Construction of a type 1 human immunodeficiency virus that maintains a primer binding site complementary to tRNA(His).
The initiation of human immunodeficiency virus type 1 reverse transcription occurs by extension of a tRNA(Lys3) primer bound near the 5' end of the viral RNA genome which is designated the primer binding site (PBS). Sequences within the viral genome upstream of the PBS which are complementary to the anticodon loop (USUU) and the T psi C loop and arm (AGGGTm psi) of tRNA(Lys3) are postulated to play a role in maintaining the selective use of tRNA(Lys3) in reverse transcription. To investigate this possibility, proviral genomes 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 [CCACAA; pHXB2(His-AC)] or T psi C loop [GACCGAGG; pHXB2(His-T psi C)] of tRNA(His) were constructed. Infectious virus was recovered upon transfection into COS-1 cells of pHXB2(His), pHXB2(His-AC), or pHXB2(His-T psi C). The appearance of infectious virus after cocultivation with SupT1 cells was delayed for the proviruses containing a PBS complementary to tRNA(His) compared with that obtained by transfection of the wild-type provirus [pHXB2(WT)]. However, by several passages in SupT1 cells, the mutant viruses demonstrated replication kinetics similar to those of the wild-type virus. A DNA sequence analysis of the PBS region from integrated proviruses revealed that by day 15 of culture, the PBS of viruses derived from pHXB2(His) and pHXB2(His-T psi C) reverted back to the wild-type PBS complementary to tRNA(Lys3). In contrast, viruses derived from pHXB2(His-AC) maintained a PBS complementary to tRNA(His) for over 4 months in culture encompassing 12 serial passages. This study, then, is the first report of a stable human immunodeficiency virus type 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.
Wakefield
J K
JK
Department of Microbiology, University of Alabama at Birmingham 35294, USA.
Kang
S M
SM
Morrow
C D
CD
D000818:Animals; D000926:Anticodon / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D016679:Genome, Viral; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011533:Proviruses / Q000235:genetics; D012354:RNA, Transfer, His / Q000378:metabolism*; D012357:RNA, Transfer, Lys / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism; D014779:Virus Replication
D000926:Anticodon; D017931:DNA Primers; D004279:DNA, Viral; D012354:RNA, Transfer, His; D012357:RNA, Transfer, Lys; D012367:RNA, Viral; D014764:Viral Proteins