Your browser doesn't support javascript.
loading
Structural requirements for initiation of translation by internal ribosome entry within genome-length hepatitis C virus RNA.
Honda, M; Ping, L H; Rijnbrand, R C; Amphlett, E; Clarke, B; Rowlands, D; Lemon, S M.
Afiliação
  • Honda M; Department of Medicine, University of North Carolina at Chapel Hill 27599-7030, USA.
Virology ; 222(1): 31-42, 1996 Aug 01.
Article em En | MEDLINE | ID: mdl-8806485
ABSTRACT
Cap-independent translation of hepatitis C virus (HCV) RNA is mediated by an internal ribosomal entry segment (IRES) located within the 5' nontranslated RNA (5'NTR), but previous studies provide conflicting views of the viral sequences which are required for translation initiation. These discrepancies could have resulted from the inclusion of less than full-length 5'NTR in constructs studied for translation or destabilization of RNA secondary structure due to fusion of the 5'NTR to heterologous reporter sequences. In an effort to resolve this confusion, we constructed a series of mutations within the 5'NTR of a nearly full-length 9.5-kb HCV cDNA clone and examined the impact of these mutations on HCV translation in vitro in rabbit reticulocyte lysates and in transfected Huh-T7 cells. The inclusion of the entire open reading frame in HCV transcripts did not lead to an increase in IRES-directed translation of the capsid and E1 proteins, suggesting that the nonstructural proteins of HCV do not include a translational transactivator. However, in reticulocyte lysates programmed with full-length transcripts, there were multiple aberrent translation initiation sites resembling those identified in some picornaviruses. The deletion of nucleotides (nt) 28-69 of the 5'NTR (stem-loop IIa) sharply reduced capsid translation both in vitro and in vivo. A small deletion mutation involving nt 328-334, immediately upstream of the initiator AUG at nt 342, also resulted in a nearly complete inhibition of translation, as did the deletion of multiple intervening structural elements. An in-frame 12-nt insertion placed within the capsid-coding region 9 nt downstream of the initiator AUG strongly inhibited translation both in vitro and in vivo, while multiple silent mutations within the first 42 nt of the open reading frame also reduced translation in reticulocyte lysates. Thus, domains II and III of the 5'NTR are both essential to activity of the IRES, while conservation of sequence downstream of the initiator AUG is required for optimal IRES-directed translation.
Assuntos
Buscar no Google
Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Iniciação Traducional da Cadeia Peptídica / RNA Viral / Hepacivirus Limite: Animals / Humans Idioma: En Revista: Virology Ano de publicação: 1996 Tipo de documento: Article País de afiliação: Estados Unidos
Buscar no Google
Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Iniciação Traducional da Cadeia Peptídica / RNA Viral / Hepacivirus Limite: Animals / Humans Idioma: En Revista: Virology Ano de publicação: 1996 Tipo de documento: Article País de afiliação: Estados Unidos