Identification and site-directed mutagenesis of the primary (2A/2B) cleavage site of the hepatitis A virus polyprotein: functional impact on the infectivity of HAV RNA transcripts.
The junction between 2A and 2B proteins of the hepatitis A virus (HAV) polyprotein is processed by the virus-encoded 3C protease to liberate the precursor for capsid proteins, but details of this cleavage remain poorly defined. We identified the location of this primary cleavage by a novel approach involving expression of HAV polypeptides in eukaryotic cells via recombinant vaccinia viruses. A substrate polyprotein spanning the putative HAV 2A/2B site was fused at its C-terminus to a poliovirus VP1 reporter sequence. This substrate was cleaved efficiently in trans by protease 3C derived from another recombinant vaccinia virus expressing a 3C precursor protein. N-terminal sequencing of the 2B-poliovirus VP1 fusion product identified the site of cleavage as the Gln836/Ala837 dipeptide, 144 residues upstream of the originally predicted site. Two mutations were introduced at the P1 position of the 2A/2B site. Gln836-->Asn, and Gln836-->Arg. Asn substitution at the P1 residue reduced the efficiency of cleavage in the vaccinia expression system and resulted in a small replication focus phenotype of virus rescued from infectious HAV RNA transcripts. Arg substitution abolished cleavage and was lethal to HAV replication. In addition to identifying the site of the primary HAV polyprotein cleavage, these results shed light on the in vivo specificities of the HAV 3C protease.
D000086042:3C Viral Proteases; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002213:Capsid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D004279:DNA, Viral / Q000235:genetics; D006367:HeLa Cells / Q000821:virology; D006507:Hepatovirus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D007668:Kidney / Q000821:virology; D008251:Macaca; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000032:analysis; D012367:RNA, Viral / Q000235:genetics*; D014158:Transcription, Genetic; D014616:Vaccinia virus / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins; D014779:Virus Replication
D017931:DNA Primers; D004279:DNA, Viral; D011498:Protein Precursors; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; D012313:RNA; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases