Biology subjects

Marecki, J. C.

Publications and source records attributed to Marecki, J. C..

3 recordsLinked to original sources

bioRxiv2025

Template switching by coronavirus polymerase requires helicase activity and is stimulated by remdesivir and molnupiravir

Polymerase template switching is an essential mechanism in coronaviruses (CoVs) that enables both sub-genomic (sg) RNA synthesis and increases genomic diversity via RNA recombination. Despite its importance, the molecular mechanism of CoV polymerase template switching remains unclear. Using magnetic tweezers, we show that the CoV non-structural protein (nsp) 13-helicase drives polymerase template switching, followed by copy-back RNA synthesis. This activity requires nsp13-helicase ATPase activity and a duplex RNA downstream of the CoV polymerase. This novel function of nsp13-helicase is targeted by the nucleotide analogs remdesivir and molnupiravir, whose incorporation in the nascent strand increases CoV polymerase template switching probability, leading to defective RNA production. We propose a novel mechanism of action where incorporation of these analogs dramatically reduces full length genome copy number by stimulating polymerase template switching. Our study further demonstrates nsp13-helicases central role in CoV replication and how this enzyme function can be indirectly targeted by analogs.

microbiology↗

bioRxiv2025

The coronavirus helicase synergizes with the viral RNA polymerase to enable rapid RNA synthesis through duplex RNA

Positive-sense (+) RNA viruses often encode helicases presumed to support replication. Their precise role remains unresolved though, especially in coronaviruses (CoV) where the helicase translocates in the opposite direction to the polymerase. Using high-throughput single-molecule magnetic tweezers, we show that the coronavirus helicase enhances RNA synthesis through duplex RNA by tenfold, forming a directional complex with the viral polymerase. Despite opposing polarity, the helicase coordinates elongation by engaging the non-template strand. A detailed kinetic model derived from large datasets reveals distinct dynamic states, including fast bursting and slow, backtracking-prone modes, which are governed by helicase engagement. These results uncover an active coupling mechanism that modulates replication dynamics and provide a mechanistic basis for continuous versus discontinuous RNA synthesis in coronaviruses. Our findings establish the viral helicase as a central regulator of RNA replication rather than a passive accessory enzyme.

biophysics↗

bioRxiv2025

A post-assembly conformational change makes the SARS-CoV-2 polymerase elongation-competent

Coronaviruses (CoV) encode sixteen non-structural proteins (nsps), most of which form the replication-transcription complex (RTC). The RTC contains a core composed of one nsp12 RNA-dependent RNA polymerase (RdRp), two nsp8s and one nsp7. The core RTC recruits other nsps to synthesize all viral RNAs within the infected cell. While essential for viral replication, the mechanism by which the core RTC assembles into a processive polymerase remains poorly understood. We show that the core RTC preferentially assembles by first having nsp12-polymerase bind to the RNA template, followed by the subsequent association of nsp7 and nsp8. Once assembled on the RNA template, the core RTC requires hundreds of seconds to undergo a conformational change that enables processive elongation. In the absence of RNA, the (apo-)RTC requires several hours to adopt its elongation-competent conformation. We propose that this obligatory activation step facilitates the recruitment of additional nsps essential for efficient viral RNA synthesis and may represent a promising target for therapeutic interventions.

biophysics↗