Up Close and Personal With SARS-CoV-2's Replication Machinery
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When a viral pathogen invades, it enters the host cells and must rapidly divide to survive. In order to divide, it is required to multiply its genetic material – long strands of RNA. The machinery responsible for performing this task is known as the viral polymerase.
Scientists are working to understand the structure and function of the viral polymerase; block the replication machinery, block the virus's ability to continue to infect the host. This is the premise behind antiviral drugs which bind to and inhibit viral polymerases.
Structural biology can lend a hand here. Advances in techniques such as cryo-electron microscopy (Cryo-EM) are progressing the at a rapid pace, enabling scientists to accurately determine the 3D structures of proteins in a more efficient manner. Of course, in a pandemic, this is key.
A preprint study published via the online server bioRxiv outlines the efforts of a team of scientists, led by Patrick Cramer at the Max Planck Institute for Biophysical Chemistry in Göttingen, in determining the 3D structure of the novel coronavirus polymerase.
Technology Networks spoke with Cramer to learn more about the study, it's relevance in developing therapeutics against SARS-CoV-2 and to gain his insights on the value of preprints in a pandemic.
Molly Campbell (MC): For our readers that may be unfamiliar, please can you tell us about your lab's research focus?
Patrick Cramer: We are combining in vitro and in vivo approaches to study gene transcription and the regulation of genes in human cells. In particular, we develop and use integrated structural biology to analyze the structure of large assemblies of protein that are involved in transcription.
MC: You recently published a preprint study in which you have determined the 3D structures of the corona polymerase. Why it was important to you to determine the polymerase's structure?
PC: In the light of the ongoing pandemic, we wanted to help. We decided to use our experience in polymerase biology and structure-function analysis to investigate how the virus replicates its genetic material and how it transcribes its genes.
It is important that we got the polymerase structure because it is the target of antiviral substances, including remdesivir, which is currently being tested in the clinic. Next, we will investigate the mechanism that remdesivir uses to block the corona polymerase and prevent duplication of its genome.
MC: Can you tell us what you discovered about the corona polymerase through studying its structure?
PC: The core structure was known, but we did not know what the accessory factors, in particular the nsp8 subunits, really do. We resolved them here for the first time in the context of the complete replicating polymerase.
We found that they make very extensive interactions with the RNA that exits from the enzyme. Comparison with the literature suggests these long nsp8 extensions are important for holding onto RNA to make replication processive. This is important to replicate the entire genome, and to prevent premature dissociation of the polymerase from RNA.
MC: What research techniques did you utilize to determine the 3D structure of the corona polymerase?
PC: Cryo-electron microscopy. This technique does not require you to form protein crystals, as is required for X-ray analysis.
MC: What challenges did you encounter in the study, and how did you overcome them?
PC: Other groups also tried to see the entire enzyme but always got to see only its core part. We were lucky in that we accidentally assembled a longer RNA molecule that then stabilizes the newly observed regions. Hard work and making use of our experience in preparing proteins, measuring their activity and solving their structures helped us in this study.
MC: In the press release, you say: "The determination of the polymerase structure will not be the last contribution of the Göttingen researchers to tackling the pandemic" – Can you tell us about your next steps in this research space?
PC: We will investigate biochemically how the drug candidate remdesivir, an antiviral compound, will block replication. Then we try to visualize the polymerase with bound remdesivir, to understand its detailed mechanism. This will hopefully lead to new insights that can be used to try to improve antiviral substances.
MC: How has the COVID-19 outbreak impacted your laboratory's research?
PC: Most of us are still working in a home office. Many can process data from home and thus can to some extent be productive, others look forward to return to the lab, but – safety first.
MC: The study is published in its current format as a preprint. In your opinion, what is the value of sharing research prior to peer-review in the current climate?
PC: It is essential. We have already got requests for materials and data that others need for progressing in their research. I was surprised that the journal where we submitted the work for consideration even asked us to post a preprint online. Science moves at an incredible pace these days.
Patrick Cramer was speaking to Molly Campbell, Science Writer, Technology Networks.
This article is based on research findings that are yet to be peer-reviewed. Results are therefore regarded as preliminary and should be interpreted as such. Find out about the role of the peer review process in research here. For further information, please contact the cited source.