On July 3, 2001, a research paper co-authored by Carol Carter, professor in the Department of Microbiology and Immunology at the Renaissance School of Medicine at Stony Brook, and a team of fellow researchers, was published. Its findings would open a new field of investigation into how pathogens escape from infected cells and reveal new opportunities for anti-viral drug development.
This year marks the 20th anniversary of the discovery outlined in that paper, “Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L domain in HIV type 1 Pr55(Gag).” David Thanassi, professor and chair of the Department of Microbiology and Immunology, cites Carter’s work as an example of the transformative power of research in real-world applications.
“The discovery described 20 years ago in Carol’s paper illustrates the power of basic research to generate unexpected insights and open new avenues for the development of therapeutic approaches to combat viral and other diseases,” Thanassi said. “It is particularly relevant to celebrate this milestone achievement now, given the ongoing COVID-19 viral pandemic. In the search for new treatments, many researchers are pursuing the now-accepted strategy, as embodied by Carol’s work, of targeting cellular factors to develop antiviral measures.”
“I had come home from a meeting in the late 1990’s when the HIV pandemic was raging, much like COVID-19 is doing now, but with abysmal antivirals and no vaccine on the horizon,” Carter recalled. “The National Institute of Health (NIH) leadership said to researchers, ‘You need to come up with some strategies to circumvent rapidly emerging drug-resistance.’”
As the HIV crisis raged, the World Health Organization (WHO) estimated that by 1999, 33 million people were living with HIV worldwide, and that 14 million had died of AIDS. The same year, then-president Bill Clinton declared HIV/AIDS a threat to U.S. national security and issued an executive order to assist developing countries in importing and producing generic HIV treatments. (UNAIDS, a United Nations program dedicated to worldwide HIV and AIDS response, estimates that 78 million people have become infected with HIV and 35 million have died from AIDS-related illnesses since the first cases were reported more than 35 years ago.)
“Drug-resistance was emerging as a major threat because there were very few good drugs on hand,” said Carter. “It struck me that this was the time to entertain an idea that would have been laughed out of the room at an earlier time: the concept of targeting cellular instead of viral encoded gene products.”
Carter said the latter puts pressure on viral evolution to select for resistant variants that can evade the drugs, a concern that is less likely to apply to cellular gene products.
“Viruses exploit cells,” explained Carter. “Cells don’t make, maintain or change gene products for the convenience of the virus.”
Stony Brook’s laboratory screened a library of “house-keeping genes” for any that were recognized by HIV, identified several, and focused on one called Tsg101 that showed itself early on to be differentially employed by the virus and host.
In uninfected cells, Tsg101 plays an instrumental role in sending proteins the cell no longer wants to the “garbage pail,” compartments where such proteins are degraded. For example, Tsg101 ensures that proteins that signal continuous cell growth, as occurs in cancers, are removed from the cytoplasm. In contrast, in HIV-infected cells, Tsg101 is recruited to sites of virus assembly at the cell periphery. There, it still facilitates removal of the assemblages from the cytoplasm, however this recruitment permits the virus to exit into the extracellular space instead of a degradative chamber.
Tsg101 stands for ‘Tumor Suppressor Protein #101. “It was the 101st gene product reported to the Protein Data Bank with that description and initially thought to suppress tumor growth,” Carter said. “Learning that produced an ‘aha!’ moment because experiments that we had done by that point were indicating that HIV needed Tsg101 to get viral particles out of the cell and because we also knew HIV did not cause cancer. We’re still working on this, but the 20-year milestone demonstrates that HIV’s recruitment of Tsg101 was a great forecaster of the existence of virus-host interactions feasible to target for anti-viral drug development. Patents describing our translational effort have been granted or are pending.”
In football terms, Tsg101 is the “quarterback” for the cellular team effort, also known as the ESCRT (endocytic sorting complexes required for trafficking) machinery that recognizes cargo coming into the cell and directs it to the proper subcellular destination, or “end zone.”
Carter said that several laboratories both in the U.S. and around the globe are working on the ESCRT machinery and have established that several human viral pathogens encode L domains to recruit Tsg101 and depend on ESCRT for virus budding from cells.
“Some labs are doing exquisite structural characterization of the ESCRT machinery components,” said Carter. “At this point, it has been discovered that ESCRT machinery is evolutionarily conserved from yeast to man. All species encode in their genomes identical or similar Tsg101 sequences that have remained essentially unchanged throughout evolution. This conservation indicates that the Tsg101 protein is unique and essential. It plays critical roles in cell division and neuronal cell pruning. Defects in the gene are embryonically lethal.”
“This finding changed the course of the field and changed the course of the lab’s research,” said James Hurley, a structural biologist at Berkeley and one of the lead investigators of ESCRT machinery function. “I don’t think any other single paper has had as much impact on our lab’s research direction.” Hurley and Carter served together on National Institutes of Health (NIH) scientific advisory committees.
Carter describes her research as both a rewarding adventure and a never-ending learning experience.
“It’s been a fantastic journey,” said Carter. “Some of our compounds developed against HIV are able to inhibit replication of other human pathogens, including SARS CoV-2. It would be great if any of them proved to be useful therapeutics.”
— Robert Emproto