HIV neutralization and mechanisms of cellular entry

Division Of Basic Sciences - Nci

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Abstract

We have taken several important steps last year towards our goals of understanding structural aspects of HIV entry and neutralization. Over the last year, we have reported the first glimpse of the structure of trimeric gp120 on the surface of the HIV-1 membrane. The envelope glycoproteins (Env) of human and simian immunodeficiency viruses (HIV and SIV, respectively) mediate virus binding to the cell surface receptor CD4 on target cells to initiate infection. Env is a heterodimer of a transmembrane glycoprotein (gp41) and a surface glycoprotein (gp120), and forms trimers on the surface of the viral membrane. Using cryo-electron tomography combined with 3D image classification and averaging, we have reported the three-dimensional structures, at resolutions of 20 , of trimeric Env displayed on native HIV-1 in the unliganded state, in complex with the broadly neutralizing antibody b12 and in a ternary complex with CD4 and the 17b antibody. By fitting the known crystal structures of the monomeric gp120 core in the b12- and CD4/17b-bound conformations into the density maps derived by electron tomography, we derive molecular models for the native HIV-1 gp120 trimer in unliganded and CD4-bound states. We demonstrate that CD4 binding results in a major reorganization of the Env trimer, causing an outward rotation and displacement of each gp120 monomer. This appears to be coupled with a rearrangement of the gp41 region along the central axis of the trimer, leading to closer contact between the viral and target cell membranes. Our findings elucidate the structure and conformational changes of trimeric HIV-1 gp120 relevant to antibody neutralization and attachment to target cells. We are now extending these studies to a variety of SIV and HIV-1 strains, in complex with both neutralizing and non-neutralizing antibodies. We expect these studies will provide important and invaluable information helpful for effective vaccine design strategies against HIV/AIDS. In parallel with structural investigation of trimeric Env spikes, we have also initiated systematic efforts to analyze HIV distribution in antigen presenting cells such as macrophages and dendritic cells. HIV-1-containing internal compartments are readily detected in images of thin sections from infected cells using conventional transmission electron microscopy, but the origin, connectivity and 3D distribution of these compartments has remained controversial. We have now determined the 3D distribution of viruses in HIV-1-infected primary human macrophages using cryo-electron tomography and ion-abrasion scanning electron microscopy (IA-SEM), a recently developed approach for nanoscale 3D imaging of whole cells. Using IA-SEM we show the presence of an extensive network of HIV-1-containing tubular compartments in infected macrophages, with diameters of 150-200 nm, and lengths of up to 5 μm that extend from vesicular compartments that contain assembling HIV-1 virions to the cell surface. These types of surface-connected tubular compartments are not observed in T-cells infected with the 29/31 KE Gag-matrix where the virus is targeted to multi-vesicular bodies and released into the extracellular medium. IA-SEM imaging also allows visualization of large sheet-like structures that extend outward from the surfaces of macrophages, which may bend and fold back to allow continual creation of viral compartments and virion-lined channels. This potential mechanism for efficient virus trafficking between the cell surface and interior may represent a subversion of pre-existing vesicular machinery for antigen capture, processing, sequestration, and presentation. Efforts to extend these studies to other antigen-presenting cells such as dendritic cells are actively underway.