A new path for HIV entry
the-scientist.com
Posted by Edyta Zielinska, 30th April 2009
A new study overthrows a long-held theory on how HIV finds its way into host cells. Rather than fusing directly with the host cell membrane, the virus is first engulfed by it to form a vesicle that releases its contents into the cytoplasm, a study published tomorrow (May 1) in Cell reports. The findings may suggest other therapeutic avenues for targeting HIV, the researchers say.
 Image: A single virus (yellow) co-labeled with a membrane (red) and content (green) markers migrates toward the cell nucleus and releases its contents into the cytosol by fusing with the endosomal membrane.
"This is such an extensive and thorough paper that I think people are going to accept this as the dominant mode of viral entry" for HIV, said Robert Blumenthal, head of the Membrane Structure and Function Section at the National Cancer Institute in Frederick, Md., who did not participate in the research.
Enveloped viruses such as HIV can enter their host cells in one of two ways. Some invade the cell by fusing their membrane envelopes with the cell's membrane. Others are engulfed by the plasma membrane into vesicles that break off inside the cell in a process called endocytosis. The virus-containing vesicles then fuse with other vesicles which contain a highly acidic environment; the lower pH changes the shape of viral proteins in such a way that the virus releases its genetic contents into the cytoplasm.
Because HIV does not require a low pH for fusion and uses its envelope protein to bind directly to receptors on the host cell's surface, CD4 and its co-receptors CCR5 and CXCR4, researchers have long assumed that the fusion occurred at the surface. But Gregory Melikyan from the Institute of Human Virology at the University of Maryland School of Medicine, the lead author of the current study, had his doubts.
"When you start thinking about it, you realize that there's no direct evidence" for fusion at the cell membrane, said Melikyan. The interaction between receptors and membrane, as well as the fact that the process doesn't need an acidic environment, don't rule out the possibility that these particles require endocytosis, he explained.
To test this possibility, Melikyan and colleagues examined HIV fusion in the presence of two types of fusion inhibitors -- one that acts exclusively at the cell membrane and another that stops all fusion -- both in the endosomes and at the cell surface. If HIV indeed fused at the cell membrane, then Melikyan expected both inhibitors to have identical effects at every time point tested. Instead, researchers observed a lag in viral fusion, suggesting an extra step in which the virus particles were being endocytosed.
To fine-tune the picture of the process, the researchers used two fluorescent stains to label two parts of the viral particle: the envelope membrane and the contents of the virus. They then followed individual virus particles as they fused with endosomes, observing that the viral contents were not released until they were inside the endocytic compartment.
The study is "going to open up the whole question of entry once more," said Mark Marsh the director of the medical research council at the University College London, who was not involved in the work. However, he said, "I wouldn't say this closes the issue," because it needs to be independently confirmed.
The results aren't likely to affect current therapeutic approaches that target HIV entry proteins, said Blumenthal. Those agents typically target earlier steps in the process, either inhibiting the virus before it attaches to the cell membrane or after it attaches but before it completes the release of its genome. But it may lead researchers to target the host's endocytic pathway in developing new drugs to treat HIV. He cautioned, though, that these approaches may have serious side effects. "The problem of targeting the host proteins is what it does to the host."
Melikyan said that other viruses believed to fuse at the cell membrane might also fuse via the endocytic pathway. While that idea has yet to be tested, "now we have the tools" to address it, he said. Just the same, Melikyan suspects that his research may ruffle a few feathers. "I expect more hate mail than congratulations," he said.
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