The ability of viruses to evade the immune system, as well as pathogenic virulence, makes the study of viral infections and development of novel antiviral compounds essential. Now a recently published study from a group of investigators led by scientists at Penn State University and the Delft University of Technology in The Netherlands has added to the knowledge base of viral replication. The team’s research shows that a new class of antiviral drugs works by causing the virus' replication machinery to pause and backtrack, preventing the virus from replicating efficiently.

Findings from the new study—published recently in Cell Reports in an article entitled “Signatures of Nucleotide Analog Incorporation by an RNA-Dependent RNA Polymerase Revealed Using High-Throughput Magnetic Tweezers”—could help speed the development and approval of related antiviral drugs.

“Viruses are a massive threat to global public health,” noted co-senior study investigator Craig Cameron, Ph.D., professor of biochemistry and molecular biology at Penn State. “Developing broad-spectrum antiviral drugs—ones that are effective against many different viruses—is vital to our ability to prevent or respond to outbreaks. We were able to demonstrate the mechanism of a newly developed class of antiviral drugs that are potentially broad spectrum.”

The mechanism of a new class of antiviral drug. The RNA polymerase enzyme (yellow) replicates the virus' genome by incorporating one nucleotide (black) at a time. Nucleotide analogs (red) are designed as antiviral drugs that can disrupt the replication process. The antiviral drugs work by: (1) incorporating mutations, (2) stopping the replication process, or (3) a newly discovered mechanism in which the RNA polymerase enzyme pauses and then backtracks. [Penn State]
The mechanism of a new class of antiviral drug. The RNA polymerase enzyme (yellow) replicates the virus’ genome by incorporating one nucleotide (black) at a time. Nucleotide analogs (red) are designed as antiviral drugs that can disrupt the replication process. The antiviral drugs work by: (1) incorporating mutations, (2) stopping the replication process, or (3) a newly discovered mechanism in which the RNA polymerase enzyme pauses and then backtracks. [Penn State]

All RNA viruses, whose genomes are composed of RNA rather than DNA, use an enzyme called RNA-dependent RNA polymerase (RdRp) to express genes and replicate their genome to make new copies of themselves. Thus, the polymerase enzyme is an ideal target for developing broad-spectrum antivirals.

“In order to make more viruses, the RNA polymerase enzyme replicates the virus genome by incorporating nucleotides—the building blocks of RNA or DNA, which are made up of a base and a sugar—one at a time,” explained co-author Jamie Arnold, Ph.D., associate research professor at Penn State. “For many antiviral drugs, alternative versions of these building blocks are designed such that when they are incorporated during replication, they somehow disrupt the process. To understand the disruption mechanism, we used magnetic tweezers that allowed us to monitor the progression of hundreds of individual RNA polymerase enzymes during the replication process in the presence of antiviral drugs.”

The magnetic tweezer assay works by tethering one end of hundreds of individual strands of RNA to a surface and attaching a magnetic bead to the other end. A magnet then holds the strands vertically while the researchers monitor the beads under a microscope. As the RNA polymerase builds new RNA, the length of the strand changes, moving the bead up or down. Because they can monitor hundreds of these processes simultaneously, the researchers are able to build datasets and develop sound statistical backing for their observations.

We used “a high-throughput magnetic tweezers platform to monitor the elongation dynamics of a prototypical RdRp over thousands of nucleotide-addition cycles in the absence and presence of a suite of nucleotide analog inhibitors,” the authors wrote. We observed multiple RdRp-RNA elongation complexes; only a subset of which are competent for analog utilization. Incorporation of a pyrazine-carboxamide nucleotide analog, T-1106, leads to RdRp backtracking. This analysis reveals a mechanism of action for this antiviral ribonucleotide that is corroborated by cellular studies. We propose that induced backtracking represents a distinct mechanistic class of antiviral ribonucleotides.”

The researchers are excited by their findings and feel that the high-throughput magnetic tweezer assay is well poised to streamline antiviral drug development projects.

“We were particularly interested in an antiviral called T-1106. It is related to favipiravir, which was recently approved in Japan for use in the treatment of influenza, but the mechanism was unknown,” Dr, Cameron remarked. “We were able to show that these antivirals—a new class that alters the base of the RNA building block, rather than the sugar—work in a new way. Unlike other known antivirals that either incorporate mutations into the replication process or stop it completely, this new class works by causing the RNA polymerase enzyme to pause and backtrack. With this understanding, we can begin to fine-tune the design of these antivirals and speed up the process of getting them approved.”