Comparative production analysis of three phlebovirus nucleoproteins under denaturing or non-denaturing conditions for crystallographic studies.

Nucleoproteins (NPs) encapsidate the Phlebovirus genomic (-)RNA. Upon recombinant expression, NPs tend to form heterogeneous oligomers impeding characterization of the encapsidation process through crystallographic studies. To overcome this problem, we set up a standard protocol in which production under both non-denaturing and denaturing/refolding conditions can be investigated and compared. The protocol was applied for three phlebovirus NPs, allowing an optimized production strategy for each of them. Remarkably, the Rift Valley fever virus NP was purified as a trimer under native conditions and yielded protein crystals whereas the refolded version could be purified as a dimer. Yields of trimeric Toscana virus NP were higher from denaturing than from native condition and lead to crystals. The production of Sandfly Fever Sicilian virus NP failed in both protocols. The comparative protocols described here should help in rationally choosing between denaturing or non-denaturing conditions, which would finally result in the most appropriate and relevant oligomerized protein species. The structure of the Rift Valley fever virus NP has been recently published using a refolded monomeric protein and we believe that the process we devised will contribute to shed light in the genome encapsidation process, a key stage in the viral life cycle.

Improved crystallization of the coxsackievirus B3 RNA-dependent RNA polymerase.

The Picornaviridae virus family contains a large number of human pathogens such as poliovirus, hepatitis A virus and rhinoviruses. Amongst the viruses belonging to the genus Enterovirus, several serotypes of coxsackievirus coexist for which neither vaccine nor therapy is available. Coxsackievirus B3 is involved in the development of acute myocarditis and dilated cardiomyopathy and is thought to be an important cause of sudden death in young adults. Here, the first crystal of a coxsackievirus RNA-dependent RNA polymerase is reported. Standard crystallization methods yielded crystals that were poorly suited to X-ray diffraction studies, with one axis being completely disordered. Crystallization was improved by testing crystallization solutions from commercial screens as additives. This approach yielded crystals that diffracted to 2.1 A resolution and that were suitable for structure determination.

Preliminary characterization of (nucleoside-2'-O-)-methyltransferase crystals from Meaban and Yokose flaviviruses.

Viral methyltranferases (MTase) are involved in the third step of the mRNA-capping process, transferring a methyl group from S-adenosyl-L-methionine (SAM) to the capped mRNA. MTases are classified into two groups: (guanine-N7)-methyltransferases (N7MTases), which add a methyl group onto the N7 atom of guanine, and (nucleoside-2'-O-)-methyltransferases (2'OMTases), which add a methyl group to a ribose hydroxyl. The MTases of two flaviviruses, Meaban and Yokose viruses, have been overexpressed, purified and crystallized in complex with SAM. Characterization of the crystals together with details of preliminary X-ray diffraction data collection (at 2.8 and 2.7 angstroms resolution, respectively) are reported here. The sequence homology relative to Dengue virus 2'OMTase and the structural conservation of specific residues in the putative active sites suggest that both enzymes belong to the 2'OMTase subgroup.

Expression, purification and crystallization of the SARS-CoV macro domain.

Macro domains or X domains are found as modules of multidomain proteins, but can also constitute a protein on their own. Recently, biochemical and structural studies of cellular macro domains have been performed, showing that they are active as ADP-ribose-1''-phosphatases. Macro domains are also present in a number of positive-stranded RNA viruses, but their precise function in viral replication is still unknown. The major human pathogen severe acute respiratory syndrome coronavirus (SARS-CoV) encodes 16 non-structural proteins (nsps), one of which (nsp3) encompasses a macro domain. The SARS-CoV nsp3 gene region corresponding to amino acids 182-355 has been cloned, expressed in Escherichia coli, purified and crystallized. The crystals belong to space group P2(1), with unit-cell parameters a = 37.5, b = 55.6, c = 108.9 angstroms, beta = 91.4 degrees, and the asymmetric unit contains either two or three molecules. Both native and selenomethionine-labelled crystals diffract to 1.8 angstroms.

Crystallization and preliminary X-ray diffraction analysis of Nsp15 from SARS coronavirus.

The non-structural protein Nsp15 from the aetiological agent of SARS (severe acute respiratory syndrome) has recently been characterized as a uridine-specific endoribonuclease. This enzyme plays an essential role in viral replication and transcription since a mutation in the related H229E human coronavirus nsp15 gene can abolish viral RNA synthesis. SARS full-length Nsp15 (346 amino acids) has been cloned and expressed in Escherichia coli with an N-terminal hexahistidine tag and has been purified to homogeneity. The protein was subsequently crystallized using PEG 8000 or 10 000 as precipitants. Small cubic crystals of the apoenzyme were obtained from 100 nl nanodrops. They belong to space group P4(1)32 or P4(3)32, with unit-cell parameters a = b = c = 166.8 angstroms. Diffraction data were collected to a maximum resolution of 2.7 angstroms.