Template-dependent RNA polyadenylation by SARS-CoV-2 RNA-dependent RNA polymerase

The RNA genome of the human Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its mRNAs contain a poly(A) tract at the 3' end of the positive strand that is essential for their replication, translation and stability. Viral RNAs maintain poly(A) tails despite the lack of a cytoplasmic polyadenylation machinery, but the underlying mechanisms remain unknown. Here, we investigated the activity of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) with synthetic and natural primer-template RNA substrates and discovered that RdRp could extend the 3' end of the RNA primer beyond the template end. This activity depends on the presence of a homopolymeric sequence at the 5' end of the template strand and is probably explained by 'slippage' and reannealing of the primer 3' end to this sequence. In the case of viral minus-strand templates containing a homopolymeric uridylate sequence this activity can ensure the extension of the poly(A) tail in the plus-strand products synthesized by RdRp. We establish the requirements for this activity in vitro and propose that it may have an important role in SARS-CoV-2 replication and mRNA synthesis inside cells.