title: The proposed gene for VP1 of HAV encodes for a larger protein than that observed in HAV-infected cells and virions.
abstract: The termini of hepatitis A virus (HAV) mature proteins have been assigned mainly by their homology to other picornaviruses and their apparent electrophoretic mobility; the proposed coding sequence for VP1 is supposed to encompass 900 nucleotides from position 2208 to 3107 of the HAV genome. In order to further characterize this protein, we analyzed the in vitro-and in vivo-synthesized translation products of the putative VP1 gene. cDNA coding for full-length VP1 was cloned under the control of a T7 promoter in pTF7-5; the resulting plasmid (pTF7-5/VP1) was used for both synthesis of RNA to program rabbit reticulocyte lysates and construction of a recombinant vaccinia virus (rvv/T7-VP1). Immunoblot analysis and immunoprecipitation using antisera raised against a synthetic peptide corresponding to amino acids 13 of 33 of VP1 (13-33/VP1) led to identification of a 37-kDa protein in lysates of in vitro translated VP1 and rvv/T7-VP1-infected HFS cells, whereas a 33-kDa protein was detected with purified virions and in lysates of HAV-infected HFS cells. Because the antiserum used was directed against an amino-terminal part of VP1 and the amino terminus of VP1 is identified by sequence analysis, these results show that VP1 present in the HAV virions and infected cells is shorter than previously proposed and suggest that the real carboxy terminus of VP1 is approximately 40 amino acids upstream. In order to limit the possible carboxy-terminal sites in the predicted region, we investigated in vitro synthesized translation products of a set of constructs with C-termini ending at potential cleavage sites for viral proteases 3C. The construct containing the nucleotides from position 2208 to 3026 codes for a protein (1-273/VP1) which exhibits the same electrophoretic mobility as VP1 synthesized by HAV in vivo.
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics; D005814:Genes, Viral; D006507:Hepatovirus / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D015678:Viral Structural Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014771:Virion / Q000235:genetics / Q000378:metabolism
chemical_list: D000596:Amino Acids; D017931:DNA Primers; D004279:DNA, Viral; C096539:VP1 protein, hepatitis A virus; D015678:Viral Structural Proteins