Biomedical subjects

John L Miamidian

Publications and source records attributed to John L Miamidian.

4 recordsLinked to original sources

PubMed2006

Functional impact of HIV coreceptor-binding site mutations.

The bridging sheet region of the gp120 subunit of the HIV-1 Env protein interacts with the major virus coreceptors, CCR5 and CXCR4. We examined the impact of mutations in and adjacent to the bridging sheet region of an X4 tropic HIV-1 on membrane fusion and entry inhibitor susceptibility. When the V3-loop of this Env was changed so that CCR5 was used, the effects of these same mutations on CCR5 use were assayed as well. We found that coreceptor-binding site mutations had greater effects on CXCR4-mediated fusion and infection than when CCR5 was used as a coreceptor, perhaps related to differences in coreceptor affinity. The mutations also reduced use of the alternative coreceptors CCR3 and CCR8 to varying degrees, indicating that the bridging sheet region is important for the efficient utilization of both major and minor HIV coreceptors. As seen before with a primary R5 virus strain, bridging sheet mutations increased susceptibility to the CCR5 inhibitor TAK-779, which correlated with CCR5 binding efficiency. Bridging sheet mutations also conferred increased susceptibility to the CXCR4 ligand AMD-3100 in the context of the X4 tropic Env. However, these mutations had little effect on the rate of membrane fusion and little effect on susceptibility to enfuvirtide, a membrane fusion inhibitor whose activity is dependent in part on the rate of Env-mediated membrane fusion. Thus, mutations that reduce coreceptor binding and enhance susceptibility to coreceptor inhibitors can affect fusion and enfuvirtide susceptibility in an Env context-dependent manner.

Binding Sites↗

PubMed2005

Enfuvirtide resistance mutations: impact on human immunodeficiency virus envelope function, entry inhibitor sensitivity, and virus neutralization.

Enfuvirtide (ENF/T-20/Fuzeon), the first human immunodeficiency virus (HIV) entry inhibitor to be licensed, targets a structural intermediate of the entry process. ENF binds the HR1 domain in gp41 after Env has bound CD4, preventing conformational changes needed for membrane fusion. Mutations in HR1 that confer ENF resistance can arise following ENF therapy. ENF resistance mutations were introduced into an R5- and X4-tropic Env to examine their impact on fusion, infection, and sensitivity to different classes of entry inhibitors and neutralizing antibodies. HR1 mutations could reduce infection and fusion efficiency and also delay fusion kinetics, likely accounting for their negative impact on viral fitness. HR1 mutations had minimal effect on virus sensitivity to other classes of entry inhibitors, including those targeting CD4 binding (BMS-806 and a CD4-specific monoclonal antibody [MAb]), coreceptor binding (CXCR4 inhibitor AMD3100 and CCR5 inhibitor TAK-779), or fusion (T-1249), indicating that ENF-resistant viruses can remain sensitive to other entry inhibitors in vivo. Some HR1 mutations conferred increased sensitivity to a subset of neutralizing MAbs that likely target fusion intermediates or with epitopes preferentially exposed following receptor interactions (17b, 48D, 2F5, 4E10, and IgGb12), as well as sera from some HIV-positive individuals. Mechanistically, enhanced neutralization correlated with reduced fusion kinetics, indicating that, in addition to steric constraints, kinetics may also limit virus neutralization by some antibodies. Therefore, escape from ENF comes at a cost to viral fitness and may confer enhanced sensitivity to humoral immunity due to prolonged exposure of epitopes that are not readily accessible in the native Env trimer. Resistance to other entry inhibitors was not observed.

CD8-Positive T-Lymphocytes↗

PubMed2004

Impact of mutations in the coreceptor binding site on human immunodeficiency virus type 1 fusion, infection, and entry inhibitor sensitivity.

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 beta21 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.

Amides↗

PubMed2002

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

HIV entry inhibitors include coreceptor antagonists and the fusion inhibitor T-20. T-20 binds the first helical region (HR1) in the gp41 subunit 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 gp120coreceptor 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.

3T3 Cells↗