INSIGHTS INTO THE INTERACTION BETWEEN TRANSFER-RNA AND PRIMER BINDING-SITE FROM CHARACTERIZATION OF A UNIQUE HIV-1 VIRUS WHICH STABLY MAINTAINS DUAL PBS COMPLEMENTARY TO TRNA(GLY) AND TRNA(HIS)
Y. Li et al., INSIGHTS INTO THE INTERACTION BETWEEN TRANSFER-RNA AND PRIMER BINDING-SITE FROM CHARACTERIZATION OF A UNIQUE HIV-1 VIRUS WHICH STABLY MAINTAINS DUAL PBS COMPLEMENTARY TO TRNA(GLY) AND TRNA(HIS), Virology, 238(2), 1997, pp. 273-282
Previously our laboratory constructed an HIV-I which stably maintained
a primer binding site (PBS) complementary to tRNA(His) by mutating th
e region of the provirus within U5 postulated to interact with the ant
icodon 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 lo
ng-term culture, we identified an unusual proviral DNA in which the U5
-PBS region contained a dual PBS complementary to tRNA(Gly) and tRNA(H
is), 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 res
ultant proviral DNA was designated as pHXB2(Gly-His). Transfection of
pHXB2(Gly-His) into cells gave rise to infectious virus. Analysis of t
he U5-PBS region revealed that the virus stably maintained the dual PB
S rather than revert back to the wild-type Pas. In addition to genomes
with the PBS complementary to tRNA(Gly) and tRNA(His), proviral genom
es were identified after extended in vitro culture which contained dua
l PBS complementary to tRNA(Gly) and tRNA(Phe). To determine which PBS
could be used for reverse transcription, we utilized an endogenous re
verse transcription/PCR method which could discriminate (based on mole
cular size of the products) between the minus strand DNA initiated fro
m the two Pass. The results of this assay demonstrated that either the
PBs complementary to tRNA(Gly) Or tRNA(His) could be used for the ini
tiation of reverse transcription. The results of our study highlight t
he complex interrelationship between U5-PBS and primer tRNA required f
or positioning the tRNA at the PBS and provides new insights into how
the tRNA primer used to initiate reverse transcription is selected. (C
) 1997 Academic Press.