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Journal article(2026)
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Pim P.B. America, Subhas C. Bera, Arnab Das, Thomas K. Anderson, John C. Marecki, Flávia S. Papini, Jamie J. Arnold, Martin Depken, David Dulin, More Authors
SummaryPositive-sense RNA ((+)RNA) viruses often encode helicases presumed to support replication. Their precise role remains unresolved, though, especially in coronaviruses (CoVs), 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 10-fold, forming a directional complex with the viral polymerase. Despite opposing polarity, the helicase coordinates elongation by engaging with 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.
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SummaryPositive-sense RNA ((+)RNA) viruses often encode helicases presumed to support replication. Their precise role remains unresolved, though, especially in coronaviruses (CoVs), 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 10-fold, forming a directional complex with the viral polymerase. Despite opposing polarity, the helicase coordinates elongation by engaging with 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.
Journal article(2025)
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Misha Klein, Arnab Das, Subhas C. Bera, Thomas K. Anderson, Dana Kocincova, Hery W. Lee, Bing Wang, Martin Depken, David Dulin
Coronaviruses (CoVs) encode 16 nonstructural 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.
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Coronaviruses (CoVs) encode 16 nonstructural 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.
