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.

scholarly journals A Stem-Loop Motif Formed by the Immediate 5′ Terminus of the Bovine Viral Diarrhea Virus Genome Modulates Translation as well as Replication of the Viral RNA

2000 ◽  
Vol 74 (13) ◽  
pp. 5825-5835 ◽  
Author(s):  
Haiying Yu ◽  
Olaf Isken ◽  
Claus W. Grassmann ◽  
Sven-Erik Behrens
Keyword(s):  
Bovine Viral Diarrhea Virus ◽  
Rna Replication ◽  
Terminal Region ◽  
Viral Rna ◽  
Bovine Viral Diarrhea ◽  
Stem Loop ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Loop Region ◽  
Viral Diarrhea Virus

ABSTRACT Bovine viral diarrhea virus (BVDV), aPestivirus member of the Flaviviridae family, has a positive-stranded RNA genome which consists of a single open reading frame (ORF) and untranslated regions (UTRs) at the 5′ and 3′ ends. The 5′ UTR harbors extensive RNA structure motifs; most of them were shown to contribute to an internal ribosomal entry site (IRES), which mediates cap-independent translation of the ORF. The extreme 5′-terminal region of the BVDV genome had so far been believed not to be required for IRES function. By structure probing techniques, we initially verified the existence of a computer-predicted stem-loop motif at the 5′ end of the viral genome (hairpin Ia) as well as at the 3′ end of the complementary negative-strand replication intermediate [termed hairpin Ia (−)]. While the stem of this structure is mainly constituted of nucleotides that are conserved among pestiviruses, the loop region is predominantly composed of variable residues. Taking a reverse genetics approach to a subgenomic BVDV replicon RNA (DI9c) which could be equally employed in a translation as well as replication assay system based on BHK-21 cells, we obtained the following results. (i) Proper folding of the Ia stem was found to be crucial for efficient translation. Thus, in the context of an authentic replication-competent viral RNA, the 5′-terminal motif operates apparently as an integral functional part of the ribosome entry. (ii) An intact loop structure and a stretch of nucleotide residues that constitute a portion of the stem of the Ia or the Ia (−) motif, respectively, were defined to represent important determinants of the RNA replication pathway. (iii) Formation of the stem structure of the Ia (−) motif was determined to be not critical for RNA replication. In summary, our findings affirmed that the 5′-terminal region of the BVDV genome encodes a bifunctional secondary structure motif which may enable the viral RNA to switch from the translation to the replicative cycle and vice versa.

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 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 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.

scholarly journals Persistence of Bovine Viral Diarrhea Virus Is Determined by a Cellular Cofactor of a Viral Autoprotease

2005 ◽  
Vol 79 (15) ◽  
pp. 9746-9755 ◽  
Author(s):  
T. Lackner ◽  
A. Müller ◽  
M. König ◽  
H.-J. Thiel ◽  
N. Tautz
Keyword(s):  
Bovine Viral Diarrhea Virus ◽  
Protease Activity ◽  
Nonstructural Protein ◽  
Cellular Protein ◽  
Viral Rna ◽  
Bovine Viral Diarrhea ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Positive Strand Rna ◽  
Viral Diarrhea Virus

ABSTRACT Polyprotein processing control is a crucial step in the life cycle of positive-strand RNA viruses. Recently, a vital autoprotease generating an essential viral replication factor was identified in such a virus, namely, the pestivirus bovine viral diarrhea virus. Surprisingly, the activity of this protease, which resides in nonstructural protein 2 (NS2), diminishes early after infection, resulting in the limitation of viral RNA replication. Here, we describe that a cellular chaperone termed Jiv (J-domain protein interacting with viral protein) acts as a cofactor of the NS2 protease. Consumption of the intracellular Jiv pool is responsible for temporal regulation of protease activity: overexpression of Jiv interfered with regulation and correlated with increased accumulation of viral RNA; downregulation of the cellular Jiv level accelerated the decline of protease activity and reduced intracellular viral RNA levels and virion production. Accordingly, the amount of a cellular protein controls pestiviral replication by limiting the generation of active viral protease molecules and replication complexes. Importantly, this unique mechanism of replication control is essential for maintenance of the noncytopathogenic phenotype of the virus and thereby for its ability to establish persistent infections. These results add an entirely novel aspect to the understanding of the molecular basis of viral persistence.

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