The discovery of a protein, fusin, on the surface of CD4+ T-cells that acts as a cofactor for the entry of T-cell tropic strains of HIV-1 was reported by Dr. E.A. Berger and co-workers, National Institute of Allergy and Infectious Diseases (NIAID; Bethesda, MD) in the May 10 issue of Science. CD4 (cluster designation 4) has long been known to be the primary receptor for HIV entry into lymphocytes and other HIV-susceptible cells bearing these receptors, but since 1986 it has been known that one or more other unidentified cofactors are also involved in HIV attachment and entry into CD4+ lymphocytes. Fusin, on the surface of CD4+ T-cells that acts as a cofactor for the entry of T-cell tropic strains of HIV-1 was reported by Dr. E.A. Berger and co-workers, National Institute of Allergy and Infectious Diseases (NIAID; Bethesda, MD) in the May 10 issue of Science. CD4 (cluster designation 4) has long been known to be the primary receptor for HIV entry into lymphocytes and other HIV-susceptible cells bearing these receptors, but since 1986 it has been known that one or more other unidentified cofactors are also involved in HIV attachment and entry into CD4+ lymphocytes. ous non-human cell types genetically modified to express surface CD4 are resistant to HIV-infection. Dr. Berger has reported, ÒOur data indicate that a second molecule, fusin, also is required for fusion and entry of certain strains of HIV into cells. With this new information on a fusion cofactor in hand, we hope to determine whether similar cofactors exist for other HIV isolates, and the relevance of these cofactors to the HIV disease process.Ó The discovery of fusin provides another target for anti-HIV drug development, particularly drugs to block fusin, and new approaches to developing animal models for HIV-infection. Several other labs have already confirmed fusin as a cofactor for HIV entry into CD4+ cells.

The researchers used a sensitive assay (described in the September 1994 issue of the Journal of Virology) to identify fusin as the CD4 cofactor. This assay involves use of vaccinia virus vector transformed murine NIH 3T3 cells expressing CD4 to determine whether other transformed cells expressing HIV-1 envelope and a reporter gene have fused with CD4. Cell fusion results in expression of the reporter gene (E. coli lacZ), turning the fused cells a visible blue color when exposed to a special stain. Vaccinia virus vectors were used to transform NIH 3T3 cells with human CD4 cDNA and a library of all cDNA sequences from a human cell line known to be ÒpermissiveÓ for HIV-1 fusion and infection. The transformed CD4+ murine cells containing the entire library of cDNA sequences were shown to weakly fuse with HIV-1, indicating that one (or more) of the component cDNAs encoded a fusion cofactor. The library was divided into fractions, screened, and a single cDNA sequence (about 1.7 kb; encoding fusin) enabling HIV fusion when accompanied by CD4 was identified. Fusin has seven transmembrane segments and is similar in sequence to the well-known superfamily of G protein-coupled receptors that have various functions in human cells. Other G protein-coupled receptor proteins are used for cell entry by different pathogens, including Plasmodium vivax (which causes malaria). These receptors are complexed with G proteins that function to transfer signals into the interior of cells and are already the targets for various drugs.

Once isolated, fusin was demonstrated to confer susceptibility to HIV-1 fusion and infection in transformed monkey and mink CD4-expressing cells and certain human cells, each of which by themselves lack this ability. Antibodies to a synthetic peptide representing the predicted NH2-terminal extracellular portion of fusin were shown to block HIV-1 infection of permissive CD4+ lymphocytes obtained from healthy volunteers. Fusin was found to be most active using T-cell line-tropic HIV-1 laboratory strains that preferentially infect CD4+ T-cells, while the activity of fusin was considerably less pronounced using macrophage-tropic HIV-1 isolates, with the differences in activity probably due to variability of HIV-1 envelope protein. Fusin variants or other cofactors are probably involved in fusion and infection of CD4+ cells by other retroviruses, such as HIV-2 and SIV. The mechanism of fusin is not known and could involve direct interaction with CD4 and HIV envelope protein or indirect mechanisms, perhaps involving G protein signaling within the cell.

One of the first applications of the discovery of fusin will likely be the development of animal models for the study of HIV-1 infection, and use of these for screening and developing anti-HIV drugs and vaccines. Transgenic mice and rabbits that express human CD4 have been developed, but these poorly support HIV-1 replication. Presumably, transgenic animals expressing both human CD4 and fusin should support HIV-1 replication and infection. Another line for investigation into the pathogenesis of HIV-infection has opened up now that a putative G protein-coupled receptor has been shown to be involved. Since T-cell line-tropic strains of HIV-1 tend to arise during later, symptomatic stages of HIV-infection and are associated with declines in CD4+ T-cell count, aberrant G protein signaling may be involved in CD4+ T-cell death. Fusin may also be involved in the binding and function of chemokines (see December 1995 Bulletin, p. 358), endogenous factors which have been shown to suppress HIV-infection. Fusin shares similarities (37% amino acid homology) with a chemokine (interleukin-8) receptor. Defective fusin or other cofactors, perhaps interacting with chemokines, may be one of the reasons why some people have not become infected with HIV-1 despite repeated exposures and explain the vast range of individual differences in time to disease advancement observed in HIV-infected persons. The NIAID technology transfer office reports that patent applications have already been filed and there is considerable interest from industry in licensing fusin for drug discovery and screening. The availability of fusin for licensing is expected to be announced soon in the Federal Register. Although the discovery of fusin is a major advance, it may not easily translate into new drugs and vaccines since various strains of HIV commonly infect other human cells that lack fusin, such as macrophages. One or more fusin variants and/or other unidentified cofactors are likely involved in HIV pathogenesis.