Sensitivity of HIV-1 to entry inhibitors correlates with envelope/coreceptor affinity, receptor density, and fusion kinetics

Abstract

HIV entry inhibitors include coreceptor antagonists and the fusion inhibitor T-20. T-20 binds the first helical region (HR1) in the gp41subunit of the viral envelope (Env) protein and prevents conformational changes required for membrane fusion. HR1 appears to become accessible to T-20 after Env binds CD4, whereas coreceptor binding is thought to induce the final conformational changes that lead to membrane fusion. Thus, T-20 binds to a structural intermediate of the fusion process. Primary viruses exhibit considerable variability in T-20 sensitivity, and determinants outside of HR1 can affect sensitivity by unknown mechanisms. We studied chimeric Env proteins containing different V3 loop sequences and found that gp120͞coreceptor affinity correlated with T-20 and coreceptor antagonist sensitivity, with greater affinity resulting in increased resistance to both classes of entry inhibitors. Enhanced affinity resulted in more rapid fusion kinetics, reducing the time during which Env is sensitive to T-20. Reduced coreceptor expression levels also delayed fusion kinetics and enhanced virus sensitivity to T-20, whereas increased coreceptor levels had the opposite effect. A single amino acid change (K421D) in the bridging sheet region of the primary virus strain YU2 reduced affinity for CCR5 and increased T-20 sensitivity by about 30-fold. Thus, mutations in Env that affect receptor engagement and membrane fusion rates can alter entry inhibitor sensitivity. Because coreceptor expression levels are typically limiting in vivo, individuals who express lower coreceptor levels may respond more favorably to entry inhibitors such as T-20, whose effectiveness we show depends in part on fusion kinetics.

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

“…However, we along with others have shown that baseline susceptibility to ENF is quite variable among viruses from different patients (9,20,39), and variations in domains of Env other than HR1 are thought to account for these differences in baseline susceptibility (8,15,34). 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).…”

“…It is noteworthy, however, that in cases where ENF resistance mutations produced only suboptimal resistance, Env replicative capacity was usually maintained, suggesting that the impact of resistance mutations on viral resistance and viral fitness was not tightly linked (see below). The identification of viral sequences that determine whether a given context is favorable or unfavorable for the expression of high resistance by ENF resistance mutations may prove to be a difficult task, given that multiple discontinuous envelope domains participate in the entry process and can modulate phenotypic resistance to ENF (8,15,34).…”

“…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.…”

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

“…To identify the mechanisms of membrane fusion, both continuities should be followed and the temporal order in which they occur identified. In the case of the HIV-1 fusion protein Env, mechanistic studies have largely relied on fusion of cells expressing the protein to cells expressing CD4 and cognate chemokine receptors (e.g., Munoz-Barroso et al, 1998;Melikyan et al, 2000;Reeves et al, 2002;Abrahamyan et al, 2003;Gallo et al, 2003;Markosyan et al, 2003). But monitoring lipid dye spread in HIV-1 Env-mediated cell-cell fusion has not proved to be very useful in following membrane continuity, for several reasons.…”

HIV-1 envelope glycoprotein-mediated fusion and pathogenesis: Implications for therapy and vaccine development

“…By examining fusion kinetics of various HIV strains we found that the sensitivity of HIV to entry inhibitors correlates with envelope:coreceptor affinity, receptor density and fusion kinetics [25]. The kinetic studies allowed us to determine parameters that govern fusion and inhibition.…”