Interference with HIV-1 primer selection by siRNA directed to the HIV-1 primer binding site

The replication of Human immunodeficiency virus type 1 (HIV-1) is characterized by the process of reverse transcription, which converts the viral RNA genome into a DNA intermediate prior to integration into the host cell chromosome. The initiation of reverse transcription needs a cellular tRNA primer, which binds to a region in the viral RNA genome, designated as the primer binding site (PBS). The 18-nucleotide PBS region of HIV-1, which is complementary to the 3' terminal 18 nucleotides of tRNA^Lys,3, is absolutely conserved among all HIV-1. The highly conserved PBS is a very good target for HIV-1 therapy. My dissertation research focuses on the interference with HIV-1 primer selection by small interference RNA (siRNA) directed to the PBS. Experiments were carried out to investigate the effect of siRNA duplexes directed to HIV-1 PBS on HIV-1 primer selection and replication.

Transfection of in vitro transcribed 21-nucleotide siRNA complementary to HIV-1 PBS inhibited HIV-1 replication. A small hairpin RNA (shRNA) complementary to the HIV-1 PBS expressed from adeno-associated virus (AAV) vector also inhibited HIV-1 replication. To investigate the mechanism of inhibition of HIV-1 replication, we determined that siRNA targeted to HIV-1 PBS did not induce degradation of mRNA containing the 5' untranslated region of HIV-1 that contains a PBS. The results of our studies suggest that siRNA targeted to the HIV-1 PBS inhibits HIV-1 replication by interference with HIV-1 primer selection rather than degradation of HIV-1 genomic RNA. Further, we investigated the capacity of siRNA directed to the HIV-1 PBS to interfere with HIV-1 primer selection. We found that siRNA directed to the HIV-1 PBS can maintain the NL4-His virus, a mutant virus that temporarily uses tRNA ^His as a primer for reverse transcription, to use tRNA^His as a primer for 2∼4 more weeks. However, this mutant virus finally reverted back to wild type virus by deleting the siRNA gene. Collectively, the results of our studies support that siRNA directed to the PBS can interfere with HIV-1 selection of tRNA^Lys,3 as a primer and underscores the evolution of HIV-1 to preferentially select tRNA^Lys,3 for replication.