THE PROPOSED GENE FOR VP1 OF HAV ENCODES FOR A LARGER PROTEIN THAN THAT OBSERVED IN HAV-INFECTED CELLS AND VIRIONS

The termini of hepatitis A virus (HAV) mature proteins have been assig ned mainly by their homology to other picomaviruses and their apparent electrophoretic mobility; the proposed coding sequence for VP1 is sup posed to encompass 900 nucleotides from position 2208 to 3107 of the H AV genome. In order to further characterize this protein, we analyzed the in vitro- and in vivo-synthesized translation products of the puta tive VP1 gene, cDNA coding for full-length VP1 was cloned under the co ntrol of a T7 promoter in pTF7-5; the resulting plasmid (pTF7-5/VP1) w as used for both synthesis of RNA to program rabbit reticulocyte lysat es and construction of a recombinant vaccinia virus (rvv/T7-VP1). Immu noblot analysis and immunoprecipitation using antisera raised against a synthetic peptide corresponding to amino acids 13 to 33 of VP1 (13-3 3/VP1) led to identification of a 37-kDa protein in lysates of in vitr o translated VP1 and rvv/T7-VP1-infected HFS cells, whereas a 33-kDa p rotein was detected with purified virions and in lysates of HAV-infect ed HFS cells. Because the antiserum used was directed against an amino -terminal part of VP1 and the amino terminus of VP1 is identified by s equence analysis, these. results show that VP1 present in the HAV viri ons 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 th e predicted region, we investigated in vitro synthesized translation p roducts of a set of constructs with C-termini ending at potential clea vage sites for viral proteases 3C. The construct containing the nucleo tides 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. (C) 1995 Academic Press, Inc.