Expression of two bovine rotavirus non-structural proteins (NSP 2, NSP 3) in the baculovirus system and production of monoclonal antibodies directed against the expressed proteins

Abstract

Studies on rotavirus non-structural proteins have been hampered in the past by difficulties in obtaining monospecific reagents. To make such reagents available, we have expressed in the baculovirus system NSP2 and NSP3 (formerly called NS35 and NS34, respectively) of the bovine rotavirus RF and produced hybridomas against these proteins. Full-length DNA copies of RNA segments 7 (coding for NSP3) and 8 (coding for NSP2) of the virus strain RF were cloned and sequenced. Each cDNA was inserted in the transfer vector pVL941 and used to transfect Spodoptera frugiperda cells (Sf9). Recombinant baculoviruses encoding these proteins were obtained. Infection of Sf9 cells with these recombinant viruses resulted in a high level of expression of NSP2 and NSP3 (range of 1 microgram per 10(6) cells). Monoclonal antibodies (MAbs) were elicited by immunization of BALB/c mice with adjuvented, unpurified recombinant proteins in the rear foot pads. Fusion was performed using lymphocytes from popliteal lymph nodes with SP2/O-Ag14 myeloma line. Screening was by differential indirect immunofluorescent staining on monolayers of Sf9 cells infected with each recombinant virus. Two MAbs proved to be reactive against NSP3 and a single one against NSP2. They showed high specificity by immunofluorescence, immunoprecipitation and Western blot. The isotype of these MAbs was IgG1. Oligomeric forms of NSP3 and NSP2 proteins were detected and the existence of intra-chain disulfide bridge in NSP2 protein was suggested. The levels of synthesis and cellular localization of NSP3 and NSP2 proteins were different as shown by immunoprecipitation and immunofluorescence.

Viral proteins VP2, VP6, and NSP2 are strongly precipitated by serum and fecal antibodies from children with rotavirus symptomatic infection

“…Immunoprecipitations with monoclonal antibodies (MAbs) to rotavirus structural and nonstructural proteins were included to identify unequivocally the corresponding rotavirus proteins. The following MAbs were used: MAb 164AE22 directed against VP2 [Roseto et al, 1983] and MAb ID3 directed against NSP3 [Aponte et al, 1993], kindly supplied by J. Cohen; MAb to VP6 (MAB 851, Chemicon International, Temecula, CA); and MAbs 5A10, 3C3, and 4C3 directed against VP7 [Gerna et al, 1988], obtained from G. Gerna of University of Pavia, Italy. To confirm the identity of VP7 in the RIP assays, radiolabeled proteins extracted from CV-1 cells infected with a recombinant vaccinia virus expressing the VP7 protein of bovine rotavirus strain RF were precipitated with the anti-VP7 monoclonal antibodies.…”

Efficient Translation of Rotavirus mRNA Requires Simultaneous Interaction of NSP3 with the Eukaryotic Translation Initiation Factor eIF4G and the mRNA 3′ End

“…1A and B) with a monoclonal antibody directed against NSP3 (2). Only a few cell clones were positive for NSP3 expression, and one (called ST-NSP3) that synthesizes a detectable amount of NSP3 by immunoprecipitation (Fig.…”

Rearranged Genomic RNA Segments Offer a New Approach to the Reverse Genetics of Rotaviruses

“…To detect NSP3 viral protein, MA-104 cell cultures were harvested at 18 h postinfection in a lysis buffer containing 50 mM Tris-HCl (pH 6.8), 2% sodium dodecyl sulfate (SDS), and 2% ␤-mercaptoethanol. SDS-PAGE and Western blotting were performed as previously described (8), using the ID3 anti-NSP3 mouse monoclonal antibody (1).…”