Uncovering the secret of viral evolution

All organisms, from fungi to mammals, have the ability to evolve and adapt to their environment. However, viruses are masters of shapeshifting and can mutate more than any other organism, allowing them to evade treatment or develop resistance to once-effective antiviral drugs.

A new study led by researchers at Harvard Medical School has used the herpes simplex virus (HSV) to shed light on one of the ways the virus develops resistance to treatments. This problem can be particularly challenging for people with weakened immune systems, including those receiving immunosuppressive treatment and those with congenital immunodeficiency.

Using a sophisticated imaging technique called cryo-electron microscopy (cryo-EM), the researchers found that the way parts of a protein responsible for viral replication migrate to different positions can affect the virus's susceptibility to drugs.

The results were announced on 27 August in cellanswer long-standing questions about why certain viruses are sensitive to antiviral drugs but not others, and how viruses become drug-resistant. The findings could lead to new approaches that limit the ability of viruses to outperform effective therapies.

Counterintuitive results

Researchers have long known that changes to the sites on a virus where antiviral drugs bind can make the virus resistant to treatment. But the HMS researchers were surprised to find that this was often not the case with HSV.

Instead, the researchers discovered that protein mutations associated with drug resistance often occur far from the drug's target site. These mutations involve changes that alter the movements of a viral protein or enzyme that allows the virus to reproduce. This raises the possibility that using drugs to block or freeze the conformational changes of these viral proteins could be a successful strategy to overcome drug resistance.

Our results show that we need to think beyond the typical drug binding sites. This really helps us see drug resistance in a new light.”

Jonathan Abraham, lead author of the study and associate professor of microbiology, Blavatnik Institute, Harvard Medical School

The new findings contribute to understanding how changes in the conformation of a viral protein – or changes in the way the different parts within that protein move as it performs its function – promote drug resistance and could be relevant to understanding drug effectiveness and drug resistance in other viruses, the researchers said.

HSV, which affects an estimated billions of people worldwide, is best known as the cause of 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.

Indications of resistance in structure and movement

A virus cannot replicate itself. To do so, viruses must enter a host cell, where they release their replication tools – proteins called polymerases – to make copies of themselves.

The current study focused on one such protein – a viral DNA polymerase – that is critical to HSV's ability to reproduce and multiply. The ability to perform its function relies on the structure of the DNA polymerase, which is often compared to a hand with three parts: palm, thumb and fingers, each of which performs important functions.

Because of their role in replication, these polymerases are important targets of antiviral drugs designed to stop viral reproduction and halt the spread of infection. The HSV polymerase is the target of acyclovir, the leading antiviral drug used to treat HSV infections, and foscarnet, a second-line drug for drug-resistant infections. Both drugs work by attacking the viral polymerase, but they do so in different ways.

Scientists have long tried to fully 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, known as their conformational dynamics, is a crucial factor in their relative susceptibility to drugs.”

Proteins, including polymerases, are not rigid, motionless objects. Instead, they are flexible and dynamic. They consist of amino acids and fold into a stable, three-dimensional shape called the native conformation – their basic structure. However, due to various binding and dispersion forces, the different parts of the proteins can move when they come into contact with other cellular components or due to external influences such as pH or temperature changes. For example, the fingers of a polymerase protein can open and close, like the fingers of a hand.

Conformational dynamics—the ability of different parts of a protein to move—allows them to efficiently perform many important functions with a limited number of ingredients. A better understanding of the conformational dynamics of polymerases is the missing link between structure and function, including whether a protein responds to a drug and whether it might become resistant to it over time.

Revealing the secret

Numerous structural studies have captured DNA polymerases in various conformations. However, a detailed understanding of the impact of polymerase conformational dynamics on drug resistance is lacking. To solve the puzzle, the researchers conducted a series of experiments, focusing on two common polymerase conformations – one open and one closed – to determine how each affects drug sensitivity.

First, they performed structural analysis using cryo-EM to obtain high-resolution visualizations of the atomic structures of HSV polymerase in multiple conformations, as well as bound to the antiviral drugs acyclovir and foscarnet. The drug-bound structures showed how the two drugs selectively bind polymerases that adopt one conformation more readily than another. One of the drugs, foscarnet, works by trapping the fingers of the DNA polymerase, causing them to become stuck in what is known as a closed configuration.

In addition, 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 DNA building block, 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 worked on HSV polymerase and acyclovir resistance for 45 years. 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,” said study co-author Donald Coen, professor of biological chemistry and molecular pharmacology at HMS. “I am pleased that this work shows that I was wrong and finally gives us at least one clear reason why HSV polymerase is selectively inhibited by the drug.”