Fusion is the third step – following attachment to the CD4 receptor and subsequent interactions with cellular coreceptors – of HIV's entry into cells. But just where the virus unleashes its contents has been a point of contention, with most experts thinking it occurs at the cell surface. Now, University of Maryland, Baltimore, researchers using imaging techniques say HIV fusion happens within the cell via fusion with an endosome, a tiny packet formed when a stretch of cell membrane engulfs the virus then pinches itself off inside the cell. (Cell 2009; 137: 433–444)
To shed light, literally, on the site of virus entry, a team lead by Gregory Melikyan performed a series of experiments using fluorescent dyes attached to HIV. One dye latched into lipids in the viral envelope, appearing red under an optical microscope. A second dye, this one fluorescing green, was loosely trapped within the viral envelope and marked the whereabouts of the viral contents. As long as the virus remained intact, the fluorescent dye molecules were tightly crammed on or within the tiny virus forming a point source of yellow light, an optical result of both dyes being so close together.
During fusion, however, the lipid-loving dye molecules on the viral envelope redistribute themselves onto the lipids in the much larger plasma membrane of the cell surface. Likewise, the dye molecules marking the viral contents disperse into the cell's interior, or cytosol. In both cases, dilution of the dye molecules causes their fluorescence to disappear.
When the team exposed HIV-infectable HeLa-derived cells to fluorescently tagged HIV-1, the researchers saw the pinpoint of light shift from yellow to red. The change in color signaled that the green dye marking the contents of the virus had diffused out but the red dye incorporated on the viral envelope remained confined to a small area. The observation suggests HIV enters cells via the formation of an endosome, and fusion occurs with the endosome, the researchers say.
According to their interpretation, HIV attaches and binds to CD4 and coreceptors on the cell surface but is simultaneously engulfed and isolated by the membrane itself as it forms an endosome. In the experiments, the fluorescence remained yellow because the dye on the viral envelope diffused onto the only-slightly-larger stretch of cell membrane, now an endosomal membrane, wrapped around the virus. Meanwhile, the vesicle was shuttled through the cytosol.
As fusion was completed near the cell nucleus, the green-tagged contents of the virus spilled into the cytosol. As a result, the point source of green fluorescence disappeared leaving behind a compact assembly of dye molecules still clinging to the endosomal membrane. The pinpoint of light changed from yellow to red.
In contrast, additional experiments showed that viral fusion with the cell plasma membrane failed to progress beyond the lipid-mixing step. This appeared as a color shift from yellow to green, which signaled that the green-dye molecules remained trapped inside the virus whereas the red dye marking the viral envelope had dispersed to lipids of the cell membrane.
The team also performed experiments using fusion inhibitors targeting viruses that were either bound to cells or those that were engulfed in endosomes. Those experiments further validated endosomes as the entry vehicle for HIV-1 into HeLa-derived cells and T cells. The researchers also found that an enzyme needed for endocytosis, dynamin, was critical to HIV-1 fusion with endosomes.
‘Endocytic entry offers several advantages,’ Melikyan says. ‘Antibodies against CD4-induced epitopes of HIV envelope glycoprotein and certain fusion inhibitors are in a race against time to catch the virus in transition due to a relatively short time of exposure on the cell surface. Endocytosis limits the time HIV spends there. Secondly, HIV gets a free ride to the nucleus and nuclear membrane. So, it's exposed for a shorter time to cellular defenses like restriction factors.’ Thus, the researchers note, the next generation of HIV entry inhibitors will have to permeate the cell membrane to efficiently block fusion.