Xg. Li et al., EFFECTS OF ALTERATIONS OF PRIMER-BINDING SITE SEQUENCES ON HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REPLICATION, Journal of virology, 68(10), 1994, pp. 6198-6206
The human immunodeficiency virus type 1 genomic RNA primer-binding sit
e (PBS) sequence comprises 18 nucleotides which are complementary to t
hose at the 3' end of the replication initiation primer tRNA(3)(Lys).
To investigate the role of the PBS in viral replication, we either del
eted the original wild-type PBS (complementary to tRNA(3)(Ly3)) or rep
laced it with DNA sequences complementary to either tRNA(1,2)(Lys) or
tRNA(Phe). Transfection of COS cells with such molecular constructs yi
elded similar levels of viral progeny that were indistinguishable with
regard to viral proteins and tRNA content. Virus particles derived fr
om PBS-deleted molecular clones were noninfectious for MT-4, Jurkat, a
nd CEM-T4 cells. However, infectious viruses were derived from constru
cts in which the PBS had been altered to sequences complementary to ei
ther tRNA(1,2)(Lys) or tRNA(Phe), although mutated forms showed signif
icant lags in replication efficiency in comparison with wild types. Mo
lecular analysis of reverse-transcribed DNA in cells infected by the m
utated viruses indicated that both tRNA(1,2)(Lys) and tRNA(Phe) could
function as primers for reverse transcription during the early stages
of infection. Sequencing of full-length proviral DNA, obtained 6 days
after infection, revealed the mutated PBS, indicating that a complete
cycle of reverse transcription had occurred. During subsequent rounds
of infection, reversion of the mutated PBS to wild-type sequences was
observed, accompanied by increased production of viral gene products.
Reversion to wild-type PBS sequences was confirmed both by specific PC
R analysis, using distinct primer pairs, and by direct sequencing of a
mplified segments. We also performed endogenous in vitro reverse trans
cription experiments in which synthesis of minus-strand strong-stop vi
ral DNA was primed from a synthetic RNA template containing a PBS comp
lementary to various tRNA isoacceptors. These results showed that tRNA
(3)(Lys) was a much more efficient primer of such reactions than eithe
r tRNA(1,2)(Lys) or tRNA(Phe).