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

2018 ◽  
Vol 92 (14) ◽  
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.

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

2006 ◽  
Vol 87 (2) ◽  
pp. 347-356 ◽  
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.

scholarly journals Immunogenicity in Swine of Orally Administered Recombinant Lactobacillus plantarum Expressing Classical Swine Fever Virus E2 Protein in Conjunction with Thymosin α-1 as an Adjuvant

2015 ◽  
Vol 81 (11) ◽  
pp. 3745-3752 ◽  
Author(s):  
Yi-Gang Xu ◽  
Xue-Ting Guan ◽  
Zhong-Mei Liu ◽  
Chang-Yong Tian ◽  
Li-Chun Cui
Keyword(s):  
Immune Responses ◽  
Lactobacillus Plantarum ◽  
Classical Swine Fever Virus ◽  
Oral Vaccine ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Economic Losses ◽  
E2 Protein ◽  
Content Type ◽  
Cellular Immune

ABSTRACTClassical swine fever, caused by classical swine fever virus (CSFV), is a highly contagious disease that results in enormous economic losses in pig industries. The E2 protein is one of the main structural proteins of CSFV and is capable of inducing CSFV-neutralizing antibodies and cytotoxic T lymphocyte (CTL) activitiesin vivo. Thymosin α-1 (Tα1), an immune-modifier peptide, plays a very important role in the cellular immune response. In this study, genetically engineeredLactobacillus plantarumbacteria expressing CSFV E2 protein alone (L. plantarum/pYG-E2) and in combination with Tα1 (L. plantarum/pYG-E2-Tα1) were developed, and the immunogenicity of each as an oral vaccine to induce protective immunity against CSFV in pigs was evaluated. The results showed that recombinantL. plantarum/pYG-E2 andL. plantarum/pYG-E2-Tα1 were both able to effectively induce protective immune responses in pigs against CSFV infection by eliciting immunoglobulin A (IgA)-based mucosal, immunoglobulin G (IgG)-based humoral, and CTL-based cellular immune responses via oral vaccination. Significant differences (P< 0.05) in the levels of immune responses were observed betweenL. plantarum/pYG-E2-Tα1 andL. plantarum/pYG-E2, suggesting a better immunogenicity ofL. plantarum/pYG-E2-Tα1 as a result of the Tα1 molecular adjuvant that can enhance immune responsiveness and augment specific lymphocyte functions. Our data suggest that the recombinantLactobacillusmicroecological agent expressing CSFV E2 protein combined with Tα1 as an adjuvant provides a promising strategy for vaccine development against CSFV.

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

2018 ◽  
Vol 92 (7) ◽  
Author(s):  
Jing Zhou ◽  
Jing Chen ◽  
Xiao-Min Zhang ◽  
Zhi-Can Gao ◽  
Chun-Chun Liu ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Rna Polymerase ◽  
Molecular Mechanism ◽  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Bimolecular Fluorescence Complementation ◽  
Rna Dependent Rna Polymerase ◽  
Wide Range

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.

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

2015 ◽  
Vol 61 (12) ◽  
pp. 948-954 ◽  
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.

scholarly journals Identification of E2 with improved secretion and immunogenicity against CSFV in piglets

2020 ◽  
Author(s):  
Huiling Xu ◽  
Yanli Wang ◽  
Guangwei Han ◽  
Weihuan Fang ◽  
fang he
Keyword(s):  
Single Dose ◽  
Signal Peptide ◽  
Classical Swine Fever Virus ◽  
Subunit Vaccine ◽  
Classical Swine Fever ◽  
Cost Effective ◽  
Fever Virus ◽  
Economic Losses ◽  
Swine Industry ◽  
Virus Challenge

Abstract Background: Outbreaks of Classical swine fever virus (CSFV) cause significant economic losses in the swine industry. Vaccination is the major method to prevent and control the disease. As live attenuated vaccines fail to elicit differentiable immunity between infected and vaccinated animals, subunit vaccine was considered as an alternative candidate to prevent and eradicate CSFV. Subunit vaccines present advantages in DIVA immunogenicity and safety. The technology was limited due to the low yield and the high cost with multiple and large doses. The native E2 signal peptide has not been well defined before. Here, the aim of this study is to develop a cost-effective and efficacious E2 vaccine candidate against CSFV with signal peptide and E2 sequence selection. Results: A novel CSFV E2 sequence (E2ZJ) was identified from an epidemic strain of Zhejiang for outstanding secretion in baculovirus and enhanced immunogenicity. E2 secretion induced with the selected signal peptide, SPZJ (SP23), increase at least 50% as compared to any other signal peptides tested. Besides, unique antigenic features were identified in E2ZJ. E2ZJ elicited CSFV antibodies at the earlier stage than other E2 types tested in mice. Moreover, higher level of neutralization antibodies against both genotypes 1 and 2 CSFV with E2ZJ was detected than other E2s with the same dosage. Further, in piglets, E2ZJ successfully elicited neutralizing immunity. A single dose of 5 μg of E2ZJ was sufficient to induce protective antibodies against CSFV in piglets and provided 100% protection against lethal virus challenge. Conclusions: Our studies provide evidence that E2ZJ guided by a novel E2 signal peptide (SPZJ) was efficiently secreted and presented significantly improved immunogenicity than conventional E2 vaccines. Moreover, a single dose of 5 μg E2ZJ is efficacious against CSFV in piglets. Keywords: Classical swine fever virus; novel signal peptide; SPZJ-E2ZJ; subunit vaccine; protective immunity

scholarly journals The N-terminal domain of Npro of classical swine fever virus determines its stability and regulates type I IFN production

2015 ◽  
Vol 96 (7) ◽  
pp. 1746-1756 ◽  
Author(s):  
Junki Mine ◽  
Tomokazu Tamura ◽  
Wasana Pinyochon ◽  
Masatoshi Okamatsu ◽  
Yoshihiro Sakoda ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Type I ◽  
Type I Ifn ◽  
Terminal Domain
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