Kinetic Factors Control Efficiencies of Cell Entry, Efficacies of Entry Inhibitors, and Mechanisms of Adaptation of Human Immunodeficiency Virus

Abstract

Replication of human immunodeficiency virus type 1 (HIV-1) in diverse conditions limiting for viral entry into cells frequently leads to adaptive mutations in the V3 loop of the gp120 envelope glycoprotein. This has suggested that the V3 loop limits the efficiencies of HIV-1 infections, possibly by directly affecting gp120-coreceptor affinities. In contrast, V3 loop mutations that enable HIV-1 JR-CSF to use the low-affinity mutant coreceptor CCR5(Y14N) are shown here to have negligible effects on the virus-coreceptor affinity but to dramatically accelerate the irreversible conformational conversion of the envelope gp41 subunits from a three-stranded coil into a six-helix bundle. This slow step is blocked irreversibly by the inhibitor T-20. To further evaluate the role of entry rates in controlling infection efficiencies and viral adaptations, we developed methods to quantitatively measure viral entry kinetics. The virions were adsorbed by spinoculation at 4°C onto HeLa-CD4/CCR5 cell clones that either had limiting or saturating concentrations of CCR5. After warming to 37°C, the completion of entry was monitored over time by the resistance of infections to the competitive CCR5 inhibitor TAK-779. Our results suggest that the efficiency of entry of cell-attached infectious HIV-1 is principally controlled by three kinetic processes. The first is a lag phase that is caused in part by the concentrationdependent reversible association of virus with CD4 and CCR5 to form an equilibrium assemblage of complexes. Second, this assembly step lowers but does not eliminate a large activation energy barrier for a rate-limiting, CCR5-dependent conformational change in gp41 that is sensitive to blockage by T-20. The rate of infection therefore depends on the fraction of infectious virions that are sufficiently saturated with CCR5 to undergo this conformational change and on the magnitude of the activation energy barrier. Although only a small fraction of fully assembled viral complexes overcome this barrier per hour, the ensuing steps of entry are rapidly completed within 5 to 10 min. Thus, this barrier limits the overall flow rate at which the attached virions enter cells, but it has no effect on the lag time that precedes this entry flow. Third, a relatively rapid and kinetically dominant process of viral inactivation, which may partly involve endocytosis, competes with infectious viral entry. Our results suggest that the V3 loop of gp120 has a major effect on the rate-limiting coreceptordependent conformational change in gp41 and that adaptive viral mutations, including V3 loop mutations, function kinetically by accelerating this inherently slow step in the entry pathway.Human immunodeficiency virus type 1 (HIV-1) invasion of cells involves a sequence of steps that ultimately cause fusion of the viral membrane with the cell surface membrane and entry of viral cores into the cytosol (3,4,17,34,48), but the rates of these steps and of the competing viral inactivation processes are unknown. Initially, the virus diffuses onto cell ...

Time-resolved Imaging of HIV-1 Env-mediated Lipid and Content Mixing between a Single Virion and Cell Membrane

“…We began illumination simultaneously with raising temperature and DiD and NC-GFP mixing was completed within ϳ5 min (Figure 6). A reporter gene assay has shown that fusion of virus does not plateau for several hours (Platt et al, 2005) and photoaffinity labeling of SIV surface proteins upon fusion with cells indicated that fusion did not saturate for at least tens of minutes (Raviv et al, 2002). The faster saturation we observed is likely due to photo-induced inactivation.…”

Reduction of CCR5 with low-dose rapamycin enhances the antiviral activity of vicriviroc against both sensitive and drug-resistant HIV-1

“…Specifically, VCV EC 50 values were ϳ 20 times higher in donors with ϳ 7 ϫ 10 3 CCR5 molecules/cell than in donors with ϳ 2 ϫ 10 3 CCR5 molecules/cell. Previous studies with cell lines have shown that CCR5 levels influence the antiviral activity of the CCR5 antagonist TAK-779, with higher CCR5 levels resulting in reduced antagonist activity (24)(25)(26). Ketas et al (27) have shown recently that donor variation in CCR5 levels (determined by mean fluorescence intensity) correlates with the antiviral activity of VCV.…”

“…In the RAPA/VCV combination, reduction of CCR5 density and partial blocking of CCR5 by VCV would effectively prevent CD4 bound viral envelopes from engaging sufficient CCR5 molecules. As a result, CD4-triggered envelopes unable to progress in the entry cascade would be exposed for prolonged periods of time and subjected to inactivation (24). We currently are investigating these mechanisms in an effort to characterize the basis for antiviral synergy by the RAPA/CCR5 antagonist combination.…”

Role of the Envelope Genetic Context in the Development of Enfuvirtide Resistance in Human Immunodeficiency Virus Type 1-Infected Patients

“…In particular, susceptibility to fusion inhibitors appears to be dependent on the affinity of gp120 for the coreceptors as well as on coreceptor density at the cell surface, both of which factors affect fusion kinetics (34). A faster fusion process will reduce the length of time during which the ENF molecular target is exposed, thus reducing the virus sensitivity to this inhibitor (28,30,34). We thus hypothesized that in patients failing ENF treatment, the biological properties of Env, and therefore the whole envelope genetic background, could influence the selection of ENF resistance mutations, thereby conditioning the emergence of ENF-resistant viruses.…”