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The 2A gene of hepatitis A virus (HAV) bears no obvious similarity to the corresponding genes of other picornaviruses and has no known function. In a preliminary effort to gain information about the HAV 2A gene product, we constructed several HAV cDNAs containing deletions of 30 or 45 nucleotides in the predicted central portion of the 2A gene. These deletions did not affect the sites of protein processing, although the rates or efficiencies of polyprotein cleavage at the surrounding cleavage junctions appeared slightly reduced. Transfection of FRhK-4 cells with RNA transcripts of the deleted HAV cDNAs generated small foci of infected cells and produced infectious virus that retained the deletion mutations. In contrast, a single amino acid insertion in the 2B coding region was lethal to virus replication despite normal protein processing. Another deletion, which included the predicted 2A/2B junction and extended into the 2B coding sequence, did not support polyprotein processing or generate viable virus. One of the viable internal 2A deletions was introduced into a wild-type HAV cDNA background, and transcripts were tested for infectivity by inoculation directly into the livers of two marmosets. Both animals seroconverted, displayed elevated serum liver enzymes, and excreted infectious virus. Thus, deletion of 10 or 15 amino acid residues from the predicted central portion of the 2A protein was tolerated with only relatively minor effects on the growth of HAV in cultured cells and in marmoset liver.

Hepatitis A viruses with deletions in the 2A gene are infectious in cultured cells and marmosets