A common hallmark of viral genome replication is a high rate of mutation, which can help viruses evade new treatments and develop resistance to once-effective antiviral drugs. A new study uncovers one of the mechanisms by which the herpes simplex virus (HSV) develops resistance to treatments. Using cryogenic electron microscopy (cryo-EM), researchers showed how conformational changes in the viral DNA polymerase alter the virus's susceptibility to drugs.
The new findings expand the understanding of how changes in the conformation of a viral protein promote drug resistance and may be relevant to understanding drug efficacy and resistance in other viruses, the researchers said. They also answer long-standing questions about why certain viruses are susceptible to antiviral drugs and others are not, and how viruses become drug-resistant.
“Our results show that we need to think beyond the typical drug binding sites,” said Dr. Jonathan Abraham, associate professor of microbiology at Harvard Medical School and HHMI investigator. “This really helps us see drug resistance in a new light.”
The results are published in cell in the paper “Viral DNA polymerase structures reveal mechanisms of resistance to antiviral drugs.”
HSV, which affects an estimated billion people worldwide, is known to cause cold sores and fever blisters, but can also cause serious eye infections, brain inflammation and liver damage in people with weakened immune systems. In addition, HSV can be transmitted from mother to baby through the birth canal during childbirth and can cause life-threatening neonatal infections.
DNA polymerases are known to be important drug targets and much work has been done to capture them in different conformations. However, a detailed understanding of the impact of polymerase conformational dynamics on drug resistance has been lacking.
HSV polymerase is the target of acyclovir, the leading antiviral drug for treating HSV infection, and foscarnet, a second-line drug for drug-resistant infections.
Abraham's group wanted to understand how changes in the polymerase make the virus insensitive to normal doses of antiviral drugs and, more generally, why acyclovir and foscarnet are not always effective against the altered forms of HSV polymerase.
“Over the years, the structures of many polymerases from different organisms have been determined, but we still do not fully understand what makes some polymerases susceptible to certain drugs but not others,” said Abraham. “Our study shows that the movement of the different parts of the polymerases, called conformational dynamics, is a crucial factor in their relative susceptibility to drugs.”
Using cryo-EM, the researchers determined structures of the DNA-bound herpes simplex virus polymerase holoenzyme in multiple conformations and interacting with antiviral agents. These structures, they say, show how “the catalytic subunit Pol and the processivity factor UL42 bind DNA to promote processive DNA synthesis.” They added: “Unexpectedly, in the absence of an incoming nucleotide, we observed Pol in multiple conformations with the closed state sensed by the finger domain.”
Structural analyses combined with computer simulations suggested that several mutations located far from the drug's binding sites confer antiviral resistance by altering the position of the polymerase fingers responsible for closing the drug to stop DNA replication.
The discovery was an unexpected twist. Until now, scientists believed that polymerases only partially close when they bind to DNA and close completely when they add a deoxynucleotide. But it turns out that the HSV polymerase can close completely just by being close to DNA. This makes it easier for acyclovir and foscarnet to attach and stop the polymerase, stopping virus replication.
“I have been working on HSV polymerase and acyclovir resistance for 45 years,” noted Dr. Donald Coen, professor of biological chemistry and molecular pharmacology at HMS. “At the time, I thought that resistance mutations would help us understand how the polymerase recognizes features of the natural molecules that the drugs mimic. I am pleased that this work shows that I was wrong and finally gives us at least a clear reason why HSV polymerase is selectively inhibited by the drug.”
