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 called3 ' 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.