A tight-binding mode of inhibition is essential for anti-human immunodeficiency virus type 1 virucidal activity of nonnucleoside reverse transcriptase inhibitors

Abstract

It was previously found that certain nonnucleoside reverse transcriptase inhibitors (NNRTI) possess virucidal activity against human immunodeficiency virus type 1 (HIV-1), and it was suggested that the tight-binding mode of inhibition of reverse transcriptase might be important for this virucidal activity (Borkow et al., J. Virol. 71:3023-3030, 1997). To test this, we compared six different NNRTI, including three tight-binding NNRTI, namely UC781, efavirenz (EFV) (Sustiva), and 5-chloro-3-phenylsulfonylindole-2-carboxamide (CSIC), and three rapid-equilibrium NNRTI, delavirdine (DLV) (Rescriptor), nevirapine (NVP) (Viramune), and UC84, in a variety of virucidal tests. Incubation of isolated HIV-1 virions with UC781, EFV, or CSIC rapidly inactivated the virus, whereas DLV, NVP, and UC84 were ineffective in this respect. Exposure of H9+ cells chronically infected by HIV-1 to the tight-binding NNRTI abolished the infectivity of nascent virus subsequently produced by these cells following removal of extracellular drug, thereby preventing cell-to-cell virus transmission in the absence of exogenous drug. In contrast, cell-to-cell transmission of HIV was blocked by DLV, NVP, and UC84 only when the drug remained in the extracellular medium. Pretreatment of uninfected lymphocytoid cells with UC781, EFV, or CSIC, but not DLV, NVP, or UC84, protected these cells from subsequent HIV-1 infection in the absence of extracellular drug. The protective effect was dependent on both the dose of NNRTI and the viral load. The overall virucidal efficacy of the tight-binding NNRTI tested was CSIC > UC781 approximately EFV. We conclude that the tight-binding mode of inhibition is an essential characteristic for virucidal NNRTI and that antiviral potency is an insufficient predictor for virucidal utility of NNRTI.

FIG. 1.

Structures of the NNRTI used in the present studies: CSIC (1); DLV (2); EFV (3); NVP (4); UC781 (5); UC84 (6).

FIG. 2.

Inactivation of isolated HIV-1 virus particles following exposure to different concentrations of NNRTI. Isolated HIV-1 particles were incubated with the indicated concentrations of various NNRTI for 2 h at 37°C. Excess drug was then removed by repeated dilution and concentration steps, as described in Materials and Methods. The exogenous drug-free virus was then used to infect MT-2 lymphocytes, and the extents of HIV-1-induced cytopathic effects were assessed 4 days postinfection. ▴, CSIC; ○, EFV; ▪, UC781; □, DLV; ▾, NVP; ▵, UC84. Data points are the means ± standard deviations of three separate determinations.

FIG. 3.

Effects of NNRTI treatment of chronically infected H9+ cells on infectivity of HIV-1 virions subsequently produced in the absence of extracellular drug. H9+ cells were incubated with 20 μM of the indicated NNRTI for 18 h at 37°C, and then the cells were washed free of exogenous drug. The H9+ cells were then cultured for 24 h in the absence of drug, and the virus produced during that period was separated from the cells by centrifugation, as described in Materials and Methods. Equal amounts of virus, corresponding to 0.25 ng of p24, were added to 5 × 10⁴ MT2 cells in a total volume of 200 μl, and the extent of infection of these cells was determined after 4 days by microscopic evaluation of syncytium formation. The data presented are the means ± standard deviations of three separate determinations.

FIG. 4.

Effects of NNRTI treatment of chronically infected H9+ cells on subsequent cell-to-cell transmission of HIV-1 in the absence of extracellular drug. H9+ chronically infected cells were incubated with the indicated concentrations of CSIC (▴), EFV (▪), UC781 (○), or DLV (□) for 18 h at 37°C, and then the cells were separated from extracellular drug and virus as described in Materials and Methods. The isolated H9+ cells were then cocultured with uninfected MT2 cells (1:300 ratio of H9+ to MT2 cells), and the extent of infection of the latter was determined 4 days later by microscopic assessment of HIV-1-induced syncytium formation.

FIG. 5.

Effects of pretreatment of uninfected MT2 cells with various concentrations of NNRTI on their subsequent infection by HIV-1 in the absence of extracellular drug. Uninfected MT2 cells were incubated with the indicated concentrations of NNRTI for 18 h and then washed to remove exogenous drug. The cells were then exposed to identical inocula of HIV-1 (MOI, 7.5 × 10⁻³). HIV-1-induced cytopathic effects were assessed daily by microscopic examination of syncytium formation as described in Materials and Methods. (A) CSIC; (B) EFV; (C) UC781; (D) DLV.

FIG. 6.

Effects of viral load on infection of MT2 cells pretreated with various concentrations of tight-binding NNRTI. Uninfected MT2 cells were incubated with the indicated concentrations of NNRTI for 18 h and then washed to remove exogenous drug. The cells were then inoculated with the indicated amounts of HIV-1 (MOI, 0.0007 to 0.18). HIV-1-induced cytopathic effects were assessed 4 days postinfection by microscopic examination of syncytium formation as described in Materials and Methods. (A) CSIC; (B) EFV; (C) UC781.