Insights into Binding of Single-Stranded Viral RNA Template to the Replication–Transcription Complex of SARS-CoV‑2 for the Priming Reaction from Molecular Dynamics Simulations

A minimal
replication–transcription complex (RTC) of SARS-CoV-2
for synthesis of viral RNAs includes the nsp12 RNA-dependent RNA polymerase
and two nsp8 RNA primase subunits for de novo primer
synthesis, one nsp8 in complex with its accessory nsp7 subunit and
the other without it. The RTC is responsible for faithfully copying
the entire (+) sense viral genome from its first 5′-end to
the last 3′-end nucleotides through a replication-intermediate
(RI) template. The single-stranded (ss) RNA template for the RI is
its 33-nucleotide 3′-poly­(A) tail adjacent to a well-characterized
secondary structure. The ssRNA template for viral transcription is
a 5′-UUUAU-3′ next to stem-loop (SL) 1′. We analyze
the electrostatic potential distribution of the nsp8 subunit within
the RTC around the template strand of the primer/template (P/T) RNA
duplex in recently published cryo-EM structures to address the priming
reaction using the viral poly­(A) template. We carried out molecular
dynamics (MD) simulations with a P/T RNA duplex, the viral poly­(A)
template, or a generic ssRNA template. We find evidence that the viral
poly­(A) template binds similarly to the template strand of the P/T
RNA duplex within the RTC, mainly through electrostatic interactions,
providing new insights into the priming reaction by the nsp8 subunit
within the RTC, which differs significantly from the existing proposal
of the nsp7/nsp8 oligomer formed outside the RTC. High-order oligomerization
of nsp8 and nsp7 for SARS-CoV observed outside the RTC of SARS-CoV-2
is not found in the RTC and not likely to be relevant to the priming
reaction.