Great news: our work on antiviral nucleotide analogs impacting the ability of the SARS-CoV-2 polymerase to elongate RNA is now out in eLife. Special congratulations to Mona and Subhas who have led the study! Thanks to the other lab members and and all the collaborators involved.

Bellow is the digest from eLife:

To multiply and spread from cell to cell, the virus responsible for COVID-19 (also known as SARS-CoV-2) must first replicate its genetic information. This process involves a ‘polymerase’ protein complex making a faithful copy by assembling a precise sequence of building blocks, or nucleotides.

The only drug approved against SARS-CoV-2 by the US Food and Drug Administration (FDA), remdesivir, consists of a nucleotide analog, a molecule whose structure is similar to the actual building blocks needed for replication. If the polymerase recognizes and integrates these analogs into the growing genetic sequence, the replication mechanism is disrupted, and the virus cannot multiply. Most approaches to study this process seem to indicate that remdesivir works by stopping the polymerase and terminating replication altogether. Yet, exactly how remdesivir and other analogs impair the synthesis of new copies of the virus remains uncertain.

To explore this question, Seifert, Bera et al. employed an approach called magnetic tweezers which uses a magnetic field to manipulate micro-particles with great precision. Unlike other methods, this technique allows analogs to be integrated under conditions similar to those found in cells, and to be examined at the level of a single molecule.

The results show that contrary to previous assumptions, remdesivir does not terminate replication; instead, it causes the polymerase to pause and backtrack (which may appear as termination in other techniques). The same approach was then applied to other nucleotide analogs, some of which were also found to target the SARS-CoV-2 polymerase. However, these analogs are incorporated differently to remdesivir and with less efficiency. They also obstruct the polymerase in distinct ways.

Taken together, the results by Seifert, Bera et al. suggest that magnetic tweezers can be a powerful approach to reveal how analogs interfere with replication. This information could be used to improve currently available analogs as well as develop new antiviral drugs that are more effective against SARS-CoV-2. This knowledge will be key at a time when treatments against COVID-19 are still lacking, and may be needed to protect against new variants and future outbreaks.

The Dulin lab wishes a very warm welcome to several new members! Sadegh Feiz comes from Iran and has joined our lab in Erlangen as a postdoc. Misha Klein is an former alumni of TU Delft where he did his PhD with our collaborator Martin Depken and joined us as postdoc in Amsterdam. Daniel Buc is master student at VU and will do his master project with us in Amsterdam. Welcome all and we wish you a great time and great science with us!

We are super excited to have our work on SARS-CoV-2 polymerase mechanochemistry now out in Cell Reports! All you wanted to know in the nucleotide addition cycle of the key enzyme is there!

Special congrats to Subhas and Mona for their fantastic work, and to all the other lab members. Also, special kudos to our great set of collaborators: Martin Depken, Robert Kirchdorffer, Bruno Canard, Jamie Arnold, and Craig Cameron!

Congrats to all the authors of the Dulin lab (in particular Subhas and Mona) and to all the collaborators for their great work! Below is the Twitter thread describing the work. Link of the preprint here

Now the science: the coronavirus core polymerase is made of the RNA-dependent RNA polymerase #RdRp nsp12 and the co-factors nsp7 + two nsp8’s. It synthesizes all the viral RNA in infected cells and is absolutely key to virus survival and therefore a great drug target.

However, we don’t know much about the stability (must synthesize 30 kb long ssRNA!) and the kinetics of the complex. Nothing about elongation on a 1 kb long ssRNA template or with the presence of secondary structures on the template. We did it. This is what we found:

1. The complex is stable once assembled and can elongate a 1 kb long template without free viral protein in solution. The observed dynamics is therefore intrinsic to the polymerase activity (no viral protein exchange).

2. We observed the response of the #SARSCoV2 polymerase at various concentration of NTPs and applied tension to investigate the nucleotide addition cycle. Conclusions:

2.1 Nucleotide addition occurs through 3 separate pathways: nucleotide addition burst (NAB), and the slow and very slow nucleotide addition (SNA and VSNA) pathways. SNA and VSNA appear as short duration pauses (1-5 seconds);

2.2 Translocation is thermally activated and occurs at the beginning of the nucleotide addition cycle. The switch between the 3 catalytic pathways happens in the pre-translocated state. Important: NTP and nucleotide analogue binding do not trigger the switch;

2.3 Following NTP binding, the nucleotide addition cycle is followed by a succession of two (nearly) irreversible steps: chemistry and a large conformational change, likely a polymerase “reset” to enable translocation and the next cycle. True for NAB, SNA and VSNA.

3. Catalytically inactive long-lived pauses are strongly stimulated by template secondary structures. Improving magnetic tweezers stability, we show these pauses are consistent with polymerase #backtrack. The #SARSCoV2 polymerase backtracks when facing secondary structures.

3.1 Our results supports a recent structure of a pre-assembled backtracking #SARSCoV2 polymerase complex https://biorxiv.org/content/10.1101/2021.03.13.435256v1

3.2 #SARSCoV2 Polymerase backtrack is likely linked to viral genome recombination and transcription. See recent work from @CramerLabhttps://biorxiv.org/content/10.1101/2021.03.23.436644v1

3.2 This opens plenty of exciting questions: what is polymerase backtracking role in coronavirus replication/transcription? Secondary structures regulatory role? How does helicase nsp13 assist #SARSCoV2 polymerase to resolve them? And other co-factors, e.g. nsp9?

Enjoy the tour!