MONOCLONAL-ANTIBODIES SPECIFIC FOR SEMLIKI-FOREST-VIRUS REPLICASE PROTEIN NSP2

A panel of monoclonal antibodies (MAbs) was raised against Semliki For est virus (SFV) nonstructural protein nsP2, which is a protease, an NT Pase, a putative RNA helicase, and a regulator of the synthesis of the subgenomic 26S mRNA encoding the structural proteins, nsP2, used for immunization, was expressed as a histidine fusion protein in Escherich ia coli and purified by metal affinity chromatography, Dot-blot assay, using a membrane fraction from SFV-infected cells as antigen, gave 33 positive clones, Of these, 30 MAbs recognized nsP2 in Western immunob lotting, and 25 showed positive indirect immunofluorescence (IFAT) in SFV-infected cells; 15 MAbs stained the cytoplasmic vacuoles (CPVI), w hich are the sites of viral RNA synthesis in alphavirus-infected cells , MAb 3B5 recognized only CPVIs, as shown by double immunofluorescence staining with polyclonal anti-nsP3 antiserum, Most of the MAbs (20/33 ) recognized the nuclear form of nsP2, which may be associated with SF V neurovirulence, Immunoprecipitation with MAbs revealed that the SFV nonstructural proteins are associated with each other, None of the MAb s recognized Sindbis virus nsP2 in immunoblotting, indicating that the y were directed to non-conserved sequences specific for SFV, Interesti ngly, these epitopes were located mostly within the N-terminal half of nsP2, Unexpectedly, the anti-nsP2 MAb 1E9 cross-reacted strongly with a host protein of 78 kDa from uninfected human, murine, avian and ins ect cells, This protein was identified as the immunoglobulin binding p rotein, BiP, by 2-D gel mapping and reaction with anti-BiP antiserum.