scholarly journals Infectious Bovine Viral Diarrhea Virus (Strain NADL) RNA from Stable cDNA Clones: a Cellular Insert Determines NS3 Production and Viral Cytopathogenicity

1998 ◽  
Vol 72 (6) ◽  
pp. 4737-4745 ◽  
Author(s):  
Ernesto Mendez ◽  
Nicolas Ruggli ◽  
Marc S. Collett ◽  
Charles M. Rice
Keyword(s):  
Bovine Viral Diarrhea Virus ◽  
Rna Replication ◽  
Bovine Viral Diarrhea ◽  
Plaque Morphology ◽  
Cdna Clones ◽  
Coding Region ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Mdbk Cells ◽  
Viral Diarrhea Virus

ABSTRACT Bovine viral diarrhea virus (BVDV), strain NADL, was originally isolated from an animal with fatal mucosal disease. This isolate is cytopathic in cell culture and produces two forms of NS3-containing proteins: uncleaved NS2-3 and mature NS3. For BVDV NADL, the production of NS3, a characteristic of cytopathic BVDV strains, is believed to be a consequence of an in-frame insertion of a 270-nucleotide cellular mRNA sequence (called cIns) in the NS2 coding region. In this study, we constructed a stable full-length cDNA copy of BVDV NADL in a low-copy-number plasmid vector. As assayed by transfection of MDBK cells, uncapped RNAs transcribed from this template were highly infectious (>105 PFU/μg). The recovered virus was similar in plaque morphology, growth properties, polyprotein processing, and cytopathogenicity to the BVDV NADL parent. Deletion of cIns abolished processing at the NS2/NS3 site and produced a virus that was no longer cytopathic for MDBK cells. This deletion did not affect the efficiency of infectious virus production or viral protein production, but it reduced the level of virus-specific RNA synthesis and accumulation. Thus, cIns not only modulates NS3 production but also upregulates RNA replication relative to an isogenic noncytopathic derivative lacking the insert. These results raise the possibility of a linkage between enhanced BVDV NADL RNA replication and virus-induced cytopathogenicity.

scholarly journals Sequence and Structural Elements at the 3′ Terminus of Bovine Viral Diarrhea Virus Genomic RNA: Functional Role during RNA Replication

1999 ◽  
Vol 73 (5) ◽  
pp. 3638-3648 ◽  
Author(s):  
Haiying Yu ◽  
Claus W. Grassmann ◽  
Sven-Erik Behrens
Keyword(s):  
Bovine Viral Diarrhea Virus ◽  
Rna Replication ◽  
Viral Rna ◽  
Bovine Viral Diarrhea ◽  
Rna Motifs ◽  
Stem Loop ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Interaction Sites ◽  
Viral Diarrhea Virus

ABSTRACT Bovine viral diarrhea virus (BVDV), a member of the genusPestivirus in the family Flaviviridae, has a positive-stranded RNA genome consisting of a single open reading frame and untranslated regions (UTRs) at the 5′ and 3′ ends. Computer modeling suggested the 3′ UTR comprised single-stranded regions as well as stem-loop structures—features that were suspected of being essentially implicated in the viral RNA replication pathway. Employing a subgenomic BVDV RNA (DI9c) that was shown to function as an autonomous RNA replicon (S.-E. Behrens, C. W. Grassmann, H. J. Thiel, G. Meyers, and N. Tautz, J. Virol. 72:2364–2372, 1998) the goal of this study was to determine the RNA secondary structure of the 3′ UTR by experimental means and to investigate the significance of defined RNA motifs for the RNA replication pathway. Enzymatic and chemical structure probing revealed mainly the conserved terminal part (termed 3′C) of the DI9c 3′ UTR containing distinctive RNA motifs, i.e., a stable stem-loop, SL I, near the RNA 3′ terminus and a considerably less stable stem-loop, SL II, that forms the 5′ portion of 3′C. SL I and SL II are separated by a long single-stranded intervening sequence, denoted SS. The 3′-terminal four C residues of the viral RNA were confirmed to be single stranded as well. Other intramolecular interactions, e.g., with upstream DI9c RNA sequences, were not detected under the experimental conditions used. Mutagenesis of the DI9c RNA demonstrated that the SL I and SS motifs do indeed play essential roles during RNA replication. Abolition of RNA stems, which ought to maintain the overall folding of SL I, as well as substitution of certain single-stranded nucleotides located in the SS region or SL I loop region, gave rise to DI9c derivatives unable to replicate. Conversely, SL I stems comprising compensatory base exchanges turned out to support replication, but mostly to a lower degree than the original structure. Surprisingly, replacement of a number of residues, although they were previously defined as constituents of a highly conserved stretch of sequence of the SS motif, had little effect on the replication ability of DI9c. In summary, these results indicate that RNA structure as well as sequence elements harbored within the 3′C region of the BVDV 3′ UTR create a common cis-acting element of the replication process. The data further point at possible interaction sites of host and/or viral proteins and thus provide valuable information for future experiments intended to identify and characterize these factors.

Bovine A20 gene overexpression during bovine viral diarrhea virus-1 infection blocks NF-κB pathway in MDBK cells

2017 ◽  
Vol 77 ◽  
pp. 23-29 ◽  
Author(s):  
Melina Villalba ◽  
Nivia Canales ◽  
Nicolas Maldonado ◽  
Carola Otth ◽  
Fernanda Fredericksen ◽  
...  
Keyword(s):  
Bovine Viral Diarrhea Virus ◽  
Bovine Viral Diarrhea ◽  
Gene Overexpression ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Mdbk Cells ◽  
Viral Diarrhea Virus ◽  
A20 Gene

scholarly journals Transdominant Inhibition of Bovine Viral Diarrhea Virus Entry

2007 ◽  
Vol 82 (5) ◽  
pp. 2427-2436 ◽  
Author(s):  
Donna M. Tscherne ◽  
Matthew J. Evans ◽  
Margaret R. MacDonald ◽  
Charles M. Rice
Keyword(s):  
Bovine Viral Diarrhea Virus ◽  
Envelope Glycoprotein ◽  
Cell Clone ◽  
Rna Virus ◽  
Bovine Viral Diarrhea ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Mdbk Cells ◽  
Positive Strand Rna ◽  
Viral Diarrhea Virus

ABSTRACT Bovine viral diarrhea virus (BVDV) is a positive-strand RNA virus and a member of the genus Pestivirus in the family Flaviviridae. To identify and characterize essential factors required for BVDV replication, a library expressing random fragments of the BVDV genome was screened for sequences that act as transdominant inhibitors of viral replication by conferring resistance to cytopathic BVDV-induced cell death. We isolated a BVDV-nonpermissive MDBK cell clone that harbored a 1.2-kb insertion spanning the carboxy terminus of the envelope glycoprotein 1 (E1), the envelope glycoprotein E2, and the amino terminus of p7. Confirming the resistance phenotype conferred by this library clone, naïve MDBK cells expressing this fragment were found to be 100- to 1,000-fold less permissive to both cytopathic and noncytopathic BVDV infection compared to parental MDBK cells, although these cells remained fully permissive to vesicular stomatitis virus. This restriction could be overcome by electroporation of BVDV RNA, indicating a block at one or more steps in viral entry prior to translation of the viral RNA. We determined that the E2 ectodomain was responsible for the inhibition to BVDV entry and that this block occurred downstream from BVDV interaction with the cellular receptor CD46 and virus binding, suggesting interference with a yet-unidentified BVDV entry factor.

scholarly journals Establishment of a Subgenomic Replicon for Bovine Viral Diarrhea Virus in Huh-7 Cells and Modulation of Interferon-Regulated Factor 3-Mediated Antiviral Response

2005 ◽  
Vol 79 (5) ◽  
pp. 2788-2796 ◽  
Author(s):  
Nigel Horscroft ◽  
Dan Bellows ◽  
Israrul Ansari ◽  
Vicky C. H. Lai ◽  
Shannon Dempsey ◽  
...  
Keyword(s):  
Viral Replication ◽  
Bovine Viral Diarrhea Virus ◽  
Sendai Virus ◽  
Rna Replication ◽  
Bovine Viral Diarrhea ◽  
Subgenomic Replicon ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Amino Terminal ◽  
Viral Diarrhea Virus

ABSTRACT We describe the development of a selectable, bi-cistronic subgenomic replicon for bovine viral diarrhea virus (BVDV) in Huh-7 cells, similar to that established for hepatitis C virus (HCV). The selection marker and reporter (Luc-Ubi-Neo) in the BVDV replicon was fused with the amino-terminal protease Npro, and expression of the nonstructural proteins (NS3 to NS5B) was driven by an encephalomyocarditis virus internal ribosome entry site. This BVDV replicon allows us to compare RNA replication of these two related viruses in a similar cellular background and to identify antiviral molecules specific for HCV RNA replication. The BVDV replicon showed similar sensitivity as the HCV replicon to interferons (alpha, beta, and gamma) and 2′-β-C-methyl ribonucleoside inhibitors. Known nonnucleoside inhibitor molecules specific for either HCV or BVDV can be easily distinguished by using the parallel replicon systems. The HCV replicon has been shown to block, via the NS3/4A serine protease, Sendai virus-induced activation of interferon regulatory factor 3 (IRF-3), a key antiviral signaling molecule. Similar suppression of IRF-3-mediated responses was also observed with the Huh-7-BVDV replicon but was independent of NS3/4A protease activity. Instead, the amino-terminal cysteine protease Npro of BVDV appears to be, at least partly, responsible for suppressing IRF-3 activation induced by Sendai virus infection. This result suggests that different viruses, including those closely related, may have developed unique mechanisms for evading host antiviral responses. The parallel BVDV and HCV replicon systems provide robust counterscreens to distinguish viral specificity of small-molecule inhibitors of viral replication and to study the interactions of the viral replication machinery with the host cell innate immune system.

scholarly journals Specific Inhibition of Bovine Viral Diarrhea Virus Replicase

2003 ◽  
Vol 77 (12) ◽  
pp. 6753-6760 ◽  
Author(s):  
Jin-Hua Sun ◽  
Julie A. Lemm ◽  
Donald R. O'Boyle ◽  
Jason Racela ◽  
Richard Colonno ◽  
...  
Keyword(s):  
Bovine Viral Diarrhea Virus ◽  
Single Amino Acid ◽  
Bovine Viral Diarrhea ◽  
Cdna Clones ◽  
Single Mutation ◽  
Resistant Virus ◽  
Replication Efficiency ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Viral Diarrhea Virus

ABSTRACT Compound-1453 was identified and characterized as a specific inhibitor of bovine viral diarrhea virus (BVDV). The concentration of compound-1453 which results in 50% protection from virus-induced cytopathic effect is ∼2.2 μM, with a therapeutic index of 60, and it is not active against a panel of RNA and DNA viruses. A time-of-addition experiment suggested that compound-1453 targets a stage of the viral life cycle after viral entry. To determine the target of compound-1453, resistant virus was generated. Resistant variants grew efficiently in the presence or absence of 33 μM compound-1453 and exhibited replication efficiency in the presence of compound-1453 approximately 1,000-fold higher than that of the wild-type (wt) virus. Functional mapping and sequence analysis of resistant cDNAs revealed a single amino acid substitution (Glu to Gly) at residue 291 in the NS5B polymerase in all eight independently generated cDNA clones. Recombinant virus containing this single mutation retained the resistance phenotype and a replication efficiency similar to that of the original isolated resistant virus. Since compound-1453 did not inhibit BVDV polymerase activity in vitro (50% inhibitory concentration > 300 μM), we developed a membrane-based assay that consisted of a BVDV RNA replicase complex isolated from virus-infected cells. Compound-1453 inhibited the activity of the wt, but not the drug-resistant, replicase in the membrane assay at concentrations similar to those observed in the viral infection assay. This work presents a novel inhibitor of a viral RNA-dependent RNA replicase.

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