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Background:  Heterosexual transmission is the leading mode of HIV-1 infection worldwide, with women particularly vulnerable to HIV infection. In the absence of an effective vaccine there is an urgent need to develop alternative prevention strategies. Potential retrovirucides or vaginal microbicides include non-nucleoside reverse transcriptase inhibitors (NNTRI). Ideally, a retrovirucidal agent should act directly on the virus and/or incorporate into nascent virion during assembly and budding. Here we evaluate the permeability of nevirapine (NVP) NNTRI inhibitor and its incorporation on nascent virus particles.

Methods:  MT4 cells were infected by NL4-3 virus. The infected cells were washed and exposed to nevirapine growing concentrations. The NVP concentration inside nascent virus particles was evaluated through of dialysis of the virus to remove the NVP present in the media. The incorporation of NVP on nascent virus was analyzed by Natural Endogenous Reverse Transcription (NERT) and HeLa Magi cells infectivity. The NERT reaction is based in reverse transcription of HIV-1 genome in intact virions exposed to dNTPs. We developed a real time polymerase chain reaction (PCR)-based assay to measure the absolute quantification of NERT activity. We compared the NERT reaction and the HeLa Magi infectivity of the nascent virus prior and after dialysis.

Results:  NVP was able to inactivate both virus in infected cells and intact particles. Our results showed a correlation between NERT activity and cell infectivity of virions exposed to NVP. The EC50 values of NVP inhibition were lower in the HeLa Magi infectivity assay (0.05 mM) when compared to the virion (25 mM NERT) suggesting low NVP permeability levels in the cell-free virions. We found a decrease of NVP- inhibition effect evaluated by NERT activity and infectivity in dialyzed virus, suggesting that the NVP concentration inside of nascent virus is lower than in the media.

Conclusions:  These data suggest that NVP penetrates the HIV-1 membrane envelope and capsid core readily, inactivating the virus. However, the characteristics of NVP inhibition, such as lower permeability in cell-free virus and exclusion of nascent virus during the budding, reduces the efficacy of NVP in microbicides strategies.

Keywords: nevirapine; permeability; NERT