The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension

te Velthuis, Aartjan J.W.; van den Worm, Sjoerd H. E.; Snijder, Eric J.

To investigate the properties of the nsp7+nsp8 [nsp(7+8)] hexadecamer in more detail, and seek answers to the above questions, we here generated and purified recombinant forms of SARS-CoV nsp8 and nsp(7+8) that have natural N-terminal residues. This technical refinement was found to greatly improve nsp8's ability to associate with nsp7. Moreover, and in contrast to previous observations (12) , exposure of the natural N-terminus proved crucial for the enzymatic activity of the complex on partially double-stranded RNA templates, demonstrating that nsp(7+8) is capable of primerdependent RdRp activity as well. Site-directed mutagenesis of nsp8 in the context of the nsp(7+8) complex identified a conserved D/ExD/E motif that is important for catalysis in vitro, possibly providing a first indication of the location of the presently unknown nsp8 active site. Overall, these results define the SARS-CoV nsp(7+8) complex as an intriguing multimeric RNA polymerase that is capable of primer extension.