NMR Studies of Genomic RNA in 3′ Untranslated Region Unveil Pseudoknot Structure that Initiates Viral RNA Replication in SARS-CoV‑2

In the 3′ untranslated region of the SARS-CoV-2 virus RNA genome, genomic RNA replication is initiated in the highly conserved region called 3′PK, containing three stem structures (P1pk, P2, and P5). According to one proposed mechanism, P1pk and distal P2 stems switch their structure to a pseudoknot through base-pairing, thereby initiating transcription by recruiting RNA-dependent RNA polymerase complexed with nonstructural proteins (nsp)­7 and nsp8. However, experimental evidence of pseudoknot formation or structural switching is unavailable. Using SARS-CoV-2 3′PK fragments, we show that 3′PK adopted stem-loop and pseudoknot forms in a mutually exclusive manner. When P1pk and P2 formed a pseudoknot, the P5 stem, which includes a sequence at the 3′ end, exited from the stem-loop structure and opened up. Interaction with the nsp7/nsp8 complex destabilized the stem-loop form but did not alter the pseudoknot form. These results suggest that the interaction between the pseudoknot and nsp7/nsp8 complex transformed the 3′ end of viral genomic RNA into single-stranded RNA ready for synthesis, presenting the unique pseudoknot structure as a potential pharmacological target.