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

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 N pro , 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-␤-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 N pro 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. Hepatitis C virus (HCV) and bovine viral diarrhea virus (BVDV) are both members of the Flaviviridae family and share many molecular and virological similarities (20, 27). They both have single-strand RNA genomes that replicate via negativestrand intermediates and produce a single polyprotein that is cleaved into individual proteins by a combination of host and viral proteases. Although HCV is a hepacivirus and BVDV is a pestivirus, there is a low degree of sequence homology between their respective nonstructural proteins and both RNAdependent RNA polymerases are structurally very similar (9).HCV is a major etiological agent for viral hepatitis. The World Health Organization estimates that 170 million people are chronically infected with HCV worldwide, and of those, 4 million are in the United States. Within 10 to 20 years of infection, 20 to 30% of chronic carriers develop cirrhosis, making HCV infection one of the major reasons for liver transplantation. Current therapies are limited to interferon treatment, either alone or in combination with ribavirin (23, 38), but patient response for certain genotypes is still unsatisfactory. This unmet medical need has created an urgent demand for the development of new dr...

The Npro product of classical swine fever virus interacts with IκBα, the NF-κB inhibitor

“…Pestiviruses such as CSFV and bovine viral diarrhea virus (BVDV) are able to cross the placenta and can establish an infection in the developing fetus, resulting in the birth of persistently infected animals (Charleston et al, 2001). In addition, pestiviruses can actively block type I interferon (IFN) induction and double-stranded (ds)RNA-mediated apoptotic responses (Baigent et al, 2002(Baigent et al, , 2004Bensaude et al, 2004;Charleston et al, 2001;Horscroft et al, 2005;Ruggli et al, 2003Ruggli et al, , 2005Schweizer & Peterhans, 2001). …”

Innate immune response to double-stranded RNA in biliary epithelial cells is associated with the pathogenesis of biliary atresia

“…For the evaluation of messenger ribonucleic acid (mRNAs) of TLR3, RIG-I, MDA-5, PKR, NF-B, interferon regulatory factor-3 (IRF-3), IFN-␤, MxA, TRAIL, TRAIL receptors (TRAIL-DR4, TRAIL-DR5, TRAIL-DcR1, and TRAIL-DcR2), CD95, and CD95 ligand in cultured BECs, total RNA was isolated from BECs, and 1 g total RNA was reverse-transcribed with an oligo-(dT) primer and reverse transcriptase to synthesize complementary deoxyribonucleic acid. Human cultured cell lines (HuCCT1, HuH7, HepG2, and WI38) and normal tissues (pancreas and liver) [18][19][20][21][22][23][24][25][26][27] were used as controls, and the complementary deoxyribonucleic acid was amplified by polymerase chain reaction (PCR) using the specific primers ( Table 1). The PCR products were subjected to electrophoresis on 1.5% agarose gels containing ethidium bromide.…”

Type I and III IFNs Produced by Plasmacytoid Dendritic Cells in Response to a Member of theFlaviviridaeSuppress Cellular Immune Responses

“…Only pestiviruses encode a novel N-terminal proteinase termed N pro , which, along with the E rns glycoprotein, block IFN production. N pro of both BVDV and CSFV antagonize the production of type 1 IFN by targeting IFN regulatory factor (IRF) 3 for proteasomal degradation (12,15,16). Despite the ability of pestiviruses to block type I IFN in the majority of host target cells, IFN can be detected in serum samples from cattle early postinfection with ncp BVDV and following CSFV infection of pigs.…”