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Insights into the interaction between tRNA and primer binding site from characterization of a unique HIV-1 virus which stably maintains dual PBS complementary to tRNA(Gly) and tRNA(His).

Abstract：

:Previously our laboratory constructed an HIV-1 which stably maintained a primer binding site (PBS) complementary to tRNA(His) by mutating the region of the provirus within U5 postulated to interact with the anticodon of tRNA(His) (J. Wakefield, S-M Kang, and C. D. Morrow, 1996, J. Virol, 70, 966-975). From the analysis of the virus obtained after long-term culture, we identified an unusual proviral DNA in which the U5-PBS region contained a dual PBS complementary to tRNA(Gly) and tRNA(His), respectively, separated by a 21-nucleotide intervening sequence. To determine if this U5-PBS region containing the dual PBS would give rise to an infectious virus, the mutant U5-PBS containing the dual PBS was subcloned into an infectious HIV-1 proviral clone, pHXB2; the resultant proviral DNA was designated as pHXB2(Gly-His). Transfection of pHXB2(Gly-His) into cells gave rise to infectious virus. Analysis of the U5-PBS region revealed that the virus stably maintained the dual PBS rather than revert back to the wild-type PBS. In addition to genomes with the PBS complementary to tRNA(Gly) and tRNA(His), proviral genomes were identified after extended in vitro culture which contained dual PBS complementary to tRNA(Gly) and tRNA(Phe). To determine which PBS could be used for reverse transcription, we utilized an endogenous reverse transcription/PCR method which could discriminate (based on molecular size of the products) between the minus strand DNA initiated from the two PBSs. The results of this assay demonstrated that either the PBS complementary to tRNA(Gly) or tRNA(His) could be used for the initiation of reverse transcription. The results of our study highlight the complex interrelationship between U5-PBS and primer tRNA required for positioning the tRNA at the PBS and provides new insights into how the tRNA primer used to initiate reverse transcription is selected.

journal_name

Virology

journal_title

Virology

authors

Li Y,Zhang Z,Kang SM,Buescher JL,Morrow CD

doi

10.1006/viro.1997.8837

subject

Has Abstract

pub_date

1997-11-24 00:00:00

pages

273-82

issue

eissn

0042-6822

issn

1096-0341

pii

S0042-6822(97)98837-1

journal_volume

238

pub_type

杂志文章

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Insights into the interaction between tRNA and primer binding site from characterization of a unique HIV-1 virus which stably maintains dual PBS complementary to tRNA(Gly) and tRNA(His).