Human Immunodeficiency Virus Type 1 Protease Inhibitor Drug-Resistant Mutants Give Discordant Results When Compared in Single-Cycle and Multiple-Cycle Fitness Assays

Abstract

The replication fitness of HIV-1 drug-resistant mutants has been measured using either multiple-cycle or single-cycle assays (MCAs or SCAs); these assays have not been systematically compared. We developed an MCA and an SCA that utilized either intact or env-deleted recombinant viral vectors, respectively, in which virus-infected cells were detected by flow cytometry of a reporter gene product. Fitness was measured using each assay for 11 protease mutants, 9 reverse transcriptase mutants, and two mutants with mutations in gag p6, which is important for the release of virus particles from the cell membrane. In the SCA, fitness (replication capacity [RC]) was defined as the proportion of cells infected by the mutant compared to the wild type 40 h after infection. MCA fitness (1؉s) was determined by comparing the changes in the relative proportions of cells infected by the mutant and the wild type between 3 and 5 days after infection. Five protease mutants showed statistically different fitness values by the MCA versus the SCA: the D30N, G48V, I50V, I54L, and I54M mutants. When all the mutants were ranked in order from most to least fit for both assays, 4 protease mutants moved more than 5 positions in rank: the D30N, I54L, I54M, and V82A mutants. There were no significant differences in fitness for the gag p6 or reverse transcriptase mutants. We propose that discordant results in the MCA and SCA are due to alterations in late events in the virus life cycle that are not captured in an SCA, such as burst size, cell-to-cell transmission, or infected-cell life span.

Novel Method for Simultaneous Quantification of Phenotypic Resistance to Maturation, Protease, Reverse Transcriptase, and Integrase HIV Inhibitors Based on 3′Gag(p2/p7/p1/p6)/PR/RT/INT-Recombinant Viruses: a Useful Tool in the Multitarget Era of Antiretroviral Therapy

“…Although this replication capacity assay remains an interesting research tool, it has not found a definitive role in patient management (39). Multiple cycle replication assays tend to amplify small differences in viral replication (17,80), which are often indistinguishable in single-cycle infection, especially VOL. 55,2011 NOVEL HIV-1 PHENOTYPING ASSAY 3739 where virus infection is monitored only until HIV-1 mRNA transcription in trans (i.e., luciferase expression) and does not account for viral protein translation, assembly, and virion maturation.…”

Nonnucleoside Reverse Transcriptase Inhibitor-Resistant HIV Is Stimulated by Efavirenz during Early Stages of Infection

“…The assays used to test stimulation were multiple-cycle assays, which include all steps of the virus life cycle, both early steps (entry, reverse transcription, and integration) and late steps (RNA production, protein expression, and viral particle production). In order to determine whether stimulation occurred during early or late steps, we modified a single-cycle assay previously designed by our group (13). We used the envelope-deleted vector, pDAT2, and with a murine leukemia virus (MLV) envelope-expressing construct, made MLV-pseudotyped virus stocks in the presence or absence of EFV.…”

“…The wild-type (WT) and mutant pAT2 and pNL4-3XX constructs were transiently transfected into either 293 cells using 40 g of plasmid DNA and Superfect (Qiagen) or 293T cells using 5 g of DNA and 25 l of Lipofectamine LTX (Invitrogen) in the absence of drug. Mutant pDAT2 constructs were transiently transfected into 293 or 293T cells using 5 g of plasmid DNA along with 5 g of pSV-A-MLV-ENV (expressing the murine leukemia virus [MLV] envelope [ENV]) and 25 l of Lipofectamine LTX (Invitrogen) as previously described (13). For all transfections, the cells were incubated for 4 h, and the transfection medium was replaced with fresh medium.…”

“…To test whether stimulation occurs during the early steps of the virus life cycle, one million PM1 cells were incubated with virus at 37°C for 1 h, washed with PBS, and cultured with medium for 48 h at 37°C, in the presence or absence of EFV (0, 400, and 800 nM), NVP (0, 50 and 200 M), or ETR (0, 1, 2, 10, and 100 nM). The cells were then stained with the anti-Thy1.1 and anti-Thy1.2 antibodies and analyzed by fluorescence-activated cell sorting (FACS) as previously described (13). Fold stimulation was calculated by dividing the percentage of infected cells in the absence of drug by the percentage of infected cells in the presence of drug.…”

HIV-1 infection renders brain vascular pericytes susceptible to the extracellular glutamate

“…DHIV was obtained from Vicente Planelles (Bosque and Planelles, 2009). Plasmid expressing the vesicular stomatitis virus G protein (VSV-G) under the control of the human cytomegalovirus promoter (pHCMV-g) was obtained from Carrie Dykes (Dykes et al , 2010). Human embryonic kidney 293T cells were used for generation of HIV-1 stocks.…”