Visualizing High-Efficiency HIV Transfer
An image of the HIV virological synapse shows a CD4-positive T cell (yellow) extending a finger-like projection into a pocket on the surface of a dendritic cell (pink) to make contact with HIV particles (red circles) sequestered there. Transfer of a virus from the dendritic cell requires HIV gp120 protein binding to its receptor, CD4, on the T cell surface. (PNAS 2010 107(30):13336-41.)
The Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immunodeficiency Syndrome (AIDS), infects and eventually kills CD4 receptor-expressing T cells, which are critical for proper immune system function. The gp120 protein on the surface of HIV particles is known to bind CD4 and a co-receptor, either CCR5 or CXCR4, leading to fusion of the virus and T cell membranes and infection of the cell. The most efficient means of viral infection occurs when an uninfected T cell interacts with a dendritic cell (DC) that has previously come in contact with HIV. Antigen presenting cells, such as DCs, normally circulate throughout the body binding or engulfing foreign material and presenting it to T cells to initiate an immune response. HIV takes advantage of this close cell-cell association to propagate, so knowing the cells’ spatial arrangement during viral transmission could elucidate novel modes of treatment.
Previous studies used two-dimensional imaging techniques to examine slices of the virological synapse, the intersection of a virus-bearing DC and a CD4-positive T cell. Since a cross-section could miss key information about the architecture of the synapse or additional interactions between the presenting cell and the T cell, Richard Felts, Ph.D., working under Sriram Subramaniam, Ph.D., in the Center for Cancer Research Laboratory of Cell Biology and their colleagues decided to obtain a more complete picture by using three-dimensional electron microscopy and high resolution light microscopy. They recently published their results in the Proceedings of the National Academy of Science.
The researchers chose to examine a very early stage of virological synapse formation before the productive infection of either the DC or the T cell. To ensure that the methods used to treat the cells and prepare them for visualization did not significantly alter their structure, the scientists used a powerful, light microscopy (two-dimensional) technique. As observed previously, the HIV particles associated with the actin cytoskeleton of the DC and blockade of actin polymer formation disrupted virus particle localization, suggesting that the treatment methods did not affect cellular morphology.
To investigate the three-dimensional structure of the DC-T cell HIV virological synapse, the scientists employed ion abrasion-scanning electron microscopy (IA-SEM). In IA-SEM, successive images are taken as an ion beam is used to remove a defined thickness of the sample, essentially taking pictures of multiple sample slices. The images are then stacked to generate a three-dimensional structure.
Via IA-SEM the researchers observed several unique features of the DC-T cell association. HIV particles taken up by DCs localized to deep membrane folds accessible to the cell surface through narrow channels. T cells were able to reach the secluded viral particles by extending finger-like membrane projections into these DC pockets. Within the HIV-containing compartment, the DCs also produced membrane protrusions allowing synapse formation by bringing viral particles within close proximity to the T cell extensions. Most surprisingly, large protrusions or sheets of DC membrane surrounded most of the remaining T cell surface, effectively sequestering the T cell from the extracellular environment. By encasing the T cell in membrane and gathering HIV into specific areas on the cell surface, DCs provide a protected location for productive synapse formation to occur.
The researchers then further examined the HIV particle distribution within the virological synapse. Two distinct confirmations were observed with approximately equal frequency. In one, viruses were found on both DC and T cell surfaces as well as at the tip and along the length of the T cell membrane extensions. In the other, contact between the DCs and T cells was detected, but viruses were only associated with the DC membrane. This indicated that more than the presence of virus and the close association of DC and T cell membranes are necessary for viral transfer.
To further investigate the requirements for viral transmission, the scientists incubated T cells with antibodies to block either the main gp120 receptor, CD4, or the co-receptor, CCR5, prior to the introduction of HIV-exposed DCs. The CD4 antibody had no effect on virological synapse formation, but caused a significant reduction in the number of viral particles transferred to the T cell surface. Treatment with the CCR5 antibody, however, did not affect HIV distribution to the surfaces of both T cells and DCs. These results suggested that HIV transfer only occurs when productive T cell infection is possible. This conclusion was confirmed when HIV failed to transfer to T cells treated with an antibody to the gp120 structure before CD4 binding, but not when cells were treated with an antibody that recognized CD4-bound gp120.
These studies by Felts, Subramaniam and colleagues provide a detailed picture of the architecture of the HIV virological synapse. They revealed several unique features of HIV transmission, including the seclusion of T cells from the extracellular environment by DC membranes, the sequestration of HIV particles in deep pockets on the DC surface, and the binding of gp120 to CD4 as the critical regulatory step in viral transfer. Future studies can put this structural information to use designing better therapeutic intervention strategies for HIV.Summary Posted: Sun, 08/01/2010
Proc Natl Acad Sci USA 2010 Jul 27; 107(30):13336-41 PubMed Link