People fear diseases such as Ebola, Marburg, Lassa fever, and rabies, for good reason; they have high mortality rates and few, if any, possible treatments. Up to 90 percent of people who contract Ebola, for instance, die of the disease.

These diseases are zoonoses—infections that can be passed between humans and animals. Humans can contract Lassa fever and rabies directly from animals, and Ebola, Marburg, and HIV are all believed to have originated in animals. It’s likely that the recent Ebola outbreak in West Africa, as with previous outbreaks of the disease, arose when a person or community came into contact with an infected great ape.

Committed to taking a One Health perspective, and facing a gaping need for therapies against these life-threatening ailments, researchers at the University of Pennsylvania School of Veterinary Medicine have been applying their expertise in infectious diseases toward understanding the molecular workings of these viruses. Their research is helping identify ways to exploit the microbes’ own sinister strategies to effectively stop the pathogens in their tracks.

Dr. Ronald N. HartyPenn Vet’s Dr. Ronald N. Harty, an associate professor of microbiology, recently teamed with colleagues to identify and develop compounds that can reduce the ability of a virus to spread infection. Reporting their findings in two studies published in the Journal of Virology, Harty and his team rounded up several prototypic compounds with the potential to one day serve as broad-spectrum anti-viral drugs.

Viruses can be powerful agents of disease, but they are relatively powerless on their own. In order to reproduce, they must take over host cell proteins and machinery. When they’re ready to exit the cell, they again use host molecules in the cell membrane in a process called “budding.” Then they move on to other cells, spreading infection.

“What happens is the virus actually hijacks or recruits different host proteins and host functions and makes use of those proteins to efficiently get out of the cell,” Harty said.

It is this step that has captured the interest of Harty and colleagues in recent years. In their two new reports, they have zeroed in on the budding process, attempting to block it and reduce viral infections to a level a person’s immune system would be able to control more easily.

In the first paper, the researchers examined the interaction between the human protein Tsg101 and the viral protein sequence known as PTAP, which is present in proteins that play important roles in the budding of Ebola and HIV. This interplay is important for the virus to break free of the host cell’s plasma membrane and continue infecting other cells. If this interaction is blocked, many of the viruses will remain tethered to the cell membrane, unable to bud out and perpetuate the infection.

Budding of the Ebola virus VP40 protein is shown in green. Cell background is shown in red.Though the viruses under study are too dangerous to manipulate in the Penn Vet lab, which is a Biosafety Level-II (BSL-II) facility, Harty’s team was nonetheless able to examine the interactions between host and viral proteins by looking at what are known as virus-like particles, or VLPs. VLPs cannot infect cells, but are useful for studying viral proteins. In this case, the researchers produced VLPs using a Junin viral matrix protein of which PTAP is a part. The pathogenic strain of Junin virus causes Argentine hemorrhagic fever and is considered a potential bioterrorism agent.

Harty’s team used the VLPs to screen dozens and dozens of small molecules to find some that would block the interaction between Tsg101 and PTAP. A promising candidate emerged called compound 0013.

Testing the effectiveness of this molecule in their VLP assay, the team found that it reduced the ability of the Junin VLP to bud off from human cells in culture by more than 90 percent. It was similarly effective against proteins that are found in Ebola and HIV.

As a final confirmation of the compound’s potential to stop a virus from spreading, they tested it against an actual virus: the nonpathogenic vaccine strain of the Junin virus. The researchers found that it significantly decreased viral budding in a dosedependent manner.

The second paper used an analogous strategy to try to block another host-virus interaction, this time examining the viral protein sequence called PPxY—found in the matrix proteins of Marburg, Ebola, and rabies viruses as well as a host of other dangerous pathogens. PPxY interacts with an enzyme in human cells called Nedd4 during budding.

TOP: Diagram illustrating interactions between host Nedd4 and the PPxY motif within the Z, VP40, and M matrix proteins of arenaviruses (e.g. Lassa Fever), filoviruses (e.g Ebola), and rhabdoviruses (e.g. rabies), respectively to facilitate virus budding.  BOTTOM: Diagram illustrating inhibition of the Nedd4-PPxY interaction by our budding inhibitors resulting in virus particles remaining tethered to the plasma membrane of the cell. (Figure by Deborah Argento).Again, after screening many small molecules to see which would best inhibit the PPxY-Nedd4 interaction, the team found two strong candidate molecules. Further testing showed that these two molecules could effectively inhibit budding of rabies virus, Marburg VLPs, and other PPxY-containing viruses.

Reining in budding even by a fraction could significantly decrease the likelihood that an infection will get out of control. According to Harty, “By slowing down virus budding, we may allow an individual’s immune system a chance to develop a robust and protective response.”

With further testing, the compounds that Harty has pinpointed may one day serve as important anti-viral therapies. Drugs that target host-virus interactions, such as the proteins involved in budding, have a distinct advantage over drugs that target viral proteins only: they make it difficult for a virus to mutate in such a way that it would develop resistance against the treatment.

“If it did that, the virus would be compromising its own ability to exit the cell and continue spreading infection,” Harty explained.

However, a focus on the host side also comes with a potential drawback: the possibility that a drug might compromise the normal function of the protein. Further refinement of the drugs could reduce this possibility, but when dealing with severe diseases such as Ebola and Argentine hemorrhagic fever, the benefits of treating an otherwise fatal condition would outweigh potential side effects, Harty said.

These drugs could also be offered in a cocktail with additional compounds that block other stages of the virus life cycle, further amplifying their power. The next steps for these potential antivirals will be to test them in animal models.

For Harty and his team of researchers, the potential clinical relevance of these early findings is especially rewarding.

“The main reason we’re excited is that if we can come up with something that’s effective, it could have a very broadspectrum appeal,” Harty said. In addition to serving people worldwide, more effective anti-viral treatments “would give us a great way to protect the military, government workers, or first responders from these very dangerous diseases.”