Cell-surface expression of influenza virus haemagglutinin in insect cells using a baculovirus vector.
A baculovirus transfer vector was constructed using a cloned copy of the Autographa californica nuclear polyhedrosis virus (AcNPV) polyhedrin gene. The ATG translation initiation codon and 32 bases of the polyhedrin mRNA 5' leader sequences were deleted and replaced with a cDNA clone of the A/PR/8/34 influenza virus haemagglutinin (HA) gene. The hybrid HA/polyhedrin gene was transferred to AcNPV and polyhedrin-negative recombinants identified which expressed HA activity. The insect cell-derived HA bound a monoclonal antibody specific for the HA gene in immunofluorescence tests and was shown to be plasma membrane-bound by haemadsorption assays. A 65 kDa protein was identified in infected cells by immunoprecipitation. The mRNA from recombinant-infected cells was analysed and revealed a major transcript of 2.95 kilobases consistent with transcription initiation and termination at the authentic polyhedrin sites. Other transcripts of different length were also detected so it was not possible to conclude which was responsible for producing the protein. The results demonstrated that AcNPV can be used to express plasma membrane-bound virus glycoproteins.
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005786:Gene Expression Regulation; D005814:Genes, Viral; D005822:Genetic Vectors; D006389:Hemagglutinins, Viral / Q000235:genetics*; D009980:Influenza A virus / Q000235:genetics*; D007304:Insect Viruses / Q000235:genetics*; D007915:Lepidoptera; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000096:biosynthesis / Q000378:metabolism
D006389:Hemagglutinins, Viral; D012333:RNA, Messenger; D012367:RNA, Viral; D014764:Viral Proteins