In vitro characterization of a simian immunodeficiency virus-human immunodeficiency virus (HIV) chimera expressing HIV type 1 reverse transcriptase to study antiviral resistance in pigtail macaques

Abstract

Antiviral resistance is a significant obstacle in the treatment of human immunodeficiency virus type 1 (HIV-1)-infected individuals. Because nonnucleoside reverse transcriptase inhibitors (NNRTIs) specifically target HIV-1 reverse transcriptase (RT) and do not effectively inhibit simian immunodeficiency virus (SIV) RT, the development of animal models to study the evolution of antiviral resistance has been problematic. To facilitate in vivo studies of NNRTI resistance, we examined whether a SIV that causes immunopathogenesis in pigtail macaques could be made sensitive to NNRTIs. Two simian-human immunodeficiency viruses (SHIVs) were derived from the genetic background of SIV(mne): SIV-RT-YY contains RT substitutions intended to confer NNRTI susceptibility (V181Y and L188Y), and RT-SHIV(mne) contains the entire HIV-1 RT coding region. Both mutant viruses grew to high titers in vitro but had reduced fitness relative to wild-type SIV(mne). Although the HIV-1 RT was properly processed into p66 and p51 subunits in RT-SHIV(mne) particles, the RT-SHIV(mne) virions had lower levels of RT per viral genomic RNA than HIV-1. Correspondingly, there was decreased RT activity in RT-SHIV(mne) and SIV-RT-YY particles. HIV-1 and RT-SHIV(mne) were similarly susceptible to the NNRTIs efavirenz, nevirapine, and UC781. However, SIV-RT-YY was less sensitive to NNRTIs than HIV-1 or RT-SHIV(mne). Classical NNRTI resistance mutations were selected in RT-SHIV(mne) after in vitro drug treatment and were monitored in a sensitive allele-specific real-time RT-PCR assay. Collectively, these results indicate that RT-SHIV(mne) may be a useful model in macaques for the preclinical evaluation of NNRTIs and for studies of the development of drug resistance in vivo.

FIG. 1.

Sequence alignment of amino acids 101 to 240 in the RTs of HIV-1_HXB2, RT-SHIV_mne, SIV_mne, and SIV-RT-YY. Dots indicate residues conserved between the different isolates. Boxed residues indicate residues 181 and 188, which are the residues that were changed to tyrosines in SIV-RT-YY.

FIG. 2.

In vitro replication kinetics of SIV isolates. (A and B) CEMx174 cell cultures were electroporated with 10 mg of SIV_mne, RT-SHIV_mne, and SIV-RT-YY DNA (A) or infected with an MOI of 0.02 with SIV_mne, RT-SHIV_mne, and SIV-RT-YY (B). SIV_mne p27 was used to monitor viral replication. Each time point represents the average of duplicates. Time points for the infections shown in panel B are the average of two independent experiments. (C) The specific infectivity (infectious units per nanogram of p27) of each virus was calculated by dividing the number of infectious units (per milliliter) as determined on JC53 BL+ cells by the amount of p27 (in nanograms per milliliter) at day 7.

FIG. 3.

Immunoblot of HIV-1 RT from HIV-1 and RT-SHIV_mne virions. HIV-1_LAI and RT-SHIV_mne virions were loaded at 0.28 × 10⁸, 0.77 × 10⁸, and 2.3 × 10⁸ RNA copies/ml per lane for HIV-1 and at 0.77 × 10⁸, 2.3 × 10⁸, and 7.0 × 10⁸ RNA copies/ml per lane for RT-SHIV_mne, as determined by real-time PCR amplification of RT. Recombinant RT was used as a positive control. The larger band is sulfhydryl-cross-linked (oxidized) RT. The gel was transferred to a PVDF membrane and probed with anti-RT monoclonal antibodies, followed by an anti-mouse immunoglobulin G-horseradish peroxidase antibody. Protein size markers are denoted on the left in kilodaltons.

FIG. 4.

In vitro RT inhibition with NNRTIs. Representative data are shown in which JC53 BL13+ cells were infected with HIV-1_NFNSX, SIV_mne, RT-SHIV_mne, or SIV-RT-YY in the presence or absence of multiple concentrations of EFV (A), NVP (B), or UC781 (C). Infections were performed in duplicate, and luciferase measurements were performed in triplicate. Results are expressed as the percent cells infected for each virus with each dilution of drug compared to infection without drug.

FIG. 5.

In vitro RT inhibition in macaque cells with EFV. sMAGI-Hi5 cells were infected with HIV-1_NFNSX, SIV_mne, and RT-SHIV_mne in the presence or absence of various concentrations of EFV. Infections were performed in duplicate, and blue cells (infectious centers) were counted. Results are expressed as the percent cells infected for each virus with each dilution of drug compared to infection without drug. IC₅₀ values in this assay were as follows: 0.4 nM for HIV-1, >150 nM for SIV_mne, <0.2 nM for RT-SHIV_mne, and 8.5 nM for SIV-RT-YY.

FIG. 6.

Proportion of specific NNRTI resistance mutations in RT-SHIV_mne selected in the presence or absence of NNRTIs. Virus selected in CEMx174 cells grown in the presence or absence of increasing concentrations of EFV (A) or NVP (B) was assayed by allele-specific real-time RT-PCR for quantitation of the mutations K103N or Y181C, respectively. The concentration (nanomolar) of each drug during the selection of the cultures is denoted at the bottom of each graph. The dashed lines indicate the background of the assays: 0.0265% ± 0.007% (K103N) and 0.169% ± 0.017% (Y181C).