Wakefield JK, Kang SM, Morrow CD; Conference on Retroviruses and Opportunistic Infections.
Program Abstr 3rd Conf Retrovir Oppor Infect Conf Retrovir Oppor Infect 3rd 1996 Wash D C. 1996 Jan 28-Feb 1; 3rd: 94.
University of Alabama at Birmingham, Birmingham, AL.
The initiation of HIV-1 reverse transcription occurs by extension of a tRNA(Lys,3) primer bound near the 5' of the viral RNA genome (the primer binding site (PBS)). Substitution of the PBS with sequences complementary to the 3'-terminal nucleotides of alternate tRNAs (e.g.tRNA(His)) results in viruses which use these tRNAs to initiate reverse transcription. After culture, each virus with the mutant PBS reverted back to a wild-type PBS complementary to the tRNA(Lys,3). Sequences within the viral genome upstream of the PBS which are complementary to the anticodon loop (USUU) and the T psi C loop/arm (AGGGT(mPsi)) of tRNA(Lys,3) are postulated to play a role in maintaining the selective use of tRNA(Lys,3) in reverse transcription. To investigate this possibility, proviral genomes were constructed which contain a PBS complementary to the 3'-terminal 18 nucleotides of tRNA (His) [pHXB2(His)]as well as sequences upstream of this PBS which are complementary to either the anticodon loop of tRNA (His) [(CCACAA); pHXB2(His-AC)] or T psi C loop [(GACCGAGG); pHXB2(His-T psi C)]. The appearance of infectious virus after recultivation with SupT1 cells was delayed for the proviruses containing a PBS complementary to tRNA(His) as compared to that obtained by transection of the wild-type provirus (pHXB2(WT)); by several passages in SupT1 cells, the mutant viruses demonstrated similar replication kinetics as the wild-type virus. PCR was used to amplify the PBS region from integrated proviruses and a DNA sequence analysis of the individual DNAs revealed that by day 15 of culture in SupT1 cells, the PBS of viruses derived from pHXB2(His) and pHXB2(His-T psi C) reverted back to that of the wild-type PBS complementary to tRNA(Lys,3). In contrast, viruses derived from pHXB2(His-AC) maintained a PBS complementary to tRNA(His) (over four months in culture encompassing 12 serial passages). This study is the first report of a stable HIV-1 which utilizes an alternative tRNA primer and suggests that interactions between the primer tRNA anticodon loop and viral sequences upstream of the PBS contribute to the specificity of the tRNA primer used in reverse transcription.
Publication Types:
Keywords:
- Anticodon
- Base Sequence
- Binding Sites
- DNA Primers
- Genome, Viral
- HIV-1
- Humans
- Polymerase Chain Reaction
- Proviruses
- RNA
- RNA, Transfer
- RNA, Viral
- RNA-Directed DNA Polymerase
- Reverse Transcription
- Transcription, Genetic
- Virion
- genetics
- reverse transcriptase, Human immunodeficiency virus 1
Other ID:
UI: 102216272
From Meeting Abstracts