Hepatitis A virus capsid protein VP1 has a heterogeneous C terminus

Hepatitis A virus (HAV) encodes a single polyprotein which is posttranslati onally processed into the functional structural and nonstructural proteins. Only one protease, viral protease 3C, has been implicated in the nine prot ein scissions. Processing of the capsid protein precursor region generates a unique intermediate, PX (VP1-2A), which accumulates in infected cells and is assumed to serve as precursor to VP1 found in virions, although the det ails of this reaction have not been determined. Coexpression in transfected cells of a variety of P1 precursor proteins with viral protease 3C demonst rated efficient production of PX, as well as VP0 and VP3; however, no matur e VP1 protein was detected. To identify the C-terminal amino acid residue o f HAV VPI, we performed peptide sequence analysis by protease-catalyzed [O- 18]H2O incorporation followed by liquid chromatography ion-trap microspray tandem mass spectrometry of HAV VP1 isolated from purified virions. Two dif ferent cell culture-adapted isolates of HAV, strains HM175pE and HM175p35, were used for these analyses. VP1 preparations from both virus isolates con tained heterogeneous C termini. The predominant C-terminal amino acid in bo th virus preparations was VP1-Ser274, which is located N terminal to a meth ionine residue in VP1-2A. In addition, the analysis of HM175pE recovered sm aller amounts of amino acids VP1-Glu273 and VP1-Thr272. In the case of HM17 5p35, which contains valine at amino acid position VP1-273, VP1-Thr272 was found in addition to VP1-Ser274. The data suggest that HAV 3C is not the pr otease responsible for generation of the VP1 C terminus. We propose the inv olvement of host cell protease(s) in the production of HAV VP1.