Monoclonal antibodies specific for Semliki Forest virus replicase protein nsP2

A panel of monoclonal antibodies (MAbs) was raised against Semliki Forest virus (SFV) nonstructural protein nsP2, which is a protease, an NTPase, 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 Escherichia 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 immunoblotting, and 25 showed positive indirect immunofluorescence (IFAT) in SFV-infected cells; 15 MAbs stained the cytoplasmic vacuoles (CPVI), which 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 SFV neurovirulence. Immunoprecipitation with MAbs revealed that the SFV nonstructural proteins are associated with each other. None of the MAbs recognized Sindbis virus nsP2 in immunoblotting, indicating that they were directed to non-conserved sequences specific for SFV. Interestingly, 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 insect cells. This protein was identified as the immunoglobulin binding protein, BiP, by 2-D gel mapping and reaction with anti-BiP antiserum.