NIAID Researchers Identify Cofactor for Entry of HIV into Cells
Researchers at the National Institute of Allergy and Infectious Diseases (NIAID) have identified a cell molecule, designated "fusin," that acts as a cofactor for the entry of HIV into immune system cells.
Edward A. Berger, Ph.D., chief of the molecular structure section of NIAID's Laboratory of Viral Diseases, and his colleagues report their findings in the May 10 issue of Science. Dr. Berger's co-authors are Yu Feng, Ph.D., Christopher C. Broder, Ph.D., and Paul E. Kennedy, B.S.
We've known since the mid-1980s that a molecule called CD4 is the primary receptor for HIV on immune system cells," says Dr. Berger. "Our recent 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."
This is a very important study. These findings provide new insights into the mechanisms of HIV's entry into cells, and further elucidate our understanding of the pathogenesis of HIV disease," says Anthony S. Fauci, M.D., NIAID director. "This provocative work also suggests possible new targets for anti-HIV therapies as well as approaches to developing better animal models of HIV infection."
Scientists have known since the early years of the AIDS epidemic that immune system cells with a specific surface molecule called CD4 ("cluster designation" 4) are the preferred targets of HIV. Cells with CD4 on their surface are called CD4+ ("CD4 positive") cells.
But CD4 alone is not sufficient to allow HIV or HIV-infected cells to fuse with target cells and infect them. Several lines of evidence have suggested that one or more cofactors, presumably molecules on the surfaces of target cells, are necessary for HIV fusion and entry. For instance, researchers have observed that although human cells that normally lack CD4 can be infected with HIV if they are genetically altered to express CD4, non-human cells generally cannot be infected with HIV, even if they are altered to express CD4.
Dr. Berger and his team zeroed in on a cofactor for the entry of HIV-1 into cells using a sensitive assay they previously described in the September 1994 Journal of Virology. (HIV-1 is the type of HIV that has caused most AIDS cases worldwide. A second type, HIV-2, found mostly in western Africa, also causes AIDS. A related virus, simian immunodeficiency virus (SIV), causes AIDS-like syndromes in monkeys and other non-human primates.)
The assay requires introducing a reporter gene into cells using a vaccinia virus vector, and allows researchers to determine whether cells expressing the HIV-1 envelope protein (Env) have fused with CD4+ target cells. When fusion occurs, the reporter gene is switched on and produces an enzyme that turns the fused cells blue when the culture dish is treated with a special stain. In the absence of cell fusion, the enzyme is not produced, and cell cultures do not turn blue upon staining.
In their experiments, the researchers used vaccinia virus to introduce into one culture dish of mouse cells the DNA sequence (cDNA) encoding human CD4, as well as a "library" containing all the cDNAs from a human cell known to be "permissive" for HIV-1 fusion and infection. Since the human cell was permissive, they predicted this library would contain a cDNA for the cofactor necessary for fusion. Into a control culture dish of cells, they introduced the CD4 cDNA and a single cDNA chosen randomly from the library.
These two groups of cells, which now expressed CD4 on their surfaces, were separately incubated with another group of cells that contained the reporter gene and expressed HIV-1 Env. Env binds to CD4; this is the first step before the fusion process that enables HIV-1 to enter cells.
The researchers found that after staining, the culture containing the entire library of cDNAs had many more blue cells than did the culture containing only the random cDNA from the library.
"This suggested that somewhere in the library was the cDNA that encoded a fusion cofactor," says Dr. Berger.
The investigators then divided the library into fractions, and screened each fraction for its ability to promote fusion. By this process, they found a single DNA sequence that enabled the CD4-expressing cells to undergo fusion with Env-expressing cells. Analysis of this DNA sequence revealed it to be similar to sequences that encode proteins of a well-known "superfamily" of receptors called G protein-coupled receptors, which have many varied functions in the body.
"This family of receptors is extraordinarily common and diverse in the body," says Dr. Berger. "Interestingly, other members of this family are exploited for entry into human cells by different pathogens, including the malaria parasite Plasmodium vivax. Most of these receptors are complexed with specialized proteins, called G proteins, that send signals to the interior of the cell, and are the specific targets of many drugs currently in use for a variety of ailments."
Additional experiments demonstrated that non-human cells, such as those from mice, monkeys and mink, became permissive for HIV-1 fusion and infection after the introduction of cDNAs for both fusin and CD4, but not after the introduction of the CD4 cDNA alone.
Certain types of human cells are resistant to HIV-1 fusion and infection even when altered to express CD4. Dr. Berger and his coworkers obtained evidence that these cells do not produce fusin; when the fusin cDNA was introduced, however, these cells became permissive for HIV-1 fusion.
The NIAID investigators also conducted experiments with permissive human CD4+ cells taken from healthy volunteers. They found that antibodies to fusin blocked HIV-1 infection of these cells, further underscoring fusin's role in fusion and entry.
Significantly, the researchers found the activity of fusin to be most pronounced in experiments using isolates of HIV-1 that preferentially infect laboratory cell lines having the properties of CD4+ T cells. Such HIV-1 isolates are called "T-cell line-tropic." Other HIV-1 isolates preferentially infect different CD4+ cells called macrophages. The investigators found the activity of fusin to be considerably less pronounced with these "macrophage-tropic" isolates of HIV-1. Differences in the tropisms of HIV-1 isolates are probably due to the variability of the Env protein.
"Many HIV-1 isolates from the body can infect both T-cell lines and macrophages," says Dr. Berger. "We don't yet know if there are only two fusin-like molecules, or a whole family of them. The search for additional fusins is ongoing in our lab and elsewhere. Other viruses, including HIV-2 and SIV, probably also use distinct fusins."
How does the fusin molecule work? "The simplest model is that fusin acts directly as a co-receptor in the fusion process, perhaps by interacting with Env or CD4 or both," says Dr. Berger. "Alternately, the role of fusin could be indirect, possibly involving a G protein signal to the cell." The identification of fusin suggests an immediate practical application: the production of a small animal model for study of HIV-1 infection. Such a model would be a valuable tool for developing anti-HIV drugs and vaccines.
"Transgenic mice and rabbits expressing human CD4 have been developed, but they support HIV-1 replication poorly," explains Dr. Berger. "Transgenic animals expressing both CD4 and fusin may be more useful." The interaction of HIV-1 with a putative G protein-coupled receptor raises many interesting questions related to HIV-1 pathogenesis, says Dr. Berger.
"Macrophage-tropic isolates are the main HIV types seen during the symptom-free stage of disease, which often lasts many years," Dr. Berger explains. "T-cell line-tropic HIV isolates appear as the infection progresses to the symptomatic phase, coincident with CD4+ T cell decline. Perhaps Env interactions with CD4 and fusin at this stage of infection trigger aberrant G protein signalling that contributes to CD4+ T cell death, either directly or indirectly. Further research is needed to address this speculative question."
Although the normal role of fusin in the body is unknown, some data suggest that the molecule may bind signalling proteins called chemokines. Several investigators have found that certain chemokines suppress HIV-1 infection of cells in the laboratory.
"At present the relationship between fusin and HIV-1 suppression by chemokines is unclear," says Dr. Berger. "However, ongoing research on fusins and chemokines may provide leads to the understanding of HIV-1 pathogenesis and possibly to the development of therapies.
Investigators also have identified a small number of people who have not become infected with HIV-1, despite repeated exposures to the virus. Other individuals show little or no decline in immune function over many years, despite being infected with HIV-1. "One explanation for these phenomena could be that CD4+ cells of these individuals are defective in fusin or related cofactors, and therefore not easily infected by HIV-1," says Dr. Berger. "Further studies may illuminate this issue."
NIAID is a component of the National Institutes of Health. NIAID conducts and supports research to prevent, diagnose and treat illnesses such as AIDS and other sexually transmitted diseases, tuberculosis, asthma and allergies. NIH is an agency of the U.S. Public Health Service, U.S. Department of Health and Human Services.
- Feng Y, Broder CC, Kennedy PE, Berger EA. HIV-1 entry co-factor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science 1996;272:872-7.
- Nussbaum O, Broder CC, Berger EA. Fusogenic mechanisms of enveloped virus glycoproteins analyzed by a novel recombinant vaccinia virus-based assay quantitating cell fusion-dependent reporter gene activation. J Virol 1994;68(9):5411-22.
NIAID press releases, fact sheets and other materials are available on the internet via the NIAID home page. The address is http://www.niaid.nih.gov.