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MICROBIOLOGY Virus Passes Acid Test for Entering CellsDiscovery Upsets a Central Dogma in Virology Some experiments are so elegantly conceived and convincingly carried out—their results so widely embraced—that few people think to repeat them years later. A team of HMS scientists has done just that, with potentially paradigm-shaking consequences for virology. Not only do their results contradict previous findings, they overturn a central dogma about how viruses invade cells.
"We were so shocked by our findings that we really had to convince ourselves," said Walther Mothes (left), shown with co-author Adrienne Boerger. "I think now that previous experiments were not wrong but assumptions at the time were too narrow." —Graham Ramsay To wangle their way into the cytoplasmic core of a cell, viruses must first fuse with the cell's outer membrane. The intruders were thought to accomplish this critical step in one of two ways and in one of two locations—either through exposure to the acidic environment inside the balloonlike endosome within the cell or by binding to receptors on the cell surface. The acid test for the first, or low pH, mode of entry was established many years ago in a classic series of experiments. Viruses such as HIV and avian leukosis virus (ALV) failed this test. So thorough seemed the experiments that a dogma quick-ly sprang up that viruses were either pH-dependent or receptor-dependent, a distinction that appeared to brook no middle ground. Walther Mothes, Adrienne Boerger, and their colleagues recently repeated those classic experiments on ALV, using methods that enable them to scrutinize their viral subjects earlier in the infection process, and under more precise conditions. By their more refined measures, ALV passes the acid test—it depends on low pH to fuse—and it also depends on receptor interaction. So it requires both. The findings appear in the Nov. 10 Cell. The discovery of a new hybrid category of viral fusion promises to shake up the field of virology. To begin, it uproots the entrenched two-part classification system, and with it, much previous work in virology. "It's a complete paradigm shift," said John Young, who recently moved from the HMS Department of Microbiology and Molecular Genetics to become the Howard Temin professor of cancer research at the University of Wisconsin at Madison. He and Jim Cunningham, a Howard Hughes investigator and HMS associate professor of medicine at Brigham and Women's Hospital, are among the co-authors on the paper. "The entire field of virology has to go back now and reclassify viruses based on the new information," said Young. If ALV fusion requires low pH, the same may be true of other so-called pH-independent viruses, he said. And some pH-dependent viruses may require receptor priming. "These things may have been missed because of the way that scientists have set up their experiments. They haven't been looking for it," Young said. There is another implication of the new discovery. Until recently most virologists took it for granted that ALV and other classic pH-independent viruses were fusing at the surface of the cell. But the new discovery suggests that ALV fusion may happen in the endosome. Mothes, who is a Jane Coffin Childs fellow at BWH, thinks the same may be true of some of the other viruses. "I think the real classification for viral fusion will turn out to be endocytosis-dependent versus endocytosis-independent," he said. Such a distinction could have far-reaching implications. ALV is a promising vehicle for introducing therapeutic genes into cells—in fact, Young is developing a gene therapy delivery system based on ALV (see Focus July 17, 1998). Understanding how the virus gets into cells could enhance such efforts. Knowing how particular viruses worm their way into cells could also aid efforts to thwart them. Intriguingly, researchers elsewhere have shown a strain of HIV to be pH-dependent. Fusion ConfusionMothes and his colleagues had no intention of scrutinizing, let alone overturning, the received doctrine in virology when they began their experiments in 1998. "The dogma was so strong I thought, 'Fusion is done,'" said Mothes. Turning instead to the step just after fusion, during which the virus sheds its coat and spills its genetic contents, he set up a PCR-based experimental system.
To enter the cytoplasm of a cell, viruses must first fuse with the cell's outer membrane. The bugs were thought to accomplish this critical step in two ways. Some, such as the influenza virus, are engulfed by the cell membrane and transported inside by a membrane-lined endosome, which becomes more and more acidic as it moves deeper into the cell. The increasingly acid environment triggers the virus to fuse with and eventually break through the endosome wall. Other viruses fuse by binding to receptors at the cell surface. ALV was thought to fall into the latter category, but researchers have shown that it fuses as a result of both receptor binding and low pH, probably in the endosome. —Courtesy of Walther Mothes He placed ALV and target cells in a buffer that prevents the acidification of the endosome. In the 1990 classic experiments, researchers had shown that inhibiting acidification of the endosome failed to affect ALV's ability to fuse, hence its classification as pH-independent. Mothes expected the same. "But we saw nothing—no fusion. That came as a shock," he said. Mothes and his colleagues decided to rerun the other classic experiments to see if their results were just a fluke. Cells infected by virus often fuse together, producing multinucleated cells, or syncytia. In the first experiment, researchers found that classic pH-dependent viruses, when exposed to low pH, quickly formed syncytia while ALV-infected cells did not. "But they looked for only one and a half minutes," said Mothes. "We looked for 10 and saw syncytia." All along the researchers had been wondering about their first experiment—why did they inhibit fusion at neutral pH when their predecessors could not? Timing, they realized, could provide an answer. Earlier researchers, following standard practice, waited days before monitoring the effects of the acid-inhibiting reagents on fusion. Because the reagents were toxic and had to be washed out after six hours, the researchers could detect only those viruses that remained inhibited after the six-hour treatment. Using their PCR assay, Mothes and his colleagues were able to monitor the fusion process much earlier, before the acid-inhibitor was washed out. They observed that ALV fusion was inhibited in the neutralized environment but recovered its ability to fuse once the neutralizing reagent was cleared—which is why it seemed unaffected by pH in the earlier assay. Convinced that low pH plays a role in ALV fusion yet wondering whether it also requires receptor priming, Boerger, a former graduate student in the Biological and Biomedical Sciences program, reran yet another of the classic experiments. In that third experiment, she exposed virus to low pH prior to infection. While classic pH-dependent viruses such as influenza virus were unable to infect target cells—presumably because their fusion apparatus had been prematurely triggered—ALV was unaffected. But when she preloaded ALV with receptor and exposed it to an acidic solution, the virus was rendered inactive—suggesting both receptor and low pH were needed to trigger fusion. As for how receptor priming and low pH conspire to provoke fusion, "we think priming may bring about a small conformational change that uncovers a pH sensor," Mothes said. It's a preliminary sketch, he said, and one that is open to revision. Ten years from now someone could define the pathways more precisely. "The entire package right now is very compelling—but it was in 1990, too," said Mothes. "Experiments that seem to give clear answers may turn out to be meaningless because you may discover there are many cofactors that go into the system. With a deeper understanding, it changes. We're still on the road here." —Misia Landau |
