f Nonstructural proteins 7 and 8 of severe acute respiratory syndrome coronavirus (SARS-CoV) have previously been shown by X-ray crystallography to form an 8:8 hexadecamer. In addition, it has been demonstrated that N-terminally His 6 -tagged SARSCoV Nsp8 is a primase able to synthesize RNA oligonucleotides with a length of up to 6 nucleotides. We present here the 2.6-Å crystal structure of the feline coronavirus (FCoV) Nsp7:Nsp8 complex, which is a 2:1 heterotrimer containing two copies of the ␣-helical Nsp7 with conformational differences between them, and one copy of Nsp8 that consists of an ␣/␤ domain and a long-␣-helix domain. The same stoichiometry is found for the Nsp7:Nsp8 complex in solution, as demonstrated by chemical crosslinking, size exclusion chromatography, and small-angle X-ray scattering. Furthermore, we show that FCoV Nsp8, like its SARSCoV counterpart, is able to synthesize short oligoribonucleotides of up to 6 nucleotides in length when carrying an N-terminal His 6 tag. Remarkably, the same protein harboring the sequence GPLG instead of the His 6 tag at its N terminus exhibits a substantially increased, primer-independent RNA polymerase activity. Upon addition of Nsp7, the RNA polymerase activity is further enhanced so that RNA up to template length (67 nucleotides) can be synthesized. Further, we show that the unprocessed intermediate polyprotein Nsp7-10 of human coronavirus (HCoV) 229E is also capable of synthesizing oligoribonucleotides up to a chain length of six. These results indicate that in case of FCoV as well as of HCoV 229E, the formation of a hexadecameric Nsp7:Nsp8 complex is not necessary for RNA polymerase activity. Further, the FCoV Nsp7:Nsp8 complex functions as a noncanonical RNA polymerase capable of synthesizing RNA of up to template length.

Non-structural protein 9 (Nsp9) of coronaviruses is believed to bind single-stranded RNA in the viral replication complex. The crystal structure of Nsp9 of human coronavirus (HCoV) 229E reveals a novel disulfide-linked homodimer, which is very different from the previously reported Nsp9 dimer of SARS coronavirus. In contrast, the structure of the Cys69Ala mutant of HCoV-229E Nsp9 shows the same dimer organization as the SARS-CoV protein. In the crystal, the wild-type HCoV-229E protein forms a trimer of dimers, whereas the mutant and SARS-CoV Nsp9 are organized in rod-like polymers. Chemical cross-linking suggests similar modes of aggregation in solution. In zone-interference gel electrophoresis assays and surface plasmon resonance experiments, the HCoV-229E wild-type protein is found to bind oligonucleotides with relatively high affinity, whereas binding by the Cys69Ala and Cys69Ser mutants is observed only for the longest oligonucleotides. The corresponding mutations in SARS-CoV Nsp9 do not hamper nucleic acid binding. From the crystal structures, a model for single-stranded RNA binding by Nsp9 is deduced. We propose that both forms of the Nsp9 dimer are biologically relevant; the occurrence of the disulfide-bonded form may be correlated with oxidative stress induced in the host cell by the viral infection.

We report on the first use of carbon-nanotube based films to produce crystals of proteins. The crystals nucleate on the surface of the film. The difficulty of crystallising proteins is a major bottleneck in the determination of the structure and function of biological molecules. The crystallisation of two model proteins and two medically relevant proteins was studied. Quantitative data on the crystallisation times of the model protein lysozyme are also presented. Two types of the nanotube film, one made with the surfactant Triton X-100 (TX-100) and one with gelatin, were tested. Both induce nucleation of the crystal phase at supersaturations at which the protein solution would otherwise remain clear, however the gelatin-based film induced nucleation down to much lower supersaturations for the two model proteins with which it was used. It appears that the interactions of gelatin with the protein molecules are particularly favourable to nucleation. Crystals of the C1 domain of the human cardiac myosin-binding protein-C that diffracted to a resolution of 1.6Å, were obtained on the TX-100 film. This is far superior to the best crystals obtained using standard techniques, which only diffracted to 3.0 Å. Thus, both our nanotube-based films are very promising candidates for future work on crystallising difficult-tocrystallise target proteins.3