Crystal Structure of Enterovirus 71 RNA-Dependent RNA Polymerase Complexed with Its Protein Primer VPg: Implication for a trans Mechanism of VPg Uridylylation

ABSTRACTClassical swine fever virus (CSFV) is the cause of classical swine fever (CSF). Nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) that is a key enzyme initiating viral RNA replication by ade novomechanism. It is also an attractive target for the development of anti-CSFV drugs. To gain a better understanding of the mechanism of CSFV RNA synthesis, here, we solved the first crystal structure of CSFV NS5B. Our studies show that the CSFV NS5B RdRp contains the characteristic finger, palm, and thumb domains, as well as a unique N-terminal domain (NTD) that has never been observed. Mutagenesis studies on NS5B validated the importance of the NTD in the catalytic activity of this novel RNA-dependent RNA polymerase. Moreover, our results shed light on CSFV infection.IMPORTANCEPigs are important domesticated animals. However, a highly contagious viral disease named classical swine fever (CSF) causes devastating economic losses. Classical swine fever virus (CSFV), the primary cause of CSF, is a positive-sense single-stranded RNA virus belonging to the genusPestivirus, familyFlaviviridae. Genome replication of CSFV depends on an RNA-dependent RNA polymerase (RdRp) known as NS5B. However, the structure of CSFV NS5B has never been reported, and the mechanism of CSFV replication is poorly understood. Here, we solve the first crystal structure of CSFV NS5B and analyze the functions of the characteristic finger, palm, and thumb domains. Additionally, our structure revealed the presence of a novel N-terminal domain (NTD). Biochemical studies demonstrated that the NTD of CSFV NS5B is very important for RdRp activity. Collectively, our studies provide a structural basis for future rational design of anti-CSFV drugs, which is critically important, as no effective anti-CSFV drugs have been developed.

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Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus

Structure ◽

10.1016/s0969-2126(00)80031-3 ◽

1999 ◽

Vol 7 (11) ◽

pp. 1417-1426 ◽

Cited By ~ 305

Author(s):

Hideo Ago ◽

Tsuyoshi Adachi ◽

Atsuhito Yoshida ◽

Masaki Yamamoto ◽

Noriyuki Habuka ◽

...

Keyword(s):

Crystal Structure ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

Rna Dependent Rna Polymerase

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Perturbation in the Conserved Methyltransferase-Polymerase Interface of Flavivirus NS5 Differentially Affects Polymerase Initiation and Elongation

Journal of Virology ◽

10.1128/jvi.02085-14 ◽

2014 ◽

Vol 89 (1) ◽

pp. 249-261 ◽

Cited By ~ 16

Author(s):

Jiqin Wu ◽

Guoliang Lu ◽

Bo Zhang ◽

Peng Gong

Keyword(s):

Crystal Structure ◽

Rna Polymerase ◽

Rna Synthesis ◽

De Novo ◽

Full Length ◽

Intramolecular Interactions ◽

Rna Dependent Rna Polymerase ◽

Hydrophobic Residues ◽

Functional Relevance

ABSTRACTThe flavivirus NS5 is a natural fusion of a methyltransferase (MTase) and an RNA-dependent RNA polymerase (RdRP). Analogous to DNA-dependent RNA polymerases, the NS5 polymerase initiates RNA synthesis through ade novomechanism and then makes a transition to a processive elongation phase. However, whether and how the MTase affects polymerase activities through intramolecular interactions remain elusive. By solving the crystal structure of the Japanese encephalitis virus (JEV) NS5, we recently identified an MTase-RdRP interface containing a set of six hydrophobic residues highly conserved among flaviviruses. To dissect the functional relevance of this interface, we made a series of JEV NS5 constructs with mutations of these hydrophobic residues and/or with the N-terminal first 261 residues and other residues up to the first 303 residues deleted. Compared to the wild-type (WT) NS5, full-length NS5 variants exhibited consistent up- or downregulation of the initiation activities in two types of polymerase assays. Five representative full-length NS5 constructs were then tested in an elongation assay, from which the apparent single-nucleotide incorporation rate constant was estimated. Interestingly, two constructs exhibited different elongation kinetics from the WT NS5, with an effect rather opposite to what was observed at initiation. Moreover, constructs with MTase and/or the linker region (residues 266 to 275) removed still retained polymerase activities, albeit at overall lower levels. However, further removal of the N-terminal extension (residues 276 to 303) abolished regular template-directed synthesis. Together, our data showed that the MTase-RdRP interface is relevant in both polymerase initiation and elongation, likely with different regulation mechanisms in these two major phases of RNA synthesis.IMPORTANCEThe flavivirus NS5 is very unique in having a methyltransferase (MTase) placed on the immediate N terminus of its RNA-dependent RNA polymerase (RdRP). We recently solved the crystal structure of the full-length NS5, which revealed a conserved interface between MTase and RdRP. Building on this discovery, here we carried outin vitropolymerase assays to address the functional relevance of the interface interactions. By explicitly probing polymerase initiation and elongation activities, we found that perturbation in the MTase-RdRP interface had different impacts on different phases of synthesis, suggesting that the roles and contribution of the interface interactions may change upon phase transitions. By comparing the N-terminal-truncated enzymes with the full-length NS5, we collected data to indicate the indispensability to regular polymerase activities of a region that was functionally unclarified previously. Taken together, we provide biochemical evidence and mechanistic insights for the cross talk between the two enzyme modules of flavivirus NS5.

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A crystal structure of alphavirus nonstructural protein 4 (nsP4) reveals an intrinsically dynamic RNA-dependent RNA polymerase

10.1101/2021.05.27.445971 ◽

2021 ◽

Author(s):

Yaw Bia Tan ◽

Laura Sandra Lello ◽

Xin Liu ◽

Yee-Song Law ◽

Congbao Kang ◽

...

Keyword(s):

Crystal Structure ◽

Rna Polymerase ◽

Nonstructural Protein ◽

3D Structure ◽

Rna Replication ◽

Viral Rna ◽

Rdrp Activity ◽

Rna Dependent Rna Polymerase ◽

Intrinsically Disordered ◽

Rdrp Domain

Alphaviruses such as Ross River virus (RRV), chikungunya virus and Venezuelan equine encephalitis virus are mosquito-borne pathogens that can cause arthritis or encephalitis diseases. Nonstructural protein 4 (nsP4) of alphaviruses possesses RNA-dependent RNA polymerase (RdRp) activity essential for viral RNA replication. No 3D structure has been available for nsP4 of any alphaviruses despite its importance for understanding alphaviral RNA replication and for the design of antiviral drugs. Here, we report a crystal structure of the RdRp domain of nsP4 from RRV determined at a resolution of 2.6 Å. The structure of the alphavirus RdRp domain appears most closely related to RdRps from pestiviruses, noroviruses and picornaviruses. Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) methods, we showed that in-solution nsP4 is highly dynamic with an intrinsically disordered N-terminal domain. Both full-length nsP4 and the RdRp domain were able to catalyze in vitro RNA polymerization. Structure-guided mutagenesis using a trans-replicase system identified nsP4 regions critical for viral RNA replication.

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A nucleobase-binding pocket in a viral RNA-dependent RNA polymerase contributes to elongation complex stability

Nucleic Acids Research ◽

10.1093/nar/gkz1170 ◽

2019 ◽

Vol 48 (3) ◽

pp. 1392-1405 ◽

Cited By ~ 6

Author(s):

Wei Shi ◽

Han-Qing Ye ◽

Cheng-Lin Deng ◽

Rui Li ◽

Bo Zhang ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Binding ◽

Vaccine Development ◽

Antiviral Drug ◽

Enterovirus 71 ◽

Binding Pocket ◽

Genome Replication ◽

Elongation Complex ◽

Rna Dependent Rna Polymerase

Abstract The enterovirus 71 (EV71) 3Dpol is an RNA-dependent RNA polymerase (RdRP) that plays the central role in the viral genome replication, and is an important target in antiviral studies. Here, we report a crystal structure of EV71 3Dpol elongation complex (EC) at 1.8 Å resolution. The structure reveals that the 5′-end guanosine of the downstream RNA template interacts with a fingers domain pocket, with the base sandwiched by H44 and R277 side chains through hydrophobic stacking interactions, and these interactions are still maintained after one in-crystal translocation event induced by nucleotide incorporation, implying that the pocket could regulate the functional properties of the polymerase by interacting with RNA. When mutated, residue R277 showed an impact on virus proliferation in virological studies with residue H44 having a synergistic effect. In vitro biochemical data further suggest that mutations at these two sites affect RNA binding, EC stability, but not polymerase catalytic rate (kcat) and apparent NTP affinity (KM,NTP). We propose that, although rarely captured by crystallography, similar surface pocket interaction with nucleobase may commonly exist in nucleic acid motor enzymes to facilitate their processivity. Potential applications in antiviral drug and vaccine development are also discussed.

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Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.96.23.13034 ◽

1999 ◽

Vol 96 (23) ◽

pp. 13034-13039 ◽

Cited By ~ 435

Author(s):

S. Bressanelli ◽

L. Tomei ◽

A. Roussel ◽

I. Incitti ◽

R. L. Vitale ◽

...

Keyword(s):

Crystal Structure ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

Rna Dependent Rna Polymerase

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Predicting Intraserotypic Recombination in Enterovirus 71

Journal of Virology ◽

10.1128/jvi.02057-18 ◽

2018 ◽

Vol 93 (4) ◽

Cited By ~ 13

Author(s):

Andrew Woodman ◽

Kuo-Ming Lee ◽

Richard Janissen ◽

Yu-Nong Gong ◽

Nynke H. Dekker ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Iii ◽

Enterovirus 71 ◽

Template Switching ◽

Rna Dependent Rna Polymerase ◽

A Cell ◽

Strain Type ◽

Species Groups ◽

Recombination Junction ◽

Recombination Assay

ABSTRACTEnteroviruses are well known for their ability to cause neurological damage and paralysis. The model enterovirus is poliovirus (PV), the causative agent of poliomyelitis, a condition characterized by acute flaccid paralysis. A related virus, enterovirus 71 (EV-A71), causes similar clinical outcomes in recurrent outbreaks throughout Asia. Retrospective phylogenetic analysis has shown that recombination between circulating strains of EV-A71 produces the outbreak-associated strains which exhibit increased virulence and/or transmissibility. While studies on the mechanism(s) of recombination in PV are ongoing in several laboratories, little is known about factors that influence recombination in EV-A71. We have developed a cell-based assay to study recombination of EV-A71 based upon previously reported assays for poliovirus recombination. Our results show that (i) EV-A71 strain type and RNA sequence diversity impacts recombination frequency in a predictable manner that mimics the observations found in nature; (ii) recombination is primarily a replicative process mediated by the RNA-dependent RNA polymerase; (iii) a mutation shown to reduce recombination in PV (L420A) similarly reduces EV-A71 recombination, suggesting conservation in mechanism(s); and (iv) sequencing of intraserotypic recombinant genomes indicates that template switching occurs by a mechanism that may require some sequence homology at the recombination junction and that the triggers for template switching may be sequence independent. The development of this recombination assay will permit further investigation on the interplay between replication, recombination and disease.IMPORTANCERecombination is a mechanism that contributes to genetic diversity. We describe the first assay to study EV-A71 recombination. Results from this assay mimic what is observed in nature and can be used by others to predict future recombination events within the enterovirus species A group. In addition, our results highlight the central role played by the viral RNA-dependent RNA polymerase (RdRp) in the recombination process. Further, our results show that changes to a conserved residue in the RdRp from different species groups have a similar impact on viable recombinant virus yields, which is indicative of conservation in mechanism.

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