Structural And Biochemical Insights Into Therna2’-o-methylation By Sars-covnsp10/Nsp16Protein Complex

Coronaviruses, which belong to the order Nidovirales, family Coronaviridae, are positive-sense single-stranded RNA viruses. The length of coronaviruses genome is about30000nucleotides, which makes it the largest genome of all RNA viruses. The genome of coronavirus contains more than7open reading frames (ORFs). The two ORFs (ORF la/ORF lab) at the5’-terminus of viral genome encode two polyproteins, which are cleaved by viral proteases into16non-structural proteins (nspl-16) co-and post-translationally. These non-structural proteins, together with several host factors and viral RNA, assemble viral replication and transcription complex to accomplish viral genome replication.In the end of2002, an emerging coronavirus broke out in Guangdong Province, P. R. China, and spread all over the world subsequently, causing about8000infections with10%lethality. This coronavirus was named as severe acute respiratory syndrome coronavirus (SARS-CoV). Because of its high pathogenicity, research about coronavirus became a hotspot. In September,2012, another emerging coronavirus broke out in the Middle East and spread to Europe, which was named as Middle East respiratory syndrome coronavirus (MERS-CoV). Until now, MERS-CoV has caused630infections and199mortalities. Coronavirus is typical zoonotic virus, which can transmitto humans from animals. This character of coronavirus makes it tend to cause emerging infection and became a threat of public health.Coronavirus viral RNAs possess5’-terminal cap1structural modification. The5’-terminal modification of RNA virus is important for viral genome replication, viral protein synthesis, virus particleassembly and escape from the recognition of host innate immune system. These functions of5’-terminal modification make it a potential target for antiviral drug discovery. The life cycle of coronavirus happens in cytoplasm, which makes it impossible to hijack the host enzymes to cap viral RNAs. Instead, coronavirus encodes viral proteins to accomplish5’-terminal capping. Previous research reveals that SARS-CoV nsp10can initiate the2’-O-methyltransferase activity of nsp16by forming nsp10/nsp16protein complex. The2’-O-methylation is the final step of cap1structure formation. However, its mechanism is still not clarified.We use protein X-ray crystallography to gain the high resolution structure of SARS-CoV nsp10/nsp16in complex with methyl donor SAM, and the resolution is2 A. The crystal structure of nsp10/nsp16protein complex reveals that, the protein surface of nspl6is covered by loop regions, especially SAM binding pocket and RNA binding cleft, the aD-helix of nsp16is too short to hold the wall of SAM binding pocket, the wall of nsp16RNA binding groove is too flexible, which makes it unable to binding guanosine group of the RNA cap structure through π-π stacking. All these structural features of nsp16make it a unique2’-O-methyltransferase compared with other2’-O-methyltransferase. These structural features make single nsp16unable to bind methyl donor SAM and substrate RNA. In the crystal structure of nsp10/nspl6, nsp10can stabilize SAM binding pocket of nsp16by two polar contacts, and also extend the positive charged surface of nsp16at its potential RNA binding groove. These two functions of nsplO help nspl6to bind methyl donor SAM and substrate RNA, thus initiate the2’-O-methyltransferase activity of nsp16. Furthermore, we use biochemical approach to confirm this hypothesis. The revelation of the mechanism that nsp10initiates the2’-O-methyltransferase activity of nsp16provides a potential target for novel specific anti-coronavirus drug design and discovery.