Identification, sequence, and transcriptional mapping of the major capsid protein gene of the baculovirus Autographa californica nuclear polyhedrosis virus.

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RESUMO

The gene encoding the major capsid protein of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) was identified, sequenced, and transcriptionally mapped. The location of the gene was determined by immunological screening of an expression library of AcMNPV open reading frame-beta-galactosidase fusions with an antibody raised to virus structural proteins. The DNA sequence of the corresponding region, which mapped within 56.6 and 58.0 map units on the AcMNPV genome, revealed a 1,040-base-pair open reading frame capable of encoding a 39-kilodalton polypeptide. The identity of the polypeptide was determined by Western blot (immunoblot) analysis of purified empty capsids with an antibody raised to the capsid-beta-galactosidase fusion protein. The identity of the peptide encoded by the gene was confirmed by immunoprecipitation of an in vitro translation product with RNA selected by hybridization to DNA sequences from the coding region of the gene. Transcripts of the capsid gene were analyzed by Northern (RNA) blots and mapped by nuclease protection and primer extension analysis. The capsid gene is transcribed maximally at 12 and 24 h postinfection but not in the presence of cycloheximide, a protein synthesis inhibitor, or aphidicolin, a viral DNA synthesis inhibitor, and is therefore classified as a late gene. The gene is transcribed in a counterclockwise direction with respect to the circular map. There are three transcriptional start sites, all containing the AGTAAG consensus sequence found at the start site of all late AcMNPV genes.

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