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Functional expression of the hemagglutinin and fusion proteins of measles virus in a baculovirus expression system
39 FUNCTIONAL EXPRESSION OF THE HEMAGGLUTININ AND FUSION PROTEINS OF MEASLES VIRUS IN A BACULOVIRUS EXPRESSION SYSTEM BRXEDIS', CHRISTOPHER D. ICHARDSONl, PHI IP MARSHALLl, DALIU 9 L~UMIE~ HALIB ALKHATIB 1 , JORGE VIALARD k 8 AND EON EBiotechnology Research Institute, NRC Canada, 6100 Royalmount Ave., Montreal, Quebec, Canada H4P 2R2 'Department of Microbiology and Immunology, McGill University, 3775 University St., Montreal, Quebec, Canada H3A 2B4 Previous work in our laboratories has concerned the molecular properties, cloning, and nucleic acid sequencing of the genes encoding the surface glycoproteins of measles virus. The membrane fusion (F) and hemagglutinin (H) proteins of measles virus were cloned and expressed using the baculovirus-insect cell expression system. Both proteins were determined to be fully functional in The proteins were hemolysis and hemagglutination assays. was proteolytically glycosylated and the fusion protein Coordinate synthesis of the two viral proteins was activated. required for optimal membrane fusion activity. Levels of recombinant viral proteins synthesized in modified expression vectors In addition a number of mutations in these were also monitored. proteins were introduced by site-specific mutagenesis and their effects on membrane fusion studied.
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EXPRESSION OF THE BUNYAVIRUS S RNA SEGMENT RICHARD M. ELLIOTT, MAW M. SMITH and ALISTAIR MCGREGOR Institute of Virology, University of Glasgow, Church Street, Glasgow Gl 1 5JR. Scotland. The nucleotide sequence of the S genome segment of Maguari virus (Bunyamwera serogroup, Bunyavirus genus) has been determined from cloned cDNA. In common with other bunyaviruses this S segment encodes two proteins, the nucleocapsid protein, N, and a nonstructural protein, NSs, in overlapping reading frames. In order to study the expression and function of the S RNA gene products the cDNA has been inserted into various plasmid and viral vectors. The S segment cDNA was placed downstream of a bacteriophage T7 promoter, and RNA was synthesized in vitro using T7 RNA polymerase and translated in a reticulocyte lysate. Both N and NSs were translated from the same RNA transcript. The cDNA was modified to remove the translational start for the N protein and the resulting RNA transcript was translated to give the NSs protein alone. We have also cloned the S cDNA into a transient expression plasmid, p91023, which contains control elements from SV40 and adenovirus. After transfection of COS cells with the recombinant plasmid synthesis of the N protein was detected by immunoprecipitation. The ability of the transfected cDNA to complement Maguari virus temperature-sensitive mutants at the nonpermissive temperature is under investigation. Lastly a recombinant baculovirus has been constructed which contains the Maguari virus S cDNA in place of the baculovirus polyhedrin gene. In infected insect cells a high level of antigenically authentic N was made and we are attempting to purify the N protein for functional analyses. Other experiments exploiting these systems will be discussed.
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EXPRESSION OF THE BUNYAVIRUS S RNA SEGMENT RICHARD M. ELLIOTT, MAW M. SMITH and ALISTAIR MCGREGOR Institute of Virology, University of Glasgow, Church Street, Glasgow Gl 1 5JR. Scotland. The nucleotide sequence of the S genome segment of Maguari virus (Bunyamwera serogroup, Bunyavirus genus) has been determined from cloned cDNA. In common with other bunyaviruses this S segment encodes two proteins, the nucleocapsid protein, N, and a nonstructural protein, NSs, in overlapping reading frames. In order to study the expression and function of the S RNA gene products the cDNA has been inserted into various plasmid and viral vectors. The S segment cDNA was placed downstream of a bacteriophage T7 promoter, and RNA was synthesized in vitro using T7 RNA polymerase and translated in a reticulocyte lysate. Both N and NSs were translated from the same RNA transcript. The cDNA was modified to remove the translational start for the N protein and the resulting RNA transcript was translated to give the NSs protein alone. We have also cloned the S cDNA into a transient expression plasmid, p91023, which contains control elements from SV40 and adenovirus. After transfection of COS cells with the recombinant plasmid synthesis of the N protein was detected by immunoprecipitation. The ability of the transfected cDNA to complement Maguari virus temperature-sensitive mutants at the nonpermissive temperature is under investigation. Lastly a recombinant baculovirus has been constructed which contains the Maguari virus S cDNA in place of the baculovirus polyhedrin gene. In infected insect cells a high level of antigenically authentic N was made and we are attempting to purify the N protein for functional analyses. Other experiments exploiting these systems will be discussed.
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