Detection of a genome-linked protein (VPg) of hepatitis A virus and its comparison with other picornaviral VPgs.

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RESUMO

The nucleotide sequence corresponding to the P3 region of the hepatitis A virus (HAV) polyprotein genome was determined from cloned cDNA and translated into an amino acid sequence. Comparison of the amino acid sequences of the genome-linked proteins (VPgs) of other picornaviruses with the predicted amino acid sequence of HAV was used to locate the primary structure of a putative VPg within the genome of HAV. The sequence of HAV VPg, like those of other picornaviral VPg molecules, contains a tyrosine residue as a potential binding site for HAV RNA in position 3 from its N terminus. The potential cleavage sites to generate VPg from a putative HAV polyprotein are between glutamic acid and glycine at the N terminus and glutamic acid and serine or glutamine and serine at the C terminus. A synthetic peptide corresponding to 10 amino acids of the predicted C terminus of HAV VPg induced anti-peptide antibodies in rabbits when it was conjugated to thyroglobulin as a carrier. These antibodies were specific for the peptide and precipitated VPg, linked to HAV RNA, from purified HAV and from lysates of HAV-infected cells. The precipitation reaction was blocked by the synthetic peptide (free in solution or coupled to carrier proteins) and prevented by pretreatment of VPg RNA with protease. Thus, our predicted amino acid sequence is colinear with the nucleotide sequence of the VPg gene in the HAV genome. From our results we concluded that HAV has the typical organization of picornavirus genes in this part of its genome. Similarity among hydrophobicity patterns of amino acid sequences of different picornaviral VPgs was revealed in hydropathy plots. Thus, the VPg of HAV appears to be closely related to VPg1 and VPg2 of foot-and-mouth disease virus. In contrast, HAV VPg has a unique isoelectric point (pI = 7.15) among the picornavirus VPgs.

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