AEGiS-GMHC: Viral Entry Discovery Suggests New Treatments

Important note: Information in this article was accurate in 1996. The state of the art may have changed since the publication date.

Viral Entry Discovery Suggests New Treatments

GMHC Treatment Issues, Volume 10, Number 5 - May 1995
Theo Smart

A central mystery surrounding the infection of CD4 cells by HIV appears to have been solved by Edward Berger, M.D., and colleagues at the National Institute of Allergy and Infectious Diseases (NIAID). Scientists have long known that the envelope protein of HIV binds to the CD4 receptor on certain types of white blood cells. But that action by itself was not adequate to fully explain how the virus fuses with and inserts its genetic material into a cell. In fact, non- human cells altered to bear the human CD4 receptor were not infectable with the virus. There had to be an unidentified co-factor on human cells to which the virus clung, and investigators have spent a decade searching for it.

The NIAID investigators now have identified the co-factor as a cell surface protein they call fusin due to its pivotal role in helping HIV fuse to CD4 cells. Their discovery reportedly rapidly has been confirmed by a number of other labs. The discovery of fusin is considered a breakthrough not only because it reveals a new target for anti-HIV drugs but because it may lead to the first true animal model for HIV infection.

Gene Libraries

Dr. Berger's team already had a model for HIV fusion using mouse cells modified to carry CD4 receptors. But, again, merely having a CD4 cell receptor did not make the mouse cell susceptible to HIV infection. To find the fusion cofactor, the researchers introduced a series of genes from a human CD4 cell line into the genetically engineered mouse CD4 cells and fusion occurred. Certain that one of these human genes was making a protein that allowed fusion, the researchers used a process of elimination to isolate the gene responsible for the fusin protein.

The fusin gene proved to be a member of the gene family that produces "G protein-coupled" cell receptors, often exploited by other viruses when entering cells. In contrast to the protruding CD4 receptor, G protein-coupled receptors weave in and out of the cell membrane. They form a depression that usually contain these receptors' critical binding region. If HIV binds to such a receptor on CD4 cells, it is already partially inside the cell.

Fusin as an Antiviral Target

To see whether fusin protein was required for infection of human CD4 cells, the researchers mixed CD4 cells with antibodies to fusin and effectively blocked infection of the CD4 cells. Antibodies to the V3 loop on the HIV envelope protein molecule gp120 also blocked fusion, suggesting that HIV binds to fusin via this highly conserved loop, although this has yet to be demonstrated. A number of other researchers have also observed that antibodies to the V3 loop neutralize the virus and block fusion.

Besides underscoring that fusion could not occur without fusin, the antibodies' antiviral activity raised the prospect that compounds interfering with this protein may block infection of CD4 cells.

But if this cofactor plays a role in the infection of CD4 cells, it would seem that simple drug screening tests would have picked up fusin blockers long ago, even if no one understood exactly how the drug worked. Indeed, other researchers screening potential anti-HIV drugs have found a few compounds believed to inhibit viral entry other than by blocking gp120 binding to the CD4 receptor. These chemicals include RhÖne-Poulenc Rorer's betulinic acid derivatives, distamycin analogs from Pharmitalia, and the bicyclam compounds owned by Johnson Mathey.

"It is possible that some of these compounds actually interfere with fusion," says William Rice, Ph.D., head of the Laboratory of Antiviral Drug Mechanisms at the National Cancer Institute (NCI). It is conceivable, too, that these compounds bind to the part of the gp120 V3 loop that may attach to the fusin receptor. A recent laboratory study of the bicyclams found that HIV reacted to these compounds precisely by acquiring resistance-conferring mutations in the V3 loop.

None of these substances have made it into human studies, and Rhone-Poulenc says it has stopped research into betulinic derivatives due to poor "pharmacodynamic properties." Rice thinks that many pharmaceutical companies have a bias against drugs that act at this stage of the infection process: "It's very hard to push forward surface-active agents [drugs that prevent the virus from entering the cell]," he says, "because of the problems with dextran sulfate and some of the other sulfated polysaccharides." These drugs blocked infection by coating the surface of the cell, but they also caused hemorrhaging in clinical trial participants.

According to Dr. Rice, the NCI currently is planning clinical trials of one of the Pharmitalia compounds, which has recently shown anti-HIV activity in an animal model. Dr. Rice believes that the discovery of the fusin protein will lead to the development of more precise laboratory tests more capable of detecting more fusion-specific inhibitors. Meanwhile, Johnson Mathey wants to launch phase I studies of JM3100 within the year. This company's biomedical group is small, though, so the company is trying to get help to run the studies from the AIDS Clinical Trials Group.

One problem with developing drugs that directly block fusin, is that no one yet knows what human protein normally binds to it. Antivirals that work by blocking fusin may interfere with its natural function. The part of the natural ligand that binds to fusin also may share homology with sequences from the V3 loop of HIV. Some researchers have found similarities between segments of the V3 loop and parts of MHC-II proteins, involved in antigen presentation to CD4 cells.

The Chemokine Connection

Another potential shortcoming of anti-fusin drugs is that the researchers found that although the protein seemed necessary for the infection of CD4 cells, anti-fusin antibodies had no effect on the infection of macrophages. Drugs that are too specific to fusin may not block infection of macrophages. [Last minute note: Anne Bousseau, RhÖne-Poulenc's AIDS Program Director, told Treatment Issues that betulinic derivatives also block HIV entry into macrophages. These compounds interact with gp120 outside the V3 loop and produce an effect more general than mere inhibition of V3 loop-fusin would produce.]

Dr. Berger's team observed that fusin closely resembles a receptor for IL-8, one of the chemokines involved in directing lymphocytes to infected tissue. The group believes that a different but possibly related cofactor allows the HIV to fuse to macrophages. This suspicion is strengthened by reports that the V3 loop of gp120 determines cell tropism -- strains of HIV that infect macrophages more efficiently have slightly different V3 loops than the HIV that concentrates on CD4 lymphocytes. Given the similarity between fusin and the IL-8 chemokine receptor, the fusin-like protein on macrophages may be the receptor for Dr. Robert Gallo's anti- HIV chemokines: Rantes, MIP-1a and MIP-1b (see January's Treatment Issues, pages 10-12). The evidence for this association is circumstantial at present.

Animal Models

Even if this breakthrough in basic science does not lead to any new therapies, it may have one other practical implication: the development of a small animal model for HIV. The lack of the fusion co-factor had made this impossible. Scientists could alter mouse or rabbit cells to carry human CD4 cell receptors, but these cells could not be infected with HIV. Now, with the same gene altering techniques that the team used to isolate the fusin gene, they could genetically modify animals to have lymphocytes with both human receptors. Such an animal model would speed the identification and development of the most promising anti-HIV compounds.

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