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J. Virol., Feb 1996, 966-975, Vol 70, No. 2
JK Wakefield, SM Kang and CD Morrow
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.
Copyright © 1996, American Society for Microbiology
Construction of a type 1 human immunodeficiency virus that maintains a primer binding site complementary to tRNA(His)
Department of Microbiology, University of Alabama at Birmingham 35294, USA.
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