Heterogeneous expression of the non-structural protein p80/p125 in cells infected with different pestiviruses

Abstract

In order to analyse the expression of the non-structural (ns) protein p80/p125 in cells infected with different pestiviruses at the protein level, radioimmunoprecipitations with the pestivirus-specific monoclonal antibody (MAb) BVD/C16 were performed. Cell lysates infected with cytopathic (cp) and non-cytopathic (ncp) bovine viral diarrhoea (BVD) virus strains and isolates, and with hog cholera (HC) virus strains were analysed. From cpBVD virus-infected cells, the MAb precipitated one or more proteins corresponding to ns p125, displaying a marked size heterogeneity. In contrast, the lower Mr ns p80 proteins from all cpBVD virus strains and isolates analysed had identical electrophoretic motility. The ncpBVD virus strains displayed either one single band or a doublet of the p 125 protein and no p80 cleavage products. The p125 proteins precipitated from HC virus-infected cells showed no size heterogeneity. The possibility is discussed that multiple recombination events, including both insertions or deletions in the genomes of ncpBVD viruses, may lead to the heterogeneous expression of the ns p125 in cpBVD virus populations.

Biosynthesis of Classical Swine Fever Virus Nonstructural Proteins

“…4B). NS3 (682 aa; calculated molecular mass, 78 kDa) has been visualized earlier in non-CP CSFV-infected cells as p75/p80 (NS3) and p120/p125 (11,30). Immunoblot analysis with NS4A-specific antibodies (GH4A1-6) allowed detection of NS4A in non-CP CSFV-infected cells.…”

Classical swine fever virus replicon particles lacking the Ernsgene: a potential marker vaccine for intradermal application

“…For titration of VRP, SK-6 cells seeded in 96-well plates were infected with tenfold dilutions of the clarified supernatant. The titer expressed in TCID 50 /mL was determined by staining the cells with mAb C16 [11] directed against viral protein NS3. The supernatant was stored as master seed at -70 • C. To confirm that the E rns deletion in the CSF-VRP genome was stable in complementing cells, the respective region was amplified by RT-PCR from total RNA obtained by Trizol (Invitrogen, Carlsbad, CA, USA) extraction of cell cultures infected with passage 9 of VRP A187delE rns .…”

Cytopathogenicity of Classical Swine Fever Virus Correlates with Attenuation in the Natural Host

“…In contrast, cp CSFV strain Alfort-Jiv described in this study combines helper virus-independent replication and genetic stability during propagation in tissue culture cells and thus is well suited to compare the properties of cp and non-cp CSFV in both susceptible cells and the natural host. The cytopathogenicity of pestiviruses including BVDV, BDV, and CSFV has been shown to correlate with the expression of large amounts of NS3, the C-terminal part of NS2-3 (5,9,20,21,23,27,30,51). The genetic background and the resulting molecular mechanisms that lead to the efficient release of free NS3 are remarkably diverse among cp pestivirus genomes, including (i) genomic deletions fusing the translation start codon (51) or the autoprotease N pro gene (72) directly to the 5Ј end of the viral NS3 gene, (ii) duplicated viral sequences located in the NS2 gene (70), (iii) point mutations in the NS2 gene (37), and (iv) the insertion of cleavage sites for cellular proteases directly upstream of NS3 (6,12,16,25,71).…”