Unraveling the mystery of virus evolution

All organizations -? from fungi to mammals -? they have the ability to evolve and adapt to their environment. But viruses are master shape shifters with the ability to mutate more than any other organism. As a result, they can avoid treatments or develop resistance to once-effective antiviral drugs.

Working with the herpes simplex virus (HSV), a new study led by Harvard Medical School researchers sheds light on one of the ways the virus becomes resistant to treatment, a problem that could be particularly challenging among people with reduced immune function, including those receiving immunosuppressive therapy and those born with immunodeficiency.

Using a sophisticated imaging technique called cryogenic electron microscopy (cryo-EM), the researchers found that how parts of a protein responsible for viral replication move to different locations can change the virus’s sensitivity to drugs.

The findings, published on August 27 in Cellanswer long-standing questions about why some viruses, but not others, are sensitive to antiviral drugs and how viruses become impervious to drugs. The results could inform new approaches that thwart the ability of viruses to overcome effective treatments.

Counterintuitive results

Researchers have long known that changes that occur in the parts of a virus where antiviral drugs bind to it can make it resistant to treatment. However, the HMS researchers found that, much to their surprise, this was often not the case with HSV.

Instead, the researchers found that protein mutations linked to drug resistance often arise 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 replicate. This raises the possibility that using drugs to block or freeze the conformational changes of these viral proteins could be a successful strategy for overcoming drug resistance.

Our findings suggest that we need to think beyond targeting typical drug binding sites. This really helps us see drug resistance in a new light.”

Jonathan Abraham, Senior Study Author and Associate Professor, Microbiology, Blavatnik Institute, Harvard Medical School

The new findings advance understanding of how changes in the conformation of a viral protein -? or changes in the way different parts within that protein move when it performs its function—drive drug resistance and may be relevant to understanding drug effectiveness and drug resistance in other viruses, the researchers noted .

HSV, which is estimated to affect billions of people worldwide, is best known as the cause of cold sores and cold sores, but it can also lead to serious eye infections, brain inflammation and liver damage in immunocompromised people. In addition, HSV can be transmitted from mother to baby through the birth canal during delivery and cause life-threatening neonatal infections.

Clues to resistance rooted in structure and movement

A virus cannot reproduce by itself. To do this, viruses must enter a host cell, where they release their tools of reproduction -? proteins called polymerases -? to make copies of themselves.

The current study focused on one such protein -? a viral DNA polymerase -? vital to HSV’s ability to reproduce and spread. Its ability to perform its function is based on the structure of DNA polymerase, which is often likened to a hand with three parts: the palm, the thumb, and the fingers, each of which performs critical functions.

Given their role in activating replication, these polymerases are critical targets of antiviral drugs, which aim to stop virus replication and stop the spread of infection. HSV polymerase is the target of acyclovir, the leading antiviral drug for the treatment of HSV infection, and foscarnet, a second-line drug used for drug-resistant infections. Both drugs work by targeting the viral polymerase, but they do so in different ways.

Scientists have long struggled to fully understand how changes in the polymerase make the virus impervious to normal doses of antiviral drugs and, more generally, why acyclovir and foscarne are not always effective against altered forms of the HSV polymerase.

“Over the years, the structures of many polymerases from various organisms have been determined, but we still do not fully understand what makes some polymerases, but not others, vulnerable to certain drugs,” Abraham said. “Our study reveals that how the different parts of the polymerases move, known as their conformational dynamics, is a critical component of their relative drug sensitivity.”

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

Configuration dynamics -? the ability of different parts of a protein to move—allowing them to efficiently manage many essential functions with a limited number of components. A better understanding of polymerase dynamics is the missing link between structure and function, including whether a protein responds to a drug and whether it could become resistant to it down the road.

Unraveling the mystery

Many structural studies have captured DNA polymerases in several distinct conformations. However, a detailed understanding of the effect of polymerase dynamics on drug resistance is lacking. To solve the puzzle, the researchers performed a series of experiments, focusing on two common polymerase conformations -? one open and one closed -? to determine how each affects drug sensitivity.

First, using cryo-EM, they conducted structural analysis to obtain high-resolution visualizations of the atomic structures of HSV polymerase in multiple conformations, as well as when bound to the antiviral drugs acyclovir and foscarnet. The drug-bound structures revealed how the two drugs selectively bind polymerases that more readily adopt one conformation over the other. One of the drugs, foscarnet, works by trapping DNA polymerase fingers so that they get stuck in a so-called closed conformation.

Further, structural analysis combined with computational simulations suggested that several mutations distant from the drug binding sites confer antiviral resistance by altering the location of the polymerase fingers responsible for closing in on the drug to stop DNA replication.

The finding was an unexpected twist. Until now, scientists believed that polymerases were only partially shut down when they attached to DNA and fully shut down only when they added a DNA building block, a deoxynucleotide. It turns out, however, that the HSV polymerase can be completely shut down just by being in close proximity to the DNA. This makes it easier for acyclovir and foscarnet to attach and stop the polymerase from working, thus stopping the virus from replicating.

“I have worked on HSV polymerase and acyclovir resistance for 45 years. At the time, I thought resistance mutations would help us understand how the polymerase recognizes features of natural drug-mimicking molecules,” said study co-author Donald Cohen, Professor. of biological chemistry and molecular pharmacology at HMS. “I’m glad this work shows me wrong and finally gives us at least one clear reason why HSV polymerase is selectively inhibited by the drug.”