Inhibition of human immunodeficiency virus type 1 replication by siRNA targeted to the highly conserved primer binding site

Abstract

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.

Inhibition of HIV-1 by a Lentiviral Vector with a Novel Tat-Inducible Expression System and a Specific Tropism to the Target Cells

“…[31][32][33][34] In most previous studies, the targets of siRNAs or shRNAs in HIV-1 transcripts are not in fact highly conserved, and only a few studies focused on targeting conserved HIV-1 sequences. 23,[35][36][37][38] As previously reported 39,40 and our results at 15-18 days postinfection show (Fig. 4A), HIV-1 can escape from inhibition by mutations in sequences targeted by a single shRNA.…”

Inhibition of Human Immunodeficiency Virus Type 1 Replication with Artificial Transcription Factors Targeting the Highly Conserved Primer-Binding Site

“…Unfortunately, long-term studies have shown that prolonged exposure to siRNAs results in mutations of the virus that allow it to escape regulation (6,10). Recently, a study showed that an siRNA targeting the PBS was able to inhibit HIV-1 infection (24). Although no mutation in the PBS was seen up to 14 days after exposure to the siRNA, our studies suggest that HIV can mutate the PBS region to escape targeting by RNAi.…”

Induction of Robust Immune Responses against Human Immunodeficiency Virus Is Supported by the Inherent Tropism of Adeno-Associated Virus Type 5 forDendritic Cells

“…In addition, AAV5 was efficiently transduced into T cells and macrophages ( Fig. 3 and 4), which may be useful for gene therapy of diseases involving hematopoietic cells (4,26,43,57,59,69).…”