Bovine viral diarrhea virus non-structural protein 5A interacts with NIK- and IKKβ-binding protein

Abstract

Bovine viral diarrhea virus (BVDV) is a positive-sense, single-stranded RNA virus that causes an economically important livestock disease worldwide. Previous studies have suggested that nonstructural protein 5A (NS5A) from hepatitis C virus (HCV) and BVDV plays a similar role during virus infection. Extensive reports are available on HCV NS5A and its interactions with the host cellular proteins; however, the role of NS5A during BVDV infection remains largely unclear. To identify the cellular proteins that interact with the N terminus of NS5A and could be involved in its function, we conducted a yeast two-hybrid screening. As a result, we identified a cellular protein termed bovine NIK-and IKKb-binding protein (NIBP), which is involved in protein trafficking and nuclear factor kappa B (NF-kB) signalling in cells. The interaction of NS5A with NIBP was confirmed both in vitro and in vivo. Complementing our glutathione S-transferase pull-down and immunoprecipitation data are the confocal immunofluorescence results, which indicate that NS5A colocalized with NIBP on the endoplasmic reticulum in the cytoplasm of BVDV-infected cells. Moreover, the minimal residues of NIBP that interact with NS5A were mapped as aa 597-623. In addition, overexpression of NS5A inhibited NF-kB activation in HEK293 and LB9.K cells as determined by luciferase reporter-gene assay. We further showed that inhibition of endogenous NIBP by small interfering RNA molecules enhanced virus replication, indicating the importance of NIBP implications in BVDV pathogenesis. Being the first reported interaction between NIBP and a viral protein, this finding suggests a novel mechanism whereby viruses may subvert host-cell machinery for mediating trafficking as well as NF-kB signalling. INTRODUCTIONPestiviruses are important livestock pathogens that belong to the family Flaviviridae, which also includes the closely related genera Hepacivirus (Hepatitis C virus; HCV) and Flavivirus (Yellow fever virus and West Nile virus). The genus Pestivirus includes the species Bovine viral diarrhea virus 1 (BVDV-1), BVDV-2, Border disease virus and Classical swine fever virus. As a well-characterized prototype member of the genus Pestivirus within the family Flaviviridae, BVDV sometimes serves as a surrogate model for HCV life-cycle studies. BVDV is an enveloped virus containing a single, positive-sense RNA of approximately 12.3 kb. The genomic RNA consists of one long open reading frame (ORF), which is flanked by untranslated regions at both ends. The ORF encodes a single polyprotein of about 4000 aa, which is processed co-and posttranslationally by both viral and cellular proteases into at least 11 mature viral proteins (N pro , C, E rns , E1, E2, p7, NS2-3, NS4A, NS4B, NS5A and NS5B) (Lindenbach et al., 2007).BVDV non-structural protein 5A (NS5A) is a phosphoprotein of 56-58 kDa that plays an essential role in BVDV replication (Tellinghuisen et al., 2006) and shares many features with its counterpart HCV NS5A, e.g. both proteins are phosphorylated by the same or simila...

A Novel Hepacivirus with an Unusually Long and Intrinsically Disordered NS5A Protein in a Wild Old World Primate

“…For HCV, the NS5A-TRAF2 protein complex has been shown to inhibit TNF-induced NF-B activation, thereby increasing cellular resistance to apoptotic stimuli (68), perhaps modulating host responses to pathogens, persistence of viral infection, and disease severity (69). An inhibitory effect by NS5A-protein complexes on TNF-induced NF-B activation has also been reported for BVDV, albeit mediated through NS5A-binding interactions with different host immunomodulatory proteins (70).…”

Understanding the Pathogenesis of Cytopathic and Noncytopathic Bovine Viral Diarrhea Virus Infection Using Proteomics

“…This interaction was suggested to play a role in the replication of BVDV (Johnson et al 2001). In addition, Y2H screening identified bovine NIK-and IKK -binding protein (NIBP), which is involved in protein trafficking and nuclear factor kappa B (NF-κB) signalling in cells (Zahoor et al 2010). The interaction of NS5A with NIBP was confirmed both in vitro and in vivo.…”

“…The same study also showed that inhibition of endogenous NIBP by RNAi enhanced virus replication, indicating the importance of NIBP in BVDV pathogenesis. This is the first reported interaction between NIBP and a viral protein, suggesting a novel mechanism whereby viruses may subvert host-cell machinery for mediating trafficking and NF-κB signaling (Zahoor et al 2010). Efficient generation of NS2-3 cleavage product NS3 is required for the cytopathogenicity of the pestiviruses.…”

Unique Localization of Bovine Viral Diarrhea Virus Non-Structural NS4B Protein in Infected Cells

“…Myc-tagged NS4B gene was subcloned into the pCAGGS mammalian expression plasmid, named pCAG-NS4B, between XhoI and BglII sites. The plasmids pME-NS3 and pCAG-NS5A were described previously Zahoor et al, 2010). The NS4A gene sequence derived from Nose strain was generated using primers with NdeI and PstI sites at the 5´ and 3´ ends, respectively, and cloned into the plasmid pME18S-myc, named pME-NS4A.…”