Inhibition of cap-dependent gene expression induced by protein 2A of hepatitis A virus.
The viral protein 2A of hepatitis A virus (HAV) lacks the conserved 18 aa sequence found in other picornavirus proteases; hence, it is unclear whether the induction of CPE by culture-adapted HAV strains is due to 2A-mediated activity. Moreover, the cleavage sites and actual borders of HAV 2A are not known. Accordingly, a nested series of cDNA sequences encoding the segment of the HAV polyprotein (aa 760-1087) were linked to the 5'-UTR of poliovirus type 2 (Lansing strain) and inserted downstream of the gene encoding human growth hormone (GH). Following transfection of COS-1 cells, levels of GH (translation of which was entirely cap dependent) were determined in culture supernatants. Expression of HAV peptides extending from aa 764, 776 or 791 to 981 strongly inhibited cap-dependent translation of GH, whereas cap-independent expression of a reporter gene (CAT) directed by the poliovirus RNA 5'-UTR was unaffected. The inhibitory effect was absent in constructs expressing either the short peptide encompassing aa 760-836 or proteins initiated downstream of the putative cleavage site 836-837, suggesting that the boundaries of a functional HAV 2A may extend from the Gln/Ser junction 791-792 to residue 981, while peptides initiated at the Gln/Ala pair 836-837 may result from alternative cleavage. Point mutations that substituted members of the triad Ser(916), His(927) and Asp(931) abolished the inhibitory effect on cap-dependent translation, suggesting that the HAV-induced CPE may be mediated by 2A protein.
Maltese
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E
Department of Cellular & Developmental Biology, University of Rome 'La Sapienza', Viale di Porta Tiburtina 28, 00185 Rome, Italy.
Bucci
M
M
Macchia
S
S
Latorre
P
P
Pagnotti
P
P
Pierangeli
A
A
Bercoff
R P
RP
D020121:5' Untranslated Regions; D000818:Animals; D019556:COS Cells; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D003588:Cytopathogenic Effect, Viral; D015870:Gene Expression; D006507:Hepatovirus / Q000235:genetics / Q000378:metabolism*; D019382:Human Growth Hormone / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012315:RNA Caps; D012333:RNA, Messenger / Q000378:metabolism; D014162:Transfection; D014764:Viral Proteins
D020121:5' Untranslated Regions; D012315:RNA Caps; D012333:RNA, Messenger; D014764:Viral Proteins; D019382:Human Growth Hormone; D003546:Cysteine Endopeptidases; C057400:picornain 2A, Picornavirus