The NPro Product of Bovine Viral Diarrhea Virus Inhibits DNA Binding by Interferon Regulatory Factor 3 and Targets It for Proteasomal Degradation

Abstract

Bovine viral diarrhea virus (BVDV) is a pestivirus that can establish a persistent infection in the developingfetus and has the ability to disable the production of type I interferon. In this report, we extend our previous observations that BVDV encodes a protein able to specifically block the activity of interferon regulatory factor 3 (IRF-3), a transcription factor essential for interferon promoter activation, by demonstrating that this is a property of the N-terminal protease fragment (NPro) of the BVDV polyprotein. Although BVDV infections cause relocalization of cellular IRF-3 from the cytoplasm to the nucleus early in infection, NPro blocks IRF-3 from binding to DNA. NPro has the additional property of targeting IRF-3 for polyubiquitination and subsequent destruction by cellular multicatalytic proteasomes. The autoprotease activity of NPro is not required for the inhibition of type I interferon induction or the targeting of IRF-3 for degradation.Bovine viral diarrhea virus (BVDV) has a global spread and is a major reproductive pathogen of cattle (12). BVDV, along with classical Swine fever virus (CSFV), Border disease virus of sheep, and a small number of isolates from undomesticated species, make up the genus Pestivirus in the family Flaviviridae. The viruses are positive-strand RNA viruses with a genome on the order of 12.5 kb in length. The RNA is translated into a single viral polyprotein that is processed by both viral and host proteases to either 11 or 12 polypeptides, depending on the virus biotype. BVDV is generally noncytopathogenic (ncp), although the virus associated with the development of mucosal disease in persistently infected animals is a cytopathogenic (cp) biotype. The result of infection of a susceptible host with BVDV is unusual; BVDV can cause an acute infection like other viruses, but infection of a pregnant animal can result in transmission of virus from the dam to the developing fetus, where the virus can replicate. Infection of the fetus prior to immune competence results in a failure of the fetus to control virus infection and can result in the birth of persistently infected offspring that fail to mount an acquired immune response to BVDV. These offspring then serve as a reservoir for further acute virus infection and are held to be critical for BVDV transmission in areas of endemicity.Although infection during fetal development takes place in the absence of a functional acquired immune response, viruses still have to evade innate immunity in order to establish a persistent infection; the ability of ncpBVDV to avoid the induction of interferon (IFN) seems critical in this regard (5). We and others have shown that ncpBVDV encodes an active block to type I IFN induction (3, 46), and there is evidence that ncpBVDV employs more than one mechanism to bring about this block. BVDV-infected cells are refractory to the addition of double-stranded RNA (dsRNA) to the medium. At least in part, the failure of this extracellular dsRNA to induce type I IFN can be explained by the action of the s...

Structural insights into phosphoinositide 3-kinase activation by the influenza A virus NS1 protein

“…S4A), an IFN antagonist provided in trans cannot rescue the phenotype of this virus. Similar results were also obtained in A549 cells using a different IFN-antagonist protein (BVDV NPro) (25) (Fig. S4B).…”

STAT2 deficiency and susceptibility to viral illness in humans

“…The latter were cultured in DMEM with 10% (vol/vol) FCS and supplemental penicillin and streptomycin. To generate control cells expressing the V protein of PIV5 and patient cells expressing functional STAT2, the PIV5 V gene and the full-length ORF of STAT2 were cloned into the bicistronic lentivirus vector (pHR-SIN-CSGW); lentiviruses were produced and the cells were transduced and selected for puromycin resistance as described previously (40). The full-length ORF of human STAT2 was amplified by using specific primers by reverse transcription (Superscript III; Invitrogen) and PCR (HiFi DNA polymerase; Kappa Biosystems), using total RNA from control patient fibroblast cells.…”

RNase-dependent inhibition of extracellular, but not intracellular, dsRNA-induced interferon synthesis by Erns of pestiviruses

“…Moreover, the fact that virus infection prevented Mx induction and at the same time led to the complete disappearance of IRF-3 suggested that the effect of intact virus may be independent of E rns . Previous work has suggested that the effect on IRF-3 of pestiviruses may be mediated through N pro , which targets IRF-3 for proteasomal degradation (Bauhofer et al, 2007;Chen et al, 2007;Hilton et al, 2006; Seago et al, 2007).…”

“…Moreover, the fact that virus infection prevented Mx induction and at the same time led to the complete disappearance of IRF-3 suggested that the effect of intact virus may be independent of E rns . Previous work has suggested that the effect on IRF-3 of pestiviruses may be mediated through N pro , which targets IRF-3 for proteasomal degradation (Bauhofer et al, 2007;Chen et al, 2007;Hilton et al, 2006; Seago et al, 2007).To assess the functions of the two proteins in the context of intact virus, rather than being expressed as single proteins, we made use of viral mutants lacking either N pro or the RNase activity of E rns (H30F). As shown previously (Schweizer & Peterhans, 2001), Mx (and hence IFN) production induced by extracellular and intracellular dsRNA was completely inhibited in cells infected with wt BVDV (Fig.…”

“…The non-structural protein N pro , a protease encoded at the 59 end of the 12.5 kb viral genome, has been shown to target the transcription factor interferon regulatory factor (IRF)-3 for proteasomal degradation (Hilton et al, 2006; Seago et al, 2007). The second protein, E rns , is a highly N-glycosylated endoRNase that forms disulfide-linked homodimers.…”