Structural And Functional Studies Of Coronavirus Replicases

Coronaviruses(CoVs)are one of the most dangerous microbiological pathogens that threaten the health of both human and animals. Especially, the newly emerging CoV member, SARS CoV, caused the breakout of"SARS"epidemic which led to worldwide attention and terror.CoV possessed complex mechanisms for self-replication: after infection, the RNA genome of CoV undergoes direction translation or/and discontinuous-transcription/translation to generate various viral proteins, including non-structural proteins(nsp), structural proteins and accessory proteins respectively. Non-structural proteins are thought to constitute the replication/transcription machinery of CoV in structural and function.Among the non-structural proteins released as functional units from the replicase poly-protein, nsp7 and nsp8 can form a primase supercomplex for de novo RNA synthesis to prime the viral RNA dependent RNA polymerase(RdRP, or nsp12 in coronavirus). The primase supercomplex is a hollow, cylindrical structure assembled from eight copies of nsp8 and tightly held together by eight copies of nsp7,which differs from any known RNA polymerases. In order to study the RNA-primase interaction and the mechanism of de novo synthesis in coronaviruses, we determined two crystal structures: the primase-GDP complex, and the C-terminal domain of nsp8 complexed with a single strand DNA oligo mimicking the RNA template. Based on these observations, we propose a model in which the template first bends into a cleft located around the rim of the central tunnel, then meets the incoming NTP along a positively-charged track constituted by the N-terminus of an adjacent nsp8. This is a new mechanism of RNA dependent RNA polymerases, and the first structural based model of coronavirus'primase.In this study, we also solved the structure of the Mpro from a newly emergent coronavirus which belongs to the group IIa family: the HCoV-HKU1 M^pro. Interestingly, although the CoV M^pros possesses similar main-chain structures as well as absolute specificity on P1 site as a Glutamine, the HCoV-HKU1 M^pro showed a second choice on histidine, which could be explained by ours structure that the smallest S1 pocket of HCoV-HKU1 M^pro might provide extra van de waals interaction to compensate the loss of one hydrogen-bond due to the mutation from glutamine to histidine. Additionally, we also discovered a group-specific feature among the M^pro structures in spite of the resemblance in their main-chain backbones.