Characterization of recombinant hepatitis A virus genomes containing exogenous sequences at the 2A/2B junction.
Hepatitis A virus (HAV) differs from other members of the family Picornaviridae in that the cleavage of the polyprotein at the 2A/2B junction, commonly considered to be the primary polyprotein cleavage by analogy with other picornaviruses, is mediated by 3C(pro), the only proteinase encoded by the virus. However, it has never been formally demonstrated that the 2A/2B junction is the site of primary cleavage, and the actual function of the 2A sequence, which lacks homology with sequence of other picornaviruses, remains unknown. To determine whether 2A functions in cis as a precursor with the nonstructural proteins, we constructed dicistronic HAV genomes in which a heterologous picornaviral internal ribosome entry site was inserted at the 2A/2B junction. Transfection of permissive FRhK-4 cells with these dicistronic RNAs failed to result in the rescue of infectious virus, indicating a possible cis replication function spanning the 2A/2B junction. However, infectious virus was recovered from recombinant HAV genomes containing exogenous protein-coding sequences inserted in-frame at the 2A/2B junction and flanked by consensus 3C(pro) cleavage sites. The replication of these recombinants was less efficient than that of the parent virus but was variable and not dependent upon the length of the inserted sequence. An HAV recombinant containing a 420-nt insertion encoding the bleomycin resistance protein Zeo was stable for up to five passages in cell culture. Inserted sequences were deleted from replicating viruses, but this did not result from homologous recombination at the flanking 3C(pro) cleavage sites, since the 5' and 3' segments of the inserted sequence were retained in the deletion mutants. These results indicate that the HAV polyprotein can tolerate an insertion at the 2A/2B junction and that the 2A polypeptide does not function in cis as a 2AB precursor. Recombinant HAV genomes containing foreign protein-coding sequences inserted at the 2A/2B junction are novel and potentially useful protein expression vectors.
Beard
M R
MR
Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1019, USA. mrbeard@utmb.edu
Cohen
L
L
Lemon
S M
SM
Martin
A
A
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004251:DNA Transposable Elements; D016679:Genome, Viral; D006507:Hepatovirus / Q000235:genetics*; D008253:Macaca mulatta; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014764:Viral Proteins / Q000378:metabolism
D004251:DNA Transposable Elements; D014764:Viral Proteins