Biosynthesis of Classical Swine Fever Virus Nonstructural Proteins

Abstract

Proteolytic processing of polyproteins is considered a crucial step in the life cycle of most positive-strand RNA viruses. An enhancement of NS2-3 processing has been described as a major difference between the noncytopathogenic (non-CP) and the cytopathogenic (CP) biotypes of pestiviruses. The effects of accelerated versus delayed NS2-3 processing on the maturation of the other nonstructural proteins (NSP) have never been compared. In this study, we analyzed the proteolytic processing of NSP in Classical swine fever virus (CSFV). Key to the investigation was a panel of newly developed monoclonal antibodies (MAbs) that facilitated monitoring of all nonstructural proteins involved in virus replication (NS2, NS3, NS4A, NS5A, and NS5B). Applying these MAbs in Western blotting and radioimmunoprecipitation allowed an unambiguous identification of the mature proteins and precursors in non-CP CSFV-infected cells. Furthermore, the kinetics of processing were determined by pulse-chase analyses for non-CP CSFV, CP CSFV, and a CP CSFV replicon. A slow but constant processing of NS4A/B-5A/B occurred in non-CP CSFV-infected cells, leading to balanced lowlevel concentrations of mature NSP. In contrast, the turnover of the polyprotein precursors was three times faster in CP CSFV-infected cells and in cells transfected with a CP CSFV replicon, causing a substantial increase of mature NSP concentrations. We conclude that a delayed processing not only of NS3 but further of all NSP represents a hallmark of regulation in non-CP pestiviruses.

Autocatalytic Cleavage within Classical Swine Fever Virus NS3 Leads to a Functional Separation of Protease and Helicase

“…Recently, we reported on the time-resolved hierarchy of CSFV nonstructural protein processing (18). As a prerequisite for studying the NS2-3 cleavage in virus-infected cells, a monoclonal antibody (moAb) was established that was directed against the protease domain of NS3 (GL3p1).…”

“…Detailed analyses demonstrated that the availability of the cellular cofactor JIV ("Jdomain protein interacting with viral protein"; DNAJC14) restricts NS2-3 cleavage to the first hours of infection in the noncytopathogenic (ncp) biotype whereas a continuous NS2-3 cleavage takes place in the cytopathogenic (cp) biotype of BVDV (17). Elevated levels of mature NS3 led to an enhanced processing of NS4-5 precursor molecules (18) and have been linked to the accelerated RNA replication of cp BVDVs (17). In a recent study, the decisive function of the NS3 helicase for virus particle formation became evident.…”

“…Hybridomas secreting the desired monoclonal antibodies against the C-terminal fragment of CSFV NS3 were generated according to a modified standard procedure as described previously (18,27). The nomencla-ture of hybridomas and properties of secreted moAbs used in this study is indicated in Table 2.…”

“…Transfection of SK-6 cells with infectious CSFV RNA was achieved by electroporation as described before (18). SK-6 cells were infected with ncp CSFV or cp CSFV-JIV progeny virus at an MOI of one.…”

Evaluation of an Erns-based enzyme-linked immunosorbent assay to distinguish Classical swine fever virus–infected pigs from pigs vaccinated with CP7_E2alf

“…24 The single open reading frame encodes for 1 polyprotein, which is cleaved during viral maturation into 4 structural (C, E rns , E1, and E2) and 9 nonstructural (N pro , p7, NS2, NS2-3, NS3, NS4a, NS4b, NS5a, and NS5b) proteins. 7,22,24 The CSFV proteins NS3, E rns , and E2 induce antibodies on viral infection. Antibodies developed against E rns and E2 have virus-neutralizing abilities and confer protective immunity against viral infection.…”