HEPATITIS-A VIRUSES WITH DELETIONS IN THE 2A GENE ARE INFECTIOUS IN CULTURED-CELLS AND MARMOSETS

The 2A gene of hepatitis A virus (HAV) bears no obvious similarity to the corresponding genes of other picornaviruses and has no known funct ion. 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. The se deletions did not affect the sites of protein processing, although the rates or efficiencies of polyprotein cleavage at the surrounding c leavage junctions appeared slightly reduced. Transfection of FRhK-4 ce lls with RNA transcripts of the deleted HAV cDNAs generated small foci of infected cells and produced infectious virus that retained the del etion mutations. In contrast, a single amino acid insertion in the 2B coding region was lethal to virus replication despite normal protein p rocessing. Another deletion, which included the predicted 2A/2B juncti on and extended into the 2B coding sequence, did not support polyprote in processing or generate viable virus. One of the viable internal 2A deletions was introduced into a wild-type HAV cDNA background, and tra nscripts were tested for infectivity by inoculation directly into the livers of two marmosets. Both animals seroconverted, displayed elevate d serum liver enzymes, and excreted infectious virus. Thus, deletion o f 10 or 15 amino acid residues from the predicted central portion of t he 2A protein was tolerated with only relatively minor effects on the growth of HAV in cultured cells and in marmoset liver.