Well over half of the world’s population is infected with herpes simplex virus (HSV) (1). Once a person is infected, the virus lives permanently in their nerve cells and can be reactivated by triggers ranging from stress to sun exposure (2). Most people with HSV infections have mild or no symptoms other than occasional blisters or ulcers, but some experience more severe effects or prolonged infections that lead to treatment resistance. When that happens, managing the disease can become a challenge.

“Although most people think of herpesviruses as causing cold sores, they can lead to severe brain infections,” said Jonathan Abraham, a virologist at Harvard Medical School. “Severe cases I’ve seen as a doctor have always had me thinking, ‘What can we do in the lab?’”

Mutations in the HSV polymerase, which the virus uses to replicate its DNA, can confer resistance to acyclovir and foscarnet, the two leading antivirals used against HSV (3). To learn how these mutations allow HSV to fight back against antivirals, Abraham and his colleagues used a technique known as cryogenic electron microscopy (cryo-EM) to capture the conformations of the virus’s DNA polymerase in the presence of DNA alone or alongside antiviral drugs in a new study (4).

Scientists often describe polymerases as “hand-shaped,” with active sites in the palm surrounded by fingers and a thumb. To observe how these mutations affect polymerase shape and movement, the researchers genetically modified cells to express the polymerase complex, purified it, and verified that it was active. Then, they used cryo-EM to observe its range of conformations, which include an “open” position with the fingers far apart from the active site on the palm and a “closed” position in which the fingers move toward the palm. Without anything to grip, the polymerase transitioned easily between conformations, but with a nucleotide in the active site — as would be the case during DNA replication — the polymerase’s fingers shifted fully into the closed conformation.

“The resistance mutations we saw were in places that influenced how the fingers of the polymerase move, rather than the specific site where the drug binds on the hand,” said Abraham. He and his colleagues found that exposing the polymerase to acyclovir produced the same closed conformation as a nucleotide in the active site — an observation that was in line with how acyclovir works to replace normal nucleotides in the active site and to block viral DNA elongation (5). Foscarnet inactivates the polymerase directly during viral replication, similarly trapping the polymerase in its closed “DNA-gripping” conformation (6). But the researchers’ simulations revealed that when mutations in the structure of HSV polymerase make the fingers more dynamic or decrease the strength of the binding to antiviral drugs, the virus can overcome the drugs’ blocking effects better.

Although most people think of herpesviruses as causing cold sores, they can lead to severe brain infections.
- Jonathan Abraham, Harvard Medical School

“It’s not necessarily the shape of the hand or where the drug binds that determines resistance,” Abraham explained. “Rather, it’s differences in how these enzymes move.” In the future, he added, drugs that trap HSV polymerase in a static conformation may help combat treatment resistance.

“Polymerase resistance via protein changes is a common way of avoiding drug potency,” said Adrian Wildfire, a virologist and drug development specialist at the pharmaceutical research company IQ-IDM, who was not involved in the research. “Will this knowledge drive new drugs for HSV? It already has for HIV and, to some extent, SARS-CoV-2. The lack of alternatives to treat disease brings about better drugs and vaccines.”

One limitation of the study was that the researchers did not have the time or resources to compare the relative effects of different mutations; they investigated four of the many possible mutations. “In the real world, resistance is rarely absolute,” Abraham said. “What are the implications of three-fold versus two-fold versus 1.5-fold inhibition? We don’t know yet.”

Currently, even the molecular modeling needed to predict whether a mutation is likely to cause treatment resistance is too complex for clinical use. “A lot of algorithms could potentially predict the structures of drug-resistant polymerases,” Abraham explained. “The problem is that they give us a static picture, and we need to understand how the proteins move. When the algorithms can accurately predict protein movement, we may be able to forecast resistance in clinical settings.”

References

1. World Health Organization. Herpes simplex virus. (2024).
2. Stoeger, T. & Adler, H. “Novel” triggers of herpesvirus reactivation and their potential health relevance. Front Microbiol   9, 3207 (2019).
3. Piret, J. & Boivin, G. DNA polymerases of herpesviruses and their inhibitors. Enzymes  50, 79–132 (2021).
4. Shankar, S. et al. Viral DNA polymerase structures reveal mechanisms of antiviral drug resistance. Cell   187, 5572 - 5586.e15 (2024).
5. McGuirt, P.V. & Furman, P.A. Acyclovir inhibition of viral DNA chain elongation in herpes simplex virus-infected cells. Am J Med   73, 67–71 (1982).
6. Crumpacker, C.S. Mechanism of action of foscarnet against viral polymerases. Am J Med   92, S3–S7 (1992).