RNA virus polymerase-helicase coupling enables rapid elongation through duplex RNA.

Abstract

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.