By David N. Leff

Science Editor

HIV - the human immunodeficiency virus - is a serial killer.

Its modus operandi is to attack its victim's police force - that is, its immune system - one T lymphocyte after another. Once an HIV particle has broken into its target T cell, the viral invader's RNA hijacks that cell's own DNA and forces it to manufacture new retroviral particles.

Then, like religious denominations sending their missionaries forth into the world to convert nonbelievers, the new viruses exit their host cell by a process called budding. On their way outward bound in search of naove neighboring T cells to take over, these fresh virions wrap themselves in a stolen souvenir fragment of their former host cell's outer membrane. That camouflage serves as a membranous envelope, which allows the virus to fuse with its next cellular victim.

This process is less simple than it seems in the telling. In fact, it took three research papers plus a commentary in today's Proceedings of the National Academy of Sciences (PNAS), dated Nov. 21, 2000, to hang some new factual flesh on the bare-bones account of this viral budding mechanism.

The first report tells the tortured tale of multistep cellular machinery - the proteasome-ubiquitin complex - plus one key HIV molecule, the gag gene and its Gag protein. The paper's title: "Proteasome inhibition interferes with Gag polyprotein processing, release, and maturation of HIV-1 and HIV-2." Its lead author is virologist Ullrich Schubert, a visiting scientist at NIAID - the National Institute of Allergy and Infectious Diseases in Bethesda, Md.

HIV's gag gene encodes the Gag protein, which heads for the target T cell's plasma membrane. It apparently honchos the new nascent viral particle's development from a horseshoe-shaped to a lollipop-shaped structure, which gets pinched off in the membrane fusion event, and released from the cell. Along the way HIV protease chops Gag into several pieces, leading to viral maturation, which makes it infectious.

Proteasomes are very complicated structures dedicated to finding and dumping proteins the cell neither wants nor needs any more. These garbage-disposal molecules constitute a good 1 percent of a cell's proteins. Ubiquitins, small, ubiquitous molecules, are informer elements that go around flagging proteins so that proteasomes can finger them for elimination. (See BioWorld Today, April 4, 2000, p. 1.)

Testing New Way To Gag Gag Protein

"What we found," Schubert told BioWorld Today, "was the effect of blocking the proteasome pathway, which with ubiquitin is a multi-enzyme complex, involved in the turnover of many proteins. We tested several proteasome inhibitors - chemical substances that can be taken up by cells and block several things. Among them, release of viral particles from the cells, and processing of the structural main component of the retrovirus, which is its Gag protein. In our in vitro experiments, those chemical inhibitors reduced the infectivity of the viral particles, which were released in lower number.

"Because maturation of the virus actually occurs after it is budded from the cell membrane," Schubert explained, "if this maturation is blocked, then the virus is less infectious, or has no infectivity at all. This basic observation," he added, "intrigued us to investigate the mechanism behind all this inhibition.

"The proteasome inhibitors," he recounted, "are various, commercially available chemicals, which all do the same thing in different ways. In one, for example, there are three peptides, which bind to and block the proteasome reversibly or irreversibly. Another inhibitor, epoxomicin, is a compound based on naturally occurring substances, produced in microorganisms. This drug, which blocks the proteasome irreversibly, was evaluated before in animals."

The same Nov. 21 issue of PNAS carries two adjacent papers, by two virologists, on the same subject: Heinrich Gvttlinger of Harvard Medical School reported on: "A role for ubiquitin ligase recruitment in retrovirus release." John Wills of Pennsylvania State University in Hershey, Pa., filed an article titled: "Ubiquitin is part of the retrovirus budding machinery."

Gvttlinger found, in HIV and other viruses, that so-called late-assembly or L-domains - particular regions in the Gag proteins - support the budding and release of particles from the T-cell surface, and that those L-domains interact with ubiquitin ligases. These are short peptides, which eventually serve as the flag for recognition by the proteasome.

"They recognize all proteins that are tagged by ubiquitin," Schubert observed, "and drag them into the digesting chamber of the huge proteasome complex. Ubiquitin's interaction with Gag's L-domain, which Gvttlinger reported, provided indirect evidence that ubiquitination of Gag is somehow involved in regulating this viral maturation - but coming from a different corner."

He added, "John Wills studied a similar phenomenon, using Rous-Sarcoma virus, which, like HIV, is another retrovirus. And he had a similar observation."

Schubert and his team are now "trying to understand the molecular mechanism - how this proteasome-ubiquitin pathway is involved in retrovirus assembly. Also we are investigating how this phenomenon can be used to develop a potential new proteasome-inhibition strategy for treating and preventing HIV infection.

"Of course," he observed, "shutting down all proteasomes in a human being would be lethal. You would think," he pointed out, "that inhibiting a proteasome - which is involved in many different cellular processes - would be incompatible with viability of the cell and its entire organism."

Millennium's New Drug To The Rescue

"But we learned just last Friday," he went on, "that there are now substances available, which under certain treatment protocols, don't harm humans at all." Schubert referred to "Phase II clinical trials now under way by Millennium Pharmaceuticals Inc,. of Cambridge, Mass., to treat cancers and other diseases, using its proprietary proteasome inhibitor, based on dipeptidyl boronic acid derivatives. They promised to give us those inhibitors, because we are about to start monkey studies of proteasome blockers," Schubert said.

"If we see any promising result - a protective effect - once we're at that stage," he concluded, "it will be very quick to go into patient studies."