Altering Expression Levels of Human Immunodeficiency Virus Type 1 gp120-gp41 Affects Efficiency but Not Kinetics of Cell-Cell Fusion

106

111

An increasingly large number of antiviral agents that prevent entry of human immunodeficiency virus (HIV) into cells are in preclinical and clinical development. The envelope (Env) protein of HIV is the major viral determinant that affects sensitivity to these compounds. To understand how changes in Env can impact entry inhibitor sensitivity, we introduced six mutations into the conserved coreceptor binding site of the R5 HIV-1 strain YU-2 and measured the effect of these changes on CD4 and coreceptor binding, membrane fusion levels and rates, virus infection, and sensitivity to the fusion inhibitors enfuvirtide (T-20) and T-1249, the CCR5 inhibitor TAK-779, and an antibody to CD4. The mutations had little effect on CD4 binding but reduced CCR5 binding to various extents. In general, reductions in coreceptor binding efficiency resulted in slower fusion kinetics and increased sensitivity to TAK-779 and enfuvirtide. In addition, low CCR5 binding usually reduced overall fusion and infection levels. However, one mutation adjacent to the bridging sheet ␤21 strand, P438A, had little effect on fusion activity, fusion rate, infectivity, or sensitivity to enfuvirtide or T-1249 despite causing a marked reduction in CCR5 binding and a significant increase in TAK-779 sensitivity. Thus, our findings indicate that changes in the coreceptor binding site of Env can modulate its fusion activity, infectivity, and entry inhibitor sensitivity by multiple mechanisms and suggest that reductions in coreceptor binding do not always result in prolonged fusion kinetics and increased sensitivity to enfuvirtide.

Section: Methodsmentioning

confidence: 99%

Impact of Mutations in the Coreceptor Binding Site on Human Immunodeficiency Virus Type 1 Fusion, Infection, and Entry Inhibitor Sensitivity

Reeves

Miamidian

Biscone

et al. 2004

106

111

“…Cells expressing high levels of ADA Env were periodically selected from the population by adding 2 mM methotrexate. The kinetics and/or extents of fusion generally increase with the densities of fusion protein (29,33) (our unpublished data). The temperature threshold for fusion consequently decreases for increased density.…”

Section: Fusion Induced By Human Immunodeficiency Virus (Hiv)mentioning

confidence: 99%

Ternary Complex Formation of Human Immunodeficiency Virus Type 1 Env, CD4, and Chemokine Receptor Captured as an Intermediate of Membrane Fusion

Mkrtchyan

Markosyan

Eadon

et al. 2005

Human immunodeficiency virus (HIV) Env-induced fusion is highly temperature dependent. When effector and target cells were coincubated at 37°C, there was a kinetic delay before fusion commenced. When effector and target cells were coincubated for varied times at 23°C, a temperature that does not permit fusion, a temperature-arrested stage was created. Raising temperature to 37°C from the 23°C intermediate eliminated the kinetic delay. Inhibitors (T22, AMD3100, and Sch-C) that block fusion by binding chemokine receptors were added after creating the intermediate so as to assess the extent of engagement between gp120 and chemokine receptors at that stage. For both CXCR4 and CCR5 as coreceptors, increasingly long times of coincubation at 23°C reduced the efficacy of the coreceptor-binding inhibitors in blocking fusion. This implies that an increasing number of ternary Env/CD4/coreceptor complexes form over time at 23°C. It also shows that ternary complex formation has a lower temperature threshold than the downstream steps that include Env folding into a six-helix bundle; this provides an experimental means to separate coreceptor binding by gp120 from the subsequent refolding of gp41 into a six-helix bundle structure. As the time of cell coincubation at 23°C was prolonged, more cells quickly fused upon the raising of the temperature to 37°C, and the increase quantitatively correlated with the greater percentage of fusion that was resistant to drugs. Therefore the pronounced kinetic delay in HIV Env-induced fusion is caused predominantly by the time needed for ternary complexes to form. Fusion induced by human immunodeficiency virus (HIV)Env is a multistep process. The gp120 subunit undergoes sequential conformational changes as it interacts with CD4 and its coreceptors (CXCR4 and/or CCR5) (see references 5, 10, and 25 and references therein). The gp120 changes directly lead to the conformational changes of the gp41 subunit that cause fusion between the viral envelope and cell membrane. When Env has engaged both CD4 and its coreceptor, this complex is called a ternary complex (24). The structure of a simplified version of a ternary complex consisting of gp120 with some of its loops deleted, the binding domain of CD4, and a monoclonal antibody that serves as a surrogate for the chemokine receptor have been determined (24). The kinetics of ternary complex formation and its relation to the kinetics of fusion are not known.Drugs that block fusion by binding to chemokine receptors-thereby preventing Env from engaging its chemokine receptors-are currently in development as therapeutics against HIV infection (10, 25). Because such drugs can be effective only prior to the formation of stable ternary complexes, they can be used to determine at which stage in the fusion process ternary complexes form. Intermediate stages of the fusion process have been captured by coincubating, under conditions suboptimal for fusion, effector (E) cells that express fusion proteins on their surfaces and target (T) cells that express appropriat...

“…The fusion kinetics of WT and mutant NiV-F proteins were determined in a ␤-lactamase reporter cell-cell fusion assay as previously described (3,34,53). For better sensitivity, the ␤-lactamase gene was also codon optimized for mammalian cell expression (Geneart, Inc., Toronto, Ontario, Canada).…”

Section: Vol 81 2007 Inside-out Signaling Of Nipah Virus Fusion Promentioning

confidence: 99%

Polybasic KKR Motif in the Cytoplasmic Tail of Nipah Virus Fusion Protein Modulates Membrane Fusion by Inside-Out Signaling

Aguilar

Matreyek

Choi

et al. 2007

185

The cytoplasmic tails of the envelope proteins from multiple viruses are known to contain determinants that affect their fusogenic capacities. Here we report that specific residues in the cytoplasmic tail of the Nipah virus fusion protein (NiV-F) modulate its fusogenic activity. Truncation of the cytoplasmic tail of NiV-F greatly inhibited cell-cell fusion. Deletion and alanine scan analysis identified a tribasic KKR motif in the membraneadjacent region as important for modulating cell-cell fusion. The K1A mutation increased fusion 5.5-fold, while the K2A and R3A mutations decreased fusion 3-to 5-fold. These results were corroborated in a reversepseudotyped viral entry assay, where receptor-pseudotyped reporter virus was used to infect cells expressing wild-type or mutant NiV envelope glycoproteins. Differential monoclonal antibody binding data indicated that hyper-or hypofusogenic mutations in the KKR motif affected the ectodomain conformation of NiV-F, which in turn resulted in faster or slower six-helix bundle formation, respectively. However, we also present evidence that the hypofusogenic phenotypes of the K2A and R3A mutants were effected via distinct mechanisms. Interestingly, the K2A mutant was also markedly excluded from lipid rafts, where ϳ20% of wild-type F and the other mutants can be found. Finally, we found a strong negative correlation between the relative fusogenic capacities of these cytoplasmic-tail mutants and the avidities of NiV-F and NiV-G interactions (P ‫؍‬ 0.007, r 2 ‫؍‬ 0.82). In toto, our data suggest that inside-out signaling by specific residues in the cytoplasmic tail of NiV-F can modulate its fusogenicity by multiple distinct mechanisms.