Porcine Mx1 Protein Inhibits Classical Swine Fever Virus Replication by Targeting Nonstructural Protein NS5B

ABSTRACTMx proteins are interferon (IFN)-induced GTPases that have broad antiviral activity against a wide range of RNA and DNA viruses; they belong to the dynamin superfamily of large GTPases. In this study, we confirmed the anti-classical swine fever virus (CSFV) activity of porcine Mx1in vitroand showed that porcine Mx2 (poMx2), human MxA (huMxA), and mouse Mx1 (mmMx1) also have anti-CSFV activityin vitro. Small interfering RNA (siRNA) experiments revealed that depletion of endogenous poMx1 or poMx2 enhanced CSFV replication, suggesting that porcine Mx proteins are responsible for the antiviral activity of interferon alpha (IFN-α) against CSFV infection. Confocal microscopy, immunoprecipitation, glutathioneS-transferase (GST) pulldown, and bimolecular fluorescence complementation (BiFC) demonstrated that poMx1 associated with NS5B, the RNA-dependent RNA polymerase (RdRp) of CSFV. We used mutations in the poMx1 protein to elucidate the mechanism of their anti-CSFV activity and found that mutants that disrupted the association with NS5B lost all anti-CSV activity. Moreover, an RdRp activity assay further revealed that poMx1 undermined the RdRp activities of NS5B. Together, these results indicate that porcine Mx proteins exert their antiviral activity against CSFV by interacting with NS5B.IMPORTANCEOur previous studies have shown that porcine Mx1 (poMx1) inhibits classical swine fever virus (CSFV) replicationin vitroandin vivo, but the molecular mechanism of action remains largely unknown. In this study, we dissect the molecular mechanism of porcine Mx1 and Mx2 against CSFVin vitro. Our results show that poMx1 associates with NS5B, the RNA-dependent RNA polymerase of CSFV, resulting in the reduction of CSFV replication. Moreover, the mutants of poMx1 further elucidate the mechanism of their anti-CSFV activities.

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Crystal Structure of Classical Swine Fever Virus NS5B Reveals a Novel N-Terminal Domain

Journal of Virology ◽

10.1128/jvi.00324-18 ◽

2018 ◽

Vol 92 (14) ◽

Cited By ~ 5

Author(s):

Weiwei Li ◽

Baixing Wu ◽

Wibowo Adian Soca ◽

Lei An

Keyword(s):

Crystal Structure ◽

Rna Polymerase ◽

Classical Swine Fever Virus ◽

Classical Swine Fever ◽

Fever Virus ◽

Structural Basis ◽

Economic Losses ◽

Rna Dependent Rna Polymerase ◽

Content Type ◽

Terminal Domain

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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Characterization of classical swine fever virus (CSFV) nonstructural protein 3 (NS3) helicase activity and its modulation by CSFV RNA-dependent RNA polymerase

Virus Research ◽

10.1016/j.virusres.2008.12.014 ◽

2009 ◽

Vol 141 (1) ◽

pp. 63-70 ◽

Cited By ~ 17

Author(s):

Guoyuan Wen ◽

Jinnuo Xue ◽

Yanping Shen ◽

Chuyu Zhang ◽

Zishu Pan

Keyword(s):

Rna Polymerase ◽

Classical Swine Fever Virus ◽

Nonstructural Protein ◽

Classical Swine Fever ◽

Fever Virus ◽

Helicase Activity ◽

Rna Dependent Rna Polymerase ◽

Nonstructural Protein 3

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In vitro antiviral activity of some uridine derivatives of 2-deoxy sugars against classical swine fever virus

Antiviral Research ◽

10.1016/j.antiviral.2010.02.314 ◽

2010 ◽

Vol 86 (2) ◽

pp. 154-162 ◽

Cited By ~ 17

Author(s):

Ewelina Krol ◽

Ilona Wandzik ◽

Wieslaw Szeja ◽

Grzegorz Grynkiewicz ◽

Boguslaw Szewczyk

Keyword(s):

Antiviral Activity ◽

Classical Swine Fever Virus ◽

Classical Swine Fever ◽

Fever Virus ◽

Derivatives Of ◽

Deoxy Sugars

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Classical swine fever virus NS3 enhances RNA-dependent RNA polymerase activity by binding to NS5B

Virus Research ◽

10.1016/j.virusres.2009.11.015 ◽

2010 ◽

Vol 148 (1-2) ◽

pp. 17-23 ◽

Cited By ~ 10

Author(s):

Ping Wang ◽

Yujing Wang ◽

Yu Zhao ◽

Zailing Zhu ◽

Jialin Yu ◽

...

Keyword(s):

Rna Polymerase ◽

Classical Swine Fever Virus ◽

Classical Swine Fever ◽

Polymerase Activity ◽

Fever Virus ◽

Rna Dependent Rna Polymerase ◽

Rna Polymerase Activity

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Episodic adaptive diversification of classical swine fever virus RNA-dependent RNA polymerase NS5B

Canadian Journal of Microbiology ◽

10.1139/cjm-2015-0334 ◽

2015 ◽

Vol 61 (12) ◽

pp. 948-954 ◽

Cited By ~ 3

Author(s):

Yan Li ◽

Zexiao Yang

Keyword(s):

Rna Polymerase ◽

Classical Swine Fever Virus ◽

Classical Swine Fever ◽

Fever Virus ◽

Adaptive Divergence ◽

Central Component ◽

Rna Dependent Rna Polymerase ◽

Evolutionary Trajectory ◽

Adaptive Diversification ◽

A Minor

Classical swine fever virus (CSFV) is the pathogen that causes a highly infectious disease of pigs and has led to disastrous losses to pig farms and related industries. The RNA-dependent RNA polymerase (RdRp) NS5B is a central component of the replicase complex (RC) in some single-stranded RNA viruses, including CSFV. On the basis of genetic variation, the CSFV RdRps could be clearly divided into 2 major groups and a minor group, which is consistent with the phylogenetic relationships and virulence diversification of the CSFV isolates. However, the adaptive signature underlying such an evolutionary profile of the polymerase and the virus is still an interesting open question. We analyzed the evolutionary trajectory of the CSFV RdRps over different timescales to evaluate the potential adaptation. We found that adaptive selection has driven the diversification of the RdRps between, but not within, CSFV major groups. Further, the major adaptive divergence-related sites are located in the surfaces relevant to the interaction with other component(s) of RC and the entrance and exit of the template-binding channel. These results might shed some light on the nature of the RdRp in virulence diversification of CSFV groups.

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De novo RNA synthesis by a recombinant classical swine fever virus RNA-dependent RNA polymerase

European Journal of Biochemistry ◽

10.1046/j.1432-1033.2003.03897.x ◽

2003 ◽

Vol 270 (24) ◽

pp. 4952-4961 ◽

Cited By ~ 16

Author(s):

Guang-Hui Yi ◽

Chu-Yu Zhang ◽

Sheng Cao ◽

Hai-Xiang Wu ◽

Yi Wang

Keyword(s):

Rna Polymerase ◽

Classical Swine Fever Virus ◽

Rna Synthesis ◽

De Novo ◽

Classical Swine Fever ◽

Fever Virus ◽

Rna Dependent Rna Polymerase ◽

Virus Rna

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Characterization of the N-terminal domain of classical swine fever virus RNA-dependent RNA polymerase

Journal of General Virology ◽

10.1099/vir.0.81385-0 ◽

2006 ◽

Vol 87 (2) ◽

pp. 347-356 ◽

Cited By ~ 24

Author(s):

Ming Xiao ◽

Huaibo Li ◽

Yujing Wang ◽

Xiaohui Wang ◽

Wei Wang ◽

...

Keyword(s):

Rna Polymerase ◽

Classical Swine Fever Virus ◽

Rna Synthesis ◽

De Novo ◽

Classical Swine Fever ◽

Fever Virus ◽

Rdrp Activity ◽

Rna Dependent Rna Polymerase ◽

Terminal Domain ◽

Ns5b Protein

To investigate RNA-dependent RNA polymerase (RdRp) further, mutational analysis of the N-terminal domain of the NS5B protein of Classical swine fever virus was performed. Results show that the N-terminal domain (positions 1–300) of the protein might be divided artificially into four different regions, N1–N4. The N1 region (positions 1–61) contained neither conserved lysine nor conserved arginine residues. NS5B protein with deletion of the N1 region has the capacity for elongative RNA synthesis, but not for de novo RNA synthesis on natural templates. All substitutions of the conserved lysines and arginines in the N2 region (positions 63–216) destroyed RdRp activity completely. Substitutions of the conserved arginines in the N3 region (positions 217–280) seriously reduced RdRp activity. However, all substitutions of the conserved lysines in this region enhanced RNA synthesis and made the mutants synthesize RNA on any template. Substitutions of the conserved arginines in the N4 region (positions 281–300) reduced elongative synthesis and destroyed de novo RNA synthesis. In contrast, substitutions of lysines in this region did not affect RdRp activity significantly. These data indicate that the N3 region might be related to the enzymic specificity for templates, and the conserved lysines and arginines in different regions have different effects on RdRp activity. In combination with the published crystal structure of bovine viral diarrhea virus NS5B, these results define the important role of the N-terminal domain of NS5B for template recognition and de novo RNA synthesis.

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