scholarly journals The Structure of Bovine Viral Diarrhea Virus RNA-Dependent RNA Polymerase and Its Amino-Terminal Domain

Structure ◽  
2006 ◽  
Vol 14 (7) ◽  
pp. 1107-1113 ◽  
Author(s):  
Kyung H. Choi ◽  
Andreas Gallei ◽  
Paul Becher ◽  
Michael G. Rossmann
Keyword(s):  
Rna Polymerase ◽  
Bovine Viral Diarrhea Virus ◽  
Bovine Viral Diarrhea ◽  
Rna Dependent Rna Polymerase ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Amino Terminal ◽  
Virus Rna ◽  
Amino Terminal Domain ◽  
Viral Diarrhea Virus

scholarly journals The Amino-Terminal Domain of Bovine Viral Diarrhea Virus Npro Protein Is Necessary for Alpha/Beta Interferon Antagonism

2006 ◽  
Vol 80 (2) ◽  
pp. 900-911 ◽  
Author(s):  
Laura H. V. G. Gil ◽  
Israrul H. Ansari ◽  
Ventzislav Vassilev ◽  
Delin Liang ◽  
Vicky C. H. Lai ◽  
...  
Keyword(s):  
Bovine Viral Diarrhea Virus ◽  
Protease Activity ◽  
Bovine Viral Diarrhea ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Amino Terminal ◽  
N Terminus ◽  
Alpha Beta ◽  
Amino Terminal Domain ◽  
Viral Diarrhea Virus

ABSTRACT The alpha/beta interferon (IFN-α/β) system is the first line of defense against viral infection and a critical link between the innate and adaptive immune responses. IFN-α/β secretion is the hallmark of cellular responses to acute RNA virus infections. As part of their survival strategy, many viruses have evolved mechanisms to counteract the host IFN-α/β response. Bovine viral diarrhea virus (BVDV) (genus Pestivirus) was reported to trigger interferon production in infected cultured cells under certain circumstances or to suppress it under others. Our studies with various cultured fibroblasts and epithelial bovine cells indicated that cytopathic (cp) BVDV induces IFN-α/β very inefficiently. Using a set of engineered cp BVDVs expressing mutant Npro and appropriate controls, we found that the IFN-α/β response to infection was dependent on Npro expression and independent of viral replication efficiency. In order to investigate whether the protease activity of Npro is required for IFN-α/β antagonism, we engineered Npro mutants lacking protease activity by replacement of amino acid E22, H49, or C69. We found that E22 and H49 substitutions abolished the ability of Npro to suppress IFN, whereas C69 had no effect, suggesting that the structural integrity of the N terminus of Npro was more important than its catalytic activity for IFN-α/β suppression. A catalytically active mutant with a change at a conserved Npro region near the N terminus (L8P) in both BVDV biotypes did not antagonize IFN-α/β production, confirming its involvement in this process. Taken together, these results not only provide direct evidence for the role of Npro in blocking IFN-α/β induction, but also implicate the amino-terminal domain of the protein in this function.

scholarly journals Mutational Analysis of Bovine Viral Diarrhea Virus RNA-Dependent RNA Polymerase

1999 ◽  
Vol 73 (12) ◽  
pp. 10129-10136 ◽  
Author(s):  
Vicky C. H. Lai ◽  
C. Cheng Kao ◽  
Eric Ferrari ◽  
Justin Park ◽  
Annette S. Uss ◽  
...  
Keyword(s):  
Amino Acids ◽  
Rna Polymerase ◽  
Bovine Viral Diarrhea Virus ◽  
De Novo ◽  
Bovine Viral Diarrhea ◽  
Rna Dependent Rna Polymerase ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
C Terminus ◽  
Viral Diarrhea Virus

ABSTRACT Recombinant bovine viral diarrhea virus (BVDV) nonstructural protein 5B (NS5B) produced in insect cells has been shown to possess an RNA-dependent RNA polymerase (RdRp) activity. Our initial attempt to produce the full-length BVDV NS5B with a C-terminal hexahistidine tag in Escherichia coli failed due to the expression of insoluble products. Prompted by a recent report that removal of the C-terminal hydrophobic domain significantly improved the solubility of hepatitis C virus (HCV) NS5B, we constructed a similar deletion of 24 amino acids at the C terminus of BVDV NS5B. The resulting fusion protein, NS5BΔCT24-His, was purified to homogeneity and demonstrated to direct RNA replication via both primer-dependent (elongative) and primer-independent (de novo) mechanisms. Furthermore, BVDV RdRp was found to utilize a circular single-stranded DNA as a template for RNA synthesis, suggesting that synthesis does not require ends in the template. In addition to the previously described polymerase motifs A, B, C, and D, alignments with other flavivirus sequences revealed two additional motifs, one N-terminal to motif A and one C-terminal to motif D. Extensive alanine substitutions showed that while most mutations had similar effects on both elongative and de novo RNA syntheses, some had selective effects. Finally, deletions of up to 90 amino acids from the N terminus did not significantly affect RdRp activities, whereas deletions of more than 24 amino acids at the C terminus resulted in either insoluble products or soluble proteins (ΔCT179 and ΔCT218) that lacked RdRp activities.

scholarly journals Quinolinecarboxamides Inhibit the Replication of the Bovine Viral Diarrhea Virus by Targeting a Hot Spot for the Inhibition of Pestivirus Replication in the RNA-Dependent RNA Polymerase

Molecules ◽  
2020 ◽  
Vol 25 (6) ◽  
pp. 1283
Author(s):  
Simone Musiu ◽  
Yunierkis Perez Castillo ◽  
Alexandra Muigg ◽  
Gerhard Pürstinger ◽  
Pieter Leyssen ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Bovine Viral Diarrhea Virus ◽  
Hot Spot ◽  
Viral Rna ◽  
Bovine Viral Diarrhea ◽  
Rna Dependent Rna Polymerase ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Viral Diarrhea Virus

The bovine viral diarrhea virus (BVDV), a pestivirus from the family of Flaviviridae is ubiquitous and causes a range of clinical manifestations in livestock, mainly cattle. Two quinolinecarboxamide analogues were identified in a CPE-based screening effort, as selective inhibitors of the in vitro bovine viral diarrhea virus (BVDV) replication, i.e., TO505-6180/CSFCI (average EC50 = 0.07 µM, SD = 0.02 µM, CC50 > 100 µM) and TO502-2403/CSFCII (average EC50 = 0.2 µM, SD = 0.06 µM, CC50 > 100 µM). The initial antiviral activity observed for both hits against BVDV was corroborated by measuring the inhibitory effect on viral RNA synthesis and the production of infectious virus. Modification of the substituents on the quinolinecarboxamide scaffold resulted in analogues that proved about 7-fold more potent (average EC50 = 0.03 with a SD = 0.01 µM) and that were devoid of cellular toxicity, for the concentration range tested (SI = 3333). CSFCII resistant BVDV variants were selected and were found to carry the F224P mutation in the viral RNA-dependent RNA polymerase (RdRp), whereas CSFCI resistant BVDV carried two mutations in the same region of the RdRp, i.e., N264D and F224Y. Likewise, molecular modeling revealed that F224P/Y and N264D are located in a small cavity near the fingertip domain of the pestivirus polymerase. CSFC-resistant BVDV proved to be cross-resistant to earlier reported pestivirus inhibitors (BPIP, AG110, LZ37, and BBP) that are known to target the same region of the RdRp. CSFC analogues did not inhibit the in vitro activity of recombinant BVDV RdRp but inhibited the activity of BVDV replication complexes (RCs). CSFC analogues likely interact with the fingertip of the pestivirus RdRp at the same position as BPIP, AG110, LZ37, and BBP. This indicates that this region is a “hot spot” for the inhibition of pestivirus replication.

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