Specific cleavages by RNase H facilitate initiation of plus-strand RNA synthesis by Moloney murine leukemia virus.
J Virol
; 77(9): 5275-85, 2003 May.
Article
em En
| MEDLINE
| ID: mdl-12692229
ABSTRACT
Successful generation, extension, and removal of the plus-strand primer is integral to reverse transcription. For Moloney murine leukemia virus, primer removal at the RNA/DNA junction leaves the 3' terminus of the plus-strand primer abutting the downstream plus-strand DNA, but this 3' terminus is not efficiently reutilized for another round of extension. The RNase H cleavage to create the plus-strand primer might similarly result in the 3' terminus of this primer abutting downstream RNA, yet efficient initiation must occur to synthesize the plus-strand DNA. We hypothesized that displacement synthesis, RNase H activity, or both must participate to initiate plus-strand DNA synthesis. Using model hybrid substrates and RNase H-deficient reverse transcriptases, we found that displacement synthesis alone did not efficiently extend the plus-strand primer at a nick with downstream RNA. However, specific cleavage sites for RNase H were identified in the sequence immediately following the 3' end of the plus-strand primer. During generation of the plus-strand primer, cleavage at these sites generated a gap. When representative gaps separated the 3' terminus of the plus-strand primer from downstream RNA, primer extension significantly improved. The contribution of RNase H to the initiation of plus-strand DNA synthesis was confirmed by comparing the effects of downstream RNA versus DNA on plus-strand primer extension by wild-type reverse transcriptase. These data suggest a model in which efficient initiation of plus-strand synthesis requires the generation of a gap immediately following the plus-strand primer 3' terminus.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
RNA Viral
/
Ribonuclease H
/
Vírus da Leucemia Murina de Moloney
Limite:
Animals
Idioma:
En
Revista:
J Virol
Ano de publicação:
2003
Tipo de documento:
Article
País de afiliação:
Estados Unidos