scholarly journals Intracellular membrane association of the N-terminal domain of classical swine fever virus NS4B determines viral genome replication and virulence

2015 ◽  
Vol 96 (9) ◽  
pp. 2623-2635 ◽  
Author(s):  
Tomokazu Tamura ◽  
Nicolas Ruggli ◽  
Naofumi Nagashima ◽  
Masatoshi Okamatsu ◽  
Manabu Igarashi ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Viral Genome ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Membrane Association ◽  
Genome Replication ◽  
Intracellular Membrane ◽  
Terminal Domain ◽  
Viral Genome Replication

scholarly journals Proline to Threonine Mutation at Position 162 of NS5B of Classical Swine Fever Virus Vaccine C Strain Promoted Genome Replication and Infectious Virus Production by Facilitating Initiation of RNA Synthesis

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1523
Author(s):  
Huining Pang ◽  
Ling Li ◽  
Hongru Liu ◽  
Zishu Pan
Keyword(s):  
Classical Swine Fever Virus ◽  
Viral Genome ◽  
Rna Synthesis ◽  
Infectious Virus ◽  
Virus Production ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Genome Replication ◽  
Rdrp Activity ◽  
Viral Genome Replication

The 3′untranslated region (3′UTR) and NS5B of classical swine fever virus (CSFV) play vital roles in viral genome replication. In this study, two chimeric viruses, vC/SM3′UTR and vC/b3′UTR, with 3′UTR substitution of CSFV Shimen strain or bovine viral diarrhea virus (BVDV) NADL strain, were constructed based on the infectious cDNA clone of CSFV vaccine C strain, respectively. After virus rescue, each recombinant chimeric virus was subjected to continuous passages in PK-15 cells. The representative passaged viruses were characterized and sequenced. Serial passages resulted in generation of mutations and the passaged viruses exhibited significantly increased genomic replication efficiency and infectious virus production compared to parent viruses. A proline to threonine mutation at position 162 of NS5B was identified in both passaged vC/SM3′UTR and vC/b3′UTR. We generated P162T mutants of two chimeras using the reverse genetics system, separately. The single P162T mutation in NS5B of vC/SM3′UTR or vC/b3′UTR played a key role in increased viral genome replication and infectious virus production. The P162T mutation increased vC/SM3′UTRP162T replication in rabbits. From RNA-dependent RNA polymerase (RdRp) assays in vitro, the NS5B containing P162T mutation (NS5BP162T) exhibited enhanced RdRp activity for different RNA templates. We further identified that the enhanced RdRp activity originated from increased initiation efficiency of RNA synthesis. These findings revealed a novel function for the NS5B residue 162 in modulating pestivirus replication.

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 Specific interaction between the classical swine fever virus NS5B protein and the viral genome

2004 ◽  
Vol 271 (19) ◽  
pp. 3888-3896 ◽  
Author(s):  
Ming Xiao ◽  
Jufang Gao ◽  
Wei Wang ◽  
Yujing Wang ◽  
Jun Chen ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Viral Genome ◽  
Specific Interaction ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Ns5b Protein

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

scholarly journals Analysis of classical swine fever virus RNA replication determinants using replicons

2013 ◽  
Vol 94 (8) ◽  
pp. 1739-1748 ◽  
Author(s):  
Peter Christian Risager ◽  
Ulrik Fahnøe ◽  
Maria Gullberg ◽  
Thomas Bruun Rasmussen ◽  
Graham J. Belsham
Keyword(s):  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Genome Replication ◽  
Coding Region ◽  
Reporter Protein ◽  
Coding Sequence ◽  
Replication Efficiency ◽  
Chimeric Rnas

Self-replicating RNAs (replicons), with or without reporter gene sequences, derived from the genome of the Paderborn strain of classical swine fever virus (CSFV) have been produced. The full-length viral cDNA, propagated within a bacterial artificial chromosome, was modified by targeted recombination within Escherichia coli. RNA transcripts were produced in vitro and introduced into cells by electroporation. The translation and replication of the replicon RNAs could be followed by the accumulation of luciferase (from Renilla reniformis or Gaussia princeps) protein expression (where appropriate), as well as by detection of CSFV NS3 protein production within the cells. Inclusion of the viral E2 coding region within the replicon was advantageous for replication efficiency. Production of chimeric RNAs, substituting the NS2 and NS3 coding regions (as a unit) from the Paderborn strain with the equivalent sequences from the highly virulent Koslov strain or the vaccine strain Riems, blocked replication. However, replacing the Paderborn NS5B coding sequence with the RNA polymerase coding sequence from the Koslov strain greatly enhanced expression of the reporter protein from the replicon. In contrast, replacement with the Riems NS5B sequence significantly impaired replication efficiency. Thus, these replicons provide a system for determining specific regions of the CSFV genome required for genome replication without the constraints of maintaining infectivity.

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.

ARFGAP1 binds to classical swine fever virus NS5A protein and enhances CSFV replication in PK-15 cells

2021 ◽  
Vol 255 ◽  
pp. 109034
Author(s):  
Liang Zhang ◽  
Mingxing Jin ◽  
Mengzhao Song ◽  
Shanchuan Liu ◽  
Tao Wang ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Ns5a Protein

Genome Variability of Classical Swine Fever Virus during the 2018–2020 Epidemic in Japan

2021 ◽  
pp. 109128
Author(s):  
Tatsuya Nishi ◽  
Katsuhiko Fukai ◽  
Tomoko Kato ◽  
Kotaro Sawai ◽  
Takehisa Yamamoto
Keyword(s):  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Genome Variability

scholarly journals A Novel E2 Glycoprotein Subunit Marker Vaccine Produced in Plant Is Able to Prevent Classical Swine Fever Virus Vertical Transmission after Double Vaccination

Vaccines ◽  
2021 ◽  
Vol 9 (5) ◽  
pp. 418
Author(s):  
Youngmin Park ◽  
Yeonsu Oh ◽  
Miaomiao Wang ◽  
Llilianne Ganges ◽  
José Alejandro Bohórquez ◽  
...  
Keyword(s):  
Vertical Transmission ◽  
Classical Swine Fever Virus ◽  
Neutralizing Antibodies ◽  
Subunit Vaccine ◽  
Vaccine Dose ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Tissue Samples ◽  
Marker Vaccine ◽  
E2 Glycoprotein

The efficacy of a novel subunit vaccine candidate, based in the CSFV E2 glycoprotein produced in plants to prevent classical swine fever virus (CSFV) vertical transmission, was evaluated. A Nicotiana benthamiana tissue culture system was used to obtain a stable production of the E2-glycoprotein fused to the porcine Fc region of IgG. Ten pregnant sows were divided into three groups: Groups 1 and 2 (four sows each) were vaccinated with either 100 μg/dose or 300 μg/dose of the subunit vaccine at 64 days of pregnancy. Group 3 (two sows) was injected with PBS. Groups 1 and 2 were boosted with the same vaccine dose. At 10 days post second vaccination, the sows in Groups 2 and 3 were challenged with a highly virulent CSFV strain. The vaccinated sows remained clinically healthy and seroconverted rapidly, showing efficient neutralizing antibodies. The fetuses from vaccinated sows did not show gross lesions, and all analyzed tissue samples tested negative for CSFV replication. However, fetuses of non-vaccinated sows had high CSFV replication in tested tissue samples. The results suggested that in vaccinated sows, the plant produced E2 marker vaccine induced the protective immunogenicity at challenge, leading to protection from vertical transmission to fetuses.

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