Highlights d Structure of SARS-CoV-2 replication-transcription complex (RTC) with nsp13 helicases d The nsp13 NTPase domains sit in front of the RCT, constraining functional models d Nsp13 may drive RTC backtracking, affecting proofreading and template switching d Structural analysis of ADP-Mg 2+-bound NiRAN domain, a potential antiviral target

RNA genome of positive polarityAn RNA genome that has mRNA polarity and, when released from viral particles, can be used directly by host ribosomes to produce viral proteins.

Molnupiravir, a wide-spectrum antiviral that is currently in phase 2/3 clinical trials for the treatment of COVID-19, is proposed to inhibit viral replication by a mechanism known as 'lethal mutagenesis'. Two recently published studies reveal the biochemical and structural bases of how molnupiravir disrupts the fidelity of SARS-CoV-2 genome replication and prevents viral propagation by fostering error accumulation in a process referred to as 'error catastrophe'.

We present an approach for preparing cryoEM grids to study short-lived molecular states. Using piezo electric dispensing, two independent streams of ~50 pL sample drops are deposited within 10 ms of each other onto a nanowire EM grid surface, and the mixing reaction stops when the grid is vitrified in liquid ethane, ~100 ms later. We demonstrate this approach for four biological systems where short-lived states are of high interest.

Backtracking, the reverse motion of the transcriptase enzyme on the nucleic acid template, is a universal regulatory feature of transcription in cellular organisms but its role in viruses is not established. Here we present evidence that backtracking extends into the viral realm, where backtracking by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) may aid viral transcription and replication. Structures of SARS-CoV-2 RdRp bound to the essential nsp13 helicase and RNA suggested the helicase facilitates backtracking. We use cryo-electron microscopy, RNA–protein cross-linking, and unbiased molecular dynamics simulations to characterize SARS-CoV-2 RdRp backtracking. The results establish that the single-stranded 3′ segment of the product RNA generated by backtracking extrudes through the RdRp nucleoside triphosphate (NTP) entry tunnel, that a mismatched nucleotide at the product RNA 3′ end frays and enters the NTP entry tunnel to initiate backtracking, and that nsp13 stimulates RdRp backtracking. Backtracking may aid proofreading, a crucial process for SARS-CoV-2 resistance against antivirals.