Journal of General Virology, Vol 78, 343-351, Copyright © 1997 by Society for General Microbiology

ARTICLES

Monoclonal antibodies specific for Semliki Forest virus replicase protein nsP2

P Kujala, M Rikkonen, T Ahola, M Kelve, M Saarma and L Kaariainen
Biocentre Viikki, Institute of Biotechnology, University of Helsinki, Finland. ptkujala@operoni.helsinki.fi

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.

This article has been cited by other articles:

				N. Tamberg, V. Lulla, R. Fragkoudis, A. Lulla, J. K. Fazakerley, and A. Merits Insertion of EGFP into the replicase gene of Semliki Forest virus results in a novel, genetically stable marker virus J. Gen. Virol., April 1, 2007; 88(4): 1225 - 1230. [Abstract] [Full Text] [PDF]

				J. K. Fazakerley, A. Boyd, M. L. Mikkola, and L. Kaariainen A Single Amino Acid Change in the Nuclear Localization Sequence of the nsP2 Protein Affects the Neurovirulence of Semliki Forest Virus J. Virol., January 1, 2002; 76(1): 392 - 396. [Abstract] [Full Text] [PDF]

				P. Kujala, A. Ikäheimonen, N. Ehsani, H. Vihinen, P. Auvinen, and L. Kääriäinen Biogenesis of the Semliki Forest Virus RNA Replication Complex J. Virol., April 15, 2001; 75(8): 3873 - 3884. [Abstract] [Full Text]

				A. Merits, L. Vasiljeva, T. Ahola, L. Kääriäinen, and P. Auvinen Proteolytic processing of Semliki Forest virus-specific non-structural polyprotein by nsP2 protease J. Gen. Virol., April 1, 2001; 82(4): 765 - 773. [Abstract] [Full Text]

				G. J. Atkins, B. J. Sheahan, and P. Liljeström The molecular pathogenesis of Semliki Forest virus: a model virus made useful? J. Gen. Virol., September 1, 1999; 80(9): 2287 - 2297. [Full Text]

				A. M. Andersson and R. F. Pettersson Targeting of a Short Peptide Derived from the Cytoplasmic Tail of the G1 Membrane Glycoprotein of Uukuniemi Virus (Bunyaviridae) to the Golgi Complex J. Virol., December 1, 1998; 72(12): 9585 - 9596. [Abstract] [Full Text] [PDF]

				M. L. Linn, L. Mateo, J. Gardner, and A. Suhrbier Alphavirus-Specific Cytotoxic T Lymphocytes Recognize a Cross-Reactive Epitope from the Capsid Protein and Can Eliminate Virus from Persistently Infected Macrophages J. Virol., June 1, 1998; 72(6): 5146 - 5153. [Abstract] [Full Text] [PDF]