Inhibition of endogenous reverse transcription of human and nonhuman primate lentiviruses: potential for development of lentivirucides

Abstract

In the current study, we extended our previous works on natural endogenous reverse transcription (NERT) and further examined its potential as a virucide molecular target in sexual transmission of primate lentiviruses. HIV-1 and SIV virions were pretreated with select nucleoside (NRTIs) and nonnucleoside RT inhibitors (NNRTIs), either alone or in combination with NERT-stimulating substances. The effects of these antiretrovirals on virion inactivation were analyzed in human T cell lines and primary cell cultures. Pretreatment of HIV-1 virions with physiologic NERT-stimulants and 3'-azido-3'-deoxythymidine 5'-triphosphate (AZT-TP) or nevirapine potently inactivated cell-free HIV-1 virions and resulted in strong inhibition of the viral infectivity. Pretreatment of chimeric SHIV-RT virions with NERT-stimulating cocktail and select antiretrovirals also resulted in virion inactivation and inhibition of viral infectivity in T cell lines. Our findings demonstrate the potential clinical utility of approaches based on inhibiting NERT in sexual transmission of HIV-1, through the development of effective anti-HIV-1 microbicides, such as NRTIs and NNRTIs.

Fig. 1

Inhibitory effects of select antiretroviral agents on NERT. Intravirion HIV-1 reverse transcripts were assayed by DNA-PCR. Virions were pretreated with nevirapine, AZT-TP and/or AZT at 4 μM, with or without NERT-stimulating cocktail. After 4 h, the DNA was extracted and subjected to semi-quantitative PCR and Southern blotting analysis. The figure is representative of two independent studies.

Fig. 2

Effects of select antiretroviral agents on NERT and HIV-1 infectivity in human primary cells. (a) Infectivity of HIV-1 virions pretreated with select antiretroviral agents. One nanogram of HIV-1_NL4-3 p24 antigen was serially diluted from pretreated virion preparations and used to infect initially quiescent PBLs. Viral infectious titer was assessed via HIV-1 p24 antigen expression (ELISA) at day 28 post-infection. The figure is representative of at least two independent studies. (b) Infectivity of HIV-1 virions pretreated with select antiretroviral agents with or without the NERT-stimulating cocktail. Ten ng of HIV-1_NL4-3 p24 antigen equivalents from virion preparations, pretreated (or nontreated) with various concentrations of select antiretroviral agents in the presence or absence of the NERT-stimulating cocktail, were used to infect human PBL cultures. Viral production was assessed via HIV-1 p24 antigen expression (ELISA) by day 12 post-infection. The figure is representative of two independent studies. NL4-3: cells infected with untreated viral preparation only; Nev: nevirapine; NERT: NERT-stimulating cocktail (100 nM dNTPs, 3 mM spermine and 0.1 mM spermidine). (c) Ten nanograms of HIV-1_ADA p24 antigen equivalents from virion preparations, pretreated (or nontreated) with various concentrations of select antiretroviral agents in the presence or absence of the NERT-stimulating cocktail, were used to infect human monocyte/macrophage cultures. Viral production was assessed via HIV-1 p24 antigen expression (ELISA) by day 5 post-infection. The figure is representative of two independent studies. ADA: cells infected with untreated HIV-1 only; Nev: nevirapine, NERT: NERT-stimulating cocktail (100 nM dNTPs, 3 mM spermine and 0.1 mM spermidine).

Fig. 3

Time-course analysis of the effects of a NERT-stimulating cocktail on HIV-1 stability and NERT-altering agents on HIV-1 infectivity at different temperature conditions. (a) HIV-1_NL4-3 virion preparations (1 ng of HIV-1 p24 antigen equivalents) were either pretreated with the NERT-stimulating cocktail, or left untreated. Subsequently, the virion preparations were incubated in RPMI-1640 medium at 37 °C for different time periods. Infectivity was assessed on initially quiescent PBLs, via HIV-1 p24 antigen expression on day 10 post-infection. The 0 h represents prompt re-isolation of virions after mixing with media containing, or not containing the NERT-stimulating cocktail. (b) HIV-1_NL4-3 virion preparations (4 ng of HIV-1 p24 antigen equivalents) were pretreated with 4 μM of AZT-TP in the presence or absence of the NERT-stimulating cocktail (100 nM dNTPs, 3 mM spermine and 0.1 mM spermidine) for 15 h at different temperature conditions (37 °C and 4 °C). After re-isolation by ultracentrifugation, the virion preparations were used to infect initially quiescent human PBLs. Viral production was assessed via HIV-1 p24 antigen expression (ELISA) by day 10 post-infection.