In this work, we successfully evaluated the use of real-time PCR for measuring intravirion DNA synthesis (the endogenous RT activity) in intact HIV-1 particles. Furthermore, RT inhibition by widely used NNRTIs in this new assay correlated well with RT inhibition measured by classical infectivity assays. This is the first time that endogenous RT activity measured directly inside viral particles was correlated with virion infectivity, and our results indicated that there is a clear linear correlation between these two variables. The real-time PCR detection of NERT activity is advantageous, since it is highly sensitive and less time-consuming than the classical infectivity assays used to measure inhibition of viral replication by antiretroviral compounds. Therefore, this new methodology is suitable for large-scale and rapid screening of potential new RT inhibitors to be used, for example, as microbicides.
The HIV-1 envelope composition renders viral particles permeable to dNTPs, which favors RT activity while still inside the virion environment. The results obtained here confirmed the findings of Zhang et al. (37
), demonstrating that the amphipathic domain of the gp41 transmembrane glycoprotein is responsible for the passage of dNTPs from the outside medium to the interior of the virions. Due to this, the increase in the basal levels of RT activity in response to an increase in the dNTP concentration was not detected in nonenveloped or even pseudotyped HIV-1 particles carrying the MLV envelope proteins. These results were important to demonstrate the specificity of our assay.
Blockage of intravirion DNA synthesis has been demonstrated to occur in the presence of both nucleoside analog inhibitors (ddTTP, ddATP, and AZT) and NNRTIs (10
). Real-time PCR detection of NERT activity was equally sensitive to the presence of NNRTIs. Furthermore, this is the first time that the effect of an RT resistance mutation was demonstrated in a NERT assay. We have also observed that a drug concentration over the IC50
calculated in cells is required to inhibit NERT activity, and for the first time, we could obtain an absolute quantification of this phenomenon. The increase in the drug concentration required to completely inhibit NERT was dependent on the drug analyzed, up to 100-fold for NVP and 10-fold for EFV; however, in both cases, the viral-membrane composition can play an important role in virion permeability to antiretroviral drugs compared to the infected-cell situation.
Other important factors are the binding and retention of NNRTI drugs in the RT molecules inside viral particles budded in the presence of an RT inhibitor. Typically, NNRTIs are hydrophobic molecules and readily traverse membrane barriers, such as that surrounding the HIV core. However, this property would also facilitate the NNRTI's efflux from the virion in the absence of sufficient extravirion levels of these inhibitors. In our experiments, we compared EFV and NVP retention inside the viral particle by dialysis procedures. NVP was more easily washed off by the dialysis process than EFV. The values of NERT inhibition for virus budded in the presence of EFV were not different before and after the wash procedure, suggesting that this NNRTI remains bound to the RT molecules inside the viral particles. These results agree with reports that named NNRTIs as rapid-equilibrium and tightly binding inhibitors (16
). NVP is a rapid-equilibrium inhibitor and requires an excess of the drug over the enzyme concentration (27
). On the other hand, EFV is a tightly binding inhibitor of HIV-1 RT (14
). Tightly binding inhibitors exhibit unique properties in their interactions with enzymes, which distinguish them from rapid-equilibrium inhibitors (31
). EFV binds rapidly to RT but, once bound, dissociates only very slowly. Thus, RT remains inhibited for prolonged periods of time after being bound, even in the absence of significant levels of unbound inhibitor or the wash procedure to remove the excess inhibitor (14
). Therefore, we suggested that tightly binding inhibition might be an important criterion for the NNRTI microbicide screening, and compounds classified as tightly binding, such as EFV, have great potential as microbicides.
Although both NVP and EFV fulfill the basic requirements for use as microbicides, our results for both the concentration required for NERT inhibition and the permeability to virions indicate that EFV is potentially a better candidate than NVP.
Of note, this is the first time that a highly sensitive methodology to quantify NERT activity has been shown to correlate well with measurements of virion infectivity. Furthermore, the profiles of RT inhibition in the presence of NVP and EFV were similar when measured both by infectivity assay and by inhibition of NERT reactions, showing a linear correlation between the two assays.
It has been demonstrated that intravirion RT activity augments virion infectivity and is important for HIV-1 to establish an infection in quiescent cells (36
); therefore, inhibiting such activity could contribute to blocking subsequent cell infection. Furthermore, our results are the first report of a direct impact of resistance mutations on the endogenous RT activities of intact viruses previously exposed to antiretroviral drugs, validating the usefulness of the NERT real-time PCR-based assay methodology in screening of new microbicide candidates able to inhibit drug-resistant variants, which can be present in semen. The methodology developed here can be used to directly evaluate the impacts of RT mutations previously described in the antiretrovirals commonly used in AIDS treatment on the microbicide candidates for which we do not have previous knowledge of resistance mutations. This work shows the usefulness of the NERT real-time PCR in screening novel antiretroviral microbicide compounds with unique mechanisms of action.