By David N. Leff
HIV1 ¿ the human immunodeficiency virus ¿ is infamous for breaking and entering its immune system target cells and pirating their genome to generate multiple new viral particles. Less notorious is how these progeny slip away from that host cell and escape to perpetuate the viral cell cycle process.
¿That process can be broken down into seven steps,¿ explained biochemist and molecular biologist Kenton Zavitz, at Myriad Genetics Inc. in Salt Lake City. ¿First, the virus attaches to its target cell and is absorbed. Then, it starts reverse transcribing from RNA to DNA. Next, the virus integrates into the host cell¿s genome, and at that point replicates copies of its genetic material through transcription. The cell¿s RNA is transcribed and translated into viral proteins. Finally, HIV¿s gag protein begins to assemble new viruses, which bud from the cell, and invade neighboring targets.
¿In the HIV world,¿ Zavitz continued, ¿there¿s really only one viral protein that¿s required to assemble the next generation of viruses, and that¿s the gag protein. You can just express gag on its own, and it will make virus-like particles. Gag is a very small protein,¿ he continued. ¿It¿s obviously doing something very complex, which is forming a virus and budding out from the target cell. HIV circles assume that gag must be commandeering some sort of cellular machinery in order to pull this off.¿
Zavitz, director of Myriad¿s Therapeutic Target Identification Program, is a co-author of the cover story in today¿s issue of the journal Cell, dated Oct. 5, 2001. Its title: ¿Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding.¿ The paper¿s senior author is biochemist and structural biologist Wesley Sundquist, at the University of Utah, also in Salt Lake City. (To see the cover, go to www.cell.com.)
¿In this article,¿ Sundquist stated, ¿we showed that the HIV1 virus can¿t bud without the Tsg101 protein. Instead, the virus gets stuck at the last stage of leaving the host cells.¿ He added: ¿We do not know if this will lead to new drugs against AIDS, but we hope it will. If it does, it will certainly be several years away.¿
The Bud Word Is The Buzz Word
Zavitz described to BioWorld Today the concatenation of viral and cellular proteins ¿ beginning with Tsg101 ¿ that combine to give the viral progeny pass keys out of the host cell:
¿Tsg101¿s function, as far as viral budding is concerned,¿ he began, ¿is its involvement in the VSP ¿ vacuolar sorting pathway. This is a pathway that the cell uses to recycle proteins that have served their function, and need to be degraded, discarded or recycled.
¿Normally the cell uses this VSP pathway to form structures called multivesicular bodies [MVB], which bud from the cytoplasm into the lysosome, carrying their viral-particle cargo. The virus adapts this machinery and turns it inside out so that the new particles are now budding from the cytoplasm out into the extracellular environment. So it makes sense that this MVB pathway is being hijacked in order to create new viruses.¿
A stubby peptide in HIV¿s budding strategy is p6. ¿It¿s a very short piece of the 52-amino-acid gag protein,¿ Zavitz pointed out. ¿P6 contains a short, four-amino-acid sequence called PTAP ¿ proline, threonine, alanine, proline. That residue is essential for viral budding. So mutations within PTAP render the virus incapable of escaping.
¿When we made those mutations in p6,¿ the Myriad scientist went on, ¿we saw that the virus got most of the way out of the cell, but couldn¿t pinch off and exit it. Inhibiting budding also prevents maturation of the gag protein, and that renders these viruses ¿ which accumulate on the cell, and get stuck there ¿ noninfectious.
¿HIV is a mutable virus ¿ constantly changing its shape, and thus frustrating antiviral drugs and vaccines,¿ Zavitz observed. ¿P6 is mutable,¿ he pointed out. ¿But this PTAP region is absolutely conserved.¿
Myriad is developing drugs against HIV1, based on this anti-budding rationale. ¿With our identification of Tsg101 as the starting point,¿ Zavitz recounted, ¿we¿ve gone on to use our protein-protein interaction technology to expand Tsg101 and find all the cellular proteins that interact and are required for that pathway downstream.
¿We¿ve identified a very broad network of proteins,¿ he went on, ¿all of which seem to be involved in these same cellular processes. Identifying them has given us a large number of putative drug targets, which we¿re looking at for their ability to thwart viral budding. We¿ve put a number of these targets into our screening procedure and have identified molecules that inhibit various aspects of this pathway, and seem to be showing antiviral activity. This pathway, which the virus is hijacking, can be disrupted by any number of means, including small molecules, so that we can inhibit the virus from escaping the cell.
¿The next step on the horizon for us,¿ Zavitz said, ¿is preclinical work in animals, mostly from a toxicological and pharmacokinetic point of view.¿
Multidrug HAART Cocktail Faces Resistance
He made the point, ¿This particular mechanism, and these clinical targets, are completely separate from those of HAART ¿ highly active antiretroviral therapy. Those cocktails are generally composed of reverse transcriptase and protease inhibitors. Our approach is looking at a whole new class of targets. We think that the potential with this is rather exciting, given that one of the problems with HAART is the emergence of cocktail-resistant mutants of the virus. So that¿s one of HIV1¿s great successes ¿ being able to be highly mutable and mutate around that.
¿Since the pathway that we¿re looking at is composed of cellular targets,¿ Zavitz continued, ¿and from the way that the virus interacts with the cellular machinery, we believe that it¿s going to be potentially more difficult for the HIV1 to mutate and therefore develop resistant mutants around this pathway. We¿re doing experiments to address that question right now, but on a theoretical basis we¿re very hopeful. And that¿s really the emerging problem for HAART drug cocktails. It¿s been estimated that within the next four years, over 40 percent of AIDS patients will have resistant viral load to the current set of cocktail therapy.¿