Protein study suggests ways to help humans thwart virusesWEST LAFAYETTE, Ind. Scientists have unraveled a genetic anomaly that protects some mice from a common cancer-causing virus.
The findings may help develop gene therapies that can be used to help humans defeat similar viruses, such as the human T-cell leukemia virus and the AIDS virus, says David A. Sanders, associate professor of biological sciences at Purdue University and lead author of the study.
Sanders and his research group at Purdue analyzed proteins in mice that are resistant to the ecotropic murine leukemia virus, a major cancer-causing virus that occurs only in mice. A very similar virus is the agent of feline leukemia, the major cause of serious illness and death in cats.
Scientists have known for years that mice that have an active Fv-4 gene are resistant to the virus, though it wasn't clear how this resistance occurred.
Their findings show how a defective protein found in mice with the Fv-4 gene works by binding with the receptor that normally serves as a doorway for viral entry, and thereby blocking the door.
"The protein looks normal, and is processed normally and incorporated into virus particles, but it is unable to promote viral entry," Sanders says. "By identifying the defect in this mouse protein and introducing it into another virus, we have found a potential new avenue for preventing viruses from entering cells."
Sanders says the method may someday be applied to block similar viruses, which belong in a class known as retroviruses. Retroviruses are a major group of cancer-causing viruses that use a unique production method to copy and insert their genetic material into a host cell.
Human cells also contain specialized receptors for retrovirus entry, Sanders says.
"Such receptors are often very specific," he says. "We know, for example, that people who don't have the receptor for HIV are resistant to HIV."
His research group is now working to apply knowledge of how this genetic defect may be used to develop genetic therapies to help block retroviruses in human cells.
In the study, Sanders and his team compared the sequence of the Fv-4 protein produced from a "blueprint" carried on the Fv-4 gene to proteins found in other leukemia viruses. They found a striking likeness between its sequence and that of the Moloney murine leukemia virus envelope protein, a protein located on the membrane of the leukemia virus. Envelope proteins allow the virus to bind to a host cell and promote the fusion of the virus and cell membranes. It is through this fusion process that the virus enters the cell.
The group then analyzed the two proteins to determine any structural differences.
"We wanted to understand how the Fv-4 protein which provides resistance to the virus differed from similar proteins that allow viral infection to occur," Sanders says.
While the analysis of the protein structures identified a number of differences, Sanders says one structural difference stood out from the rest a substitution of one type of amino acid in the Fv-4 protein for another.
In this case, arginine was substituted for glycine. Arginine and glycine are among 20 amino acids which link together to form proteins. Because each amino acid has a unique structure and properties, substituting one amino acid for another in a protein can cause the protein to perform differently, Sanders says.
"We thought it was possible that this one particular change was responsible for making it prevent infection rather than allowing infection," he says.
To test their theory, the group took a mutated section of the mouse protein and inserted it into the Moloney murine leukemia virus protein. They then used cell cultures to determine what changes occurred.
The results showed that the single change prevented viral infection by binding to the receptor used for viral entry. By blocking entry to the virus, the protein shuts down the virus' ability to propagate and make more virus.
"We found that proteins with this mutation not only prevent the virus from reproducing, but it actually prevents viruses from entering cells," Sanders says. "And if the virus does happen to get into the cell, which occurred in a few cases, it makes the virus that comes out (after the cell manufactures new viruses) less infectious. Those are all exactly the properties one would want to have in such a modified protein."
Sanders' research was funded by the National Institutes of Health.
The Purdue group is now working to apply this knowledge to develop genetic therapies to help block retroviruses in human cells.
"Ideally, we could introduce a gene for an HIV envelope protein that is defective in membrane fusion," Sanders says. "Introducing such a gene into cells would protect cells at the entry step and, if virus happened to make it in, it would reduce the infectivity of new viruses manufactured in, and released from, the cell."
Sanders says such a genetic therapy approach could be used not only in individuals who have not yet been infected with HIV, but may provide protection for those who already have been infected by the AIDS virus.
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Fv-4: Identification of the Defect in Env and the Mechanism of Resistance to Ecotropic MuLV
Gwen M. Taylor, Yi Gao, and David Avram Sanders
Mice expressing the Fv-4 gene are resistant to infection by ecotropic murine leukemia viruses (MuLV). The Fv-4 gene encodes an envelope protein whose putative receptor binding domain resembles that of ecotropic MuLV envelope protein. Resistance to ecotropic MuLVs appears to result from viral interference involving binding of the endogenously expressed Fv-4 protein to the ecotropic receptor, although the immune system also plays a role in resistance. The Fv-4 env protein is processed normally and can be incorporated into virus particles but is unable to promote viral entry. Among the many sequence variations between the TM subunit of the Fv-4 env protein and the TM subunits of other MuLV envelope proteins there is a substitution of an arginine residue in the Fv-4 env protein for a glycine residue (gly-491 in Moloney MuLV Env) that is otherwise conserved in all of the other MuLVs. This residue is present in the MuLV TM fusion-peptide sequence. In this study gly-491 of the Moloney MuLV Env has been substituted with other residues and a mutant Env bearing a substitution for gly-487 was also created. The G491R recapitulates the Fv-4 env phenotype in cell culture indicating that this substitution is sufficient for creating an Env that can establish the interference-mediated resistance to ecotropic viruses produced by the Fv-4 gene. Analysis of the mutant MuLV Envs also has implications for an understanding of the role of conserved glycine residues in fusion peptides and for engineering organismal resistance to retroviruses.