Background:  A nonhuman primate model is needed for studying the development of HIV-1 antiviral resistance in clinically relevant drug protocols. We are developing a pigtailed macaque model to examine HIV-1 resistance to non-nucleoside reverse transcriptase inhibitors (NNRTI) and the relative fitness of drug resistant viral isolates. NNRTI specifically target HIV-1 reverse transcriptase (RT) and do not inhibit simian immunodeficiency virus (SIV) RT.

Methods:  Two modified SIV_mne isolates were created by replacing the RT gene of 2 distinct SIV_mne molecular clones with HIV-1 RT. RT-SHIV_mne mutant virus replicated in CEMx174 cells and primary PBMC over multiple rounds, producing infectious virus within a week of inoculation. Viral growth kinetics were monitored by capsid protein ELISA and RT cleavage was evaluated by Western blot analysis. Inhibitory concentrations (IC₅₀) of nucleoside RT inhibitors (NRTI; AZT, ddI, d4T, 3TC, and tenofovir) and NNRTI (efavirenz, nevirapine, and UC-781) were determined for HIV-1, wild-type SIV_mne, and RT-SHIV_mne in single-cycle virus replication assays. RT-SHIV_mne was grown in the presence of escalating doses of NNRTI and evaluated by sequencing for RT resistance mutations by population sequencing and by allele specific real-time PCR.

Results:  RT-SHIV_mne replicated slower than wild type SIV_mne, displaying a 1-magnitude decrease of replicative capacity in vitro. No adaptation of the chimeric virus was observed after extensive in vitro passage. SIV_mne protease appeared to properly cleave the HIV-1 RT from the Gag-Pol polyprotein as well as produce p51 and p66 in an equimolar ratio in viral particles. HIV-1 and RT-SHIV_mne were susceptible to NNRTI as well as NRTIs, whereas wild-type SIV_mne was only susceptible to NRTIs. Classical NNRTI resistance mutations K103N, Y181C, and V179D were found in RT-SHIV_mne. The emergence of K103N and Y181C in the virus appeared over time in the presence of EFV and NVP, respectively. We currently are initiating the replication of RT-SHIV_mne in pigtailed macaques.

Conclusions:  RT-SHIV_mne should be useful for preclinical trials of NNRTI in macaques and for understanding the development of drug resistance in vivo. This RT-SHIV_mne infection model will also be used to evaluate the relative fitness of wild type and mutant HIV-1 RT under non-selective and selective conditions in vivo.

Keywords: RT; NNRTI; macaque