The initiation of HIV-1 reverse transcription occurs at an 18-nucleotide sequence in the viral genome designated as the primer binding site (PBS), which is complementary to the 3' terminal
nucleotides of
tRNA(Lys,3). Since the PBS is highly conserved among all infectious HIV-1, it represents an attractive target for the development of new
therapeutics to inhibit viral replication. In this study, we have evaluated three approaches using
small interfering RNA (siRNAs) targeted to the PBS for the capacity to inhibit HIV-1 replication. In the first, transfection of a 21-nucleotide
siRNA complementary to the PBS into cells inhibited production of HIV-1 following
infection. Control siRNAs of the same length complementary to HIV-1 gag
mRNA or to gfp
mRNA decreased the production of virus or had no effect on virus replication, respectively. Analysis of the PBS of integrated proviruses derived from viruses that ultimately grew in cultures transfected with
siRNA all contained wild-type PBS sequence, demonstrating that HIV-1 did not mutate to escape inhibition by
siRNA. In the second approach, hairpin
siRNA targeted to the wild-type PBS were expressed using an adeno-associated virus (AAV) vector. HIV-1 replication was inhibited in cells infected with AAV encoding the
siRNA to the wild-type PBS, but not in cells infected with AAV encoding an
siRNA of the same length targeted to an irrelevant PBS. Finally, studies from this laboratory have shown that alteration of the PBS to be complementary to
tRNAHis results in the production of infectious virus that rapidly reverts to utilize
tRNALys,3 following in vitro culture. A proviral genome containing a PBS complementary to
tRNAHis that encodes an
siRNA molecule complementary to the wild-type PBS under control of a U6 promoter within the nef gene was as infectious as the parent HIV-1 genome containing no insert in nef. The virus with the PBS only complementary to
tRNAHis reverted to use
tRNALys,3, coincident with rapid virus growth, while the virus encoding
siRNA grew slower than the virus without
siRNA and maintained the PBS complementary to
tRNAHis longer in culture. At later times of
infection, viruses with the PBS complementary to
tRNAHis and the
siRNA exhibited a rapid increase in p24
antigen in the culture. Analysis of the PBS revealed that it was now complementary to
tRNALys,3. Analysis of the gene encoding the
siRNA revealed that the reversion of the PBS coincided with the deletion of the gene encoding
siRNA. The results of these studies show that
siRNA targeted to the PBS of HIV-1 can inhibit virus replication, supporting the concept that HIV-1 has evolved a strong preference to select
tRNALys,3 for high-level replication and establishing the PBS and primer selection as a potential target for new
therapeutics.