Hybrid Ty1/HIV-1 elements used to detect inhibitors and monitor the activity of HIV-1 reverse transcriptase

Abstract

We previously demonstrated that hybrid retrotransposons composed of the yeast Ty1 element and the reverse transcriptase (RT) of HIV-1 are active in the yeast Saccharomyces cerevisiae. The RT activity of these hybrid Ty1/HIV-1 (his3AI/AIDS RT; HART) elements can be monitored by using a simple genetic assay. HART element reverse transcription depends on both the polymerase and RNase H domains of HIV-1 RT. Here we demonstrate that the HART assay is sensitive to inhibitors of HIV-1 RT. (-)-(S)-8-Chloro-4,5,6, 7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)imidazo[4,5,1-jk][1, 4]-benzodiazepin-2(1H)-thione monohydrochloride (8 Cl-TIBO), a well characterized non-nucleoside RT inhibitor (NNRTI) of HIV-1 RT, blocks propagation of HART elements. HART elements that express NNRTI-resistant RT variants of HIV-1 are insensitive to 8 Cl-TIBO, demonstrating the specificity of inhibition in this assay. HART elements carrying NNRTI-resistant variants of HIV-1 RT can be used to identify compounds that are active against drug-resistant viruses.

Figure 1

Hybrid Ty1/HIV-1 RT/RH elements. (A) The RT/RH domain of HIV-1 was used to replace Ty1 RT/RH, resulting in HART (his3AI AIDS Reverse Transcriptase) elements. The inducible galactose promoter-driven hybrid HIV/Ty element marked with his3AI, HART, is shown. his3AI serves as an indicator of passage through an RNA intermediate and of reverse transcription. Expression results in full-length element RNA (wavy line) carrying an antisense copy of his3AI at the 3′ end of the transcript. An artificial intron (open rectangele) interrupts the his3 coding region and is flanked by splice donor and splice acceptor sites such that RNA splicing (▿) of the intron can occur only on the antisense transcript (the sense his3AI transcript is interrupted by a “backward” intron). Splicing, followed by reverse transcription, yields a cDNA copy of the hybrid element carrying a functional HIS3 gene. Integrase (IN)-mediated insertion or cDNA (homologous) recombination of this Ty-HIS3 cDNA results in histidine prototrophy that is scored by genetic selection. Hybrid elements carrying wild-type HIV-1 RT (HART) and the NNRTI-resistant variants HART-L100I and HART-Y181C, along with Ty, can all be assayed for RT activity with this genetic selection. (B) The nucleotide and amino acid sequences of the Ty/HIV-1 RT junction and the Ty protease (PR) cleavage site are shown.

Figure 2

Scheme demonstrating that inhibition of HIV-1 RT can be monitored in yeast. To demonstrate that the HART assay is an appropriate means to screen for inhibitors of HIV-1 RT in yeast, the HART assay was carried out with a previously characterized NNRTI (8 Cl-TIBO) and controls for the specificity of inhibition. The rationale for that experiment is shown. Specific inhibitors of HIV-1 RT should not inhibit the activity of Ty elements (Ty-his3AI) because reverse transcription is carried out by Ty RT. HART elements should be inhibited by 8 Cl-TIBO. HIV-1 RT variants (L100I and Y181C) are resistant to 8 Cl-TIBO, and HART elements carrying them should be active.

Figure 3

HIV-1 reverse transcription is inhibited by 8 Cl-TIBO in yeast. Inhibition of HIV-1 RT activity in yeast was tested by using the RT inhibitor 8 Cl-TIBO and controls for the specificity of inhibition. Yeast patches carrying Ty (Ty-his3AI) or hybrid retroelements (HART, HART-L100I, and HART-Y181C) were replica-plated onto galactose plates containing no or various concentrations of 8 Cl-TIBO, to induce expression of the elements. These plates were then replica-plated to media lacking histidine, containing no or various concentrations of 8 Cl-TIBO, to select for RT-mediated events.