together, these results demonstrate that nucleolin is a nucleolar component that interacts with viral RNA and NS6/7 and is required for feline calicivirus replication.     

Interaction of cellular proteins with the 5' end of Norwalk virus genomic RNA

The lack of a susceptible cell line and an animal model for Norwalk virus (NV) infection has prompted the development of alternative strategies to generate in vitro RNAs that approximate the authentic viral genome. This approach has allowed the study of viral RNA replication and gene expression. In this study, using mobility shift and cross-linking assays, we detected several cellular proteins from HeLa and CaCo-2 cell extracts that bind to, and form stable complexes with, the first 110 nucleotides of the 5' end of NV genomic RNA, a region previously predicted to form a double stem-loop structure. These proteins had molecular weights similar to those of the HeLa cellular proteins that bind to the internal ribosomal entry site of poliovirus RNA. HeLa proteins La, PCBP-2, and PTB, which are important for poliovirus translation, and hnRNP L, which is possibly implicated in hepatitis C virus translation, interact with NV RNA. These protein-RNA interactions are likely to play a role in NV translation and/or replication.     

The structure of a protein primer-polymerase complex in the initiation of genome replication

Picornavirus RNA replication is initiated by the covalent attachment of a UMP molecule to the hydroxyl group of a tyrosine in the terminal protein VPg. This reaction is carried out by the viral RNA-dependent RNA polymerase (3D). Here, we report the X-ray structure of two complexes between foot-and-mouth disease virus 3D, VPg1, the substrate UTP and divalent cations, in the absence and in the presence of an oligoadenylate of 10 residues. In both complexes, VPg fits the RNA binding cleft of the polymerase and projects the key residue Tyr3 into the active site of 3D. This is achieved by multiple interactions with residues of motif F and helix alpha8 of the fingers domain and helix alpha13 of the thumb domain of the polymerase. The complex obtained in the presence of the oligoadenylate showed the product of the VPg uridylylation (VPg-UMP). Two metal ions and the catalytic aspartic acids of the polymerase active site, together with the basic residues of motif F, have been identified as participating in the priming reaction.     

Template recognition and formation of initiation complexes by the replicase of a segmented double-stranded RNA virus

Replication of the segmented double-stranded (ds) RNA genome of viruses belonging to the Reoviridae family requires the RNA-dependent RNA polymerase (RdRP) to use 10-12 different mRNAs as templates for (-) strand synthesis. Rotavirus serves as a model system for study of this process, since its RdRP (VP1) is catalytically active and can specifically recognize template mRNAs in vitro. Here, we have analyzed the requirements for template recognition by the rotavirus RdRP and compared those to the requirements for formation of (-) strand initiation complexes. The results show that multiple functionally independent recognition signals are present at the 3'-end of viral mRNAs, some positioned in nonconserved regions upstream of the highly conserved 3'-terminal consensus sequence. We also found that RdRP recognition signals are distinct from cis-acting signals that promote (-) strand synthesis, because deletions of portions of the 3'-consensus sequence that caused viral mRNAs to be poorly replicated in vitro did not necessarily prevent efficient recognition of the RNA by the RdRP. Although the RdRP alone can specifically bind to viral mRNAs, our analysis reveals that this interaction is not sufficient to generate initiation complexes, even in the presence of nucleotides and divalent cations. Rather, the formation of initiation complexes also requires the core lattice protein (VP2), a virion component that forms a T = 1 icosahedral shell that encapsidates the segmented dsRNA genome. The essential role that the core lattice protein has in (-) strand initiation provides a mechanism for the coordination of genome replication and virion assembly.     

Norwalk virus N-terminal nonstructural protein is associated with disassembly of the Golgi complex in transfected cells

Norwalk virus is the prototype strain for members of the genus Norovirus in the family Caliciviridae, which are associated with epidemic gastroenteritis in humans. The nonstructural protein encoded in the N-terminal region of the first open reading frame (ORF1) of the Norwalk virus genome is analogous in gene order to proteins 2A and 2B of the picornaviruses; the latter is known for its membrane-associated activities. Confocal microscopy imaging of cells transfected with a vector plasmid that provided expression of the entire Norwalk virus N-terminal protein (amino acids 1 to 398 of the ORF1 polyprotein) showed colocalization of this protein with cellular proteins of the Golgi apparatus. Furthermore, this colocalization was characteristically associated with a visible disassembly of the Golgi complex into discrete aggregates. Deletion of a predicted hydrophobic region (amino acids 360 to 379) in a potential 2B-like (2BL) region (amino acids 301 to 398) near the C terminus of the Norwalk virus N-terminal protein reduced Golgi colocalization and disassembly. Confocal imaging was conducted to examine the expression characteristics of fusion proteins in which the 2BL region from the N-terminal protein of Norwalk virus (a genogroup I norovirus) or MD145 (a genogroup II norovirus) was fused to the C terminus of enhanced green fluorescent protein. Expression of each fusion protein in cells showed evidence for its colocalization with the Golgi apparatus. These data indicate that the N-terminal protein of Norwalk virus interacts with the Golgi apparatus and may play a 2BL role in the induction of intracellular membrane rearrangements associated with positive-strand RNA virus replication in cells.     

Norovirus RNA‐dependent RNA polymerase: A computational study of metal‐binding preferences

Norovirus (NV) RNA‐dependent RNA polymerase (RdRP) is essential for replicating the genome of the virus, which makes this enzyme a key target for the development of antiviral agents against NV gastroenteritis. In this work, a complex of NV RdRP bound to manganese ions and an RNA primer‐template duplex was investigated using X‐ray crystallography and hybrid quantum chemical/molecular mechanical simulations. Experimentally, the complex crystallized in a tetragonal crystal form. The nature of the primer/template duplex binding in the resulting structure indicates that the complex is a closed back‐tracked state of the enzyme, in which the ‐end of the primer occupies the position expected for the post‐incorporated nucleotide before translocation. Computationally, it is found that the complex can accept a range of divalent metal cations without marked distortions in the active site structure. The highest binding energy is for copper, followed closely by manganese and iron, and then by zinc, nickel, and cobalt. Proteins 2017; 85:1435–1445. © 2017 Wiley Periodicals, Inc.