New Antiviral Target Revealed by the Hexameric Structure of Mouse Hepatitis Virus Nonstructural Protein nsp15

Abstract

The unique coronavirus transcription/replication machinery comprised of multiple virus-encoded nonstructural proteins (nsp) plays a vital role during initial and intermediate phases of the viral life cycle. The crystal structure of mouse hepatitis virus strain A59 (MHV-A59) nsp15 is reported at 2.15-Å resolution. nsp15 is an XendoU endoribonuclease and is the first one from this family to have its structure unveiled. The MHV-A59 nsp15 monomer structure has a novel protein fold. Two nsp15 trimers form a back-to-back hexamer that is believed to be the functional unit. The structure reveals the catalytic site including the highly conserved residues His262, His277, and Lys317, which is supported by mutagenesis analysis. Gel filtration and enzyme activity assays confirmed that the hexamer is the active form for nsp15 and demonstrate the specificity of nsp15 for uridylate. The high sequence conservation of nsp15 in coronaviruses, including that of severe acute respiratory syndrome, suggests that this protein may provide a new target for the design of antiviral therapeutics.Mouse hepatitis virus (MHV) belongs to group II of the genus Coronavirus (CoV), together with bovine coronavirus, human coronavirus strain OC43, and the recently identified severe acute respiratory syndrome coronavirus (SARS-CoV) (2). The two strains of MHV that have been extensively studied to date are A59 (MHV-A59) and JHM (MHV-JHM or MHV-4). These coronavirus strains cause a variety of diseases in susceptible mice, such as enteritis, hepatitis, and panencephalitis, with acute and chronic demyelination that is histologically similar to multiple sclerosis in humans (13,14). Much of our knowledge concerning the replication mechanism of coronaviruses has been acquired from the use of MHV as a model for pathogenesis, docking and entry, receptor usage, transcription, replication, polymerase function, assembly, and release (31). MHV-A59 in particular is used extensively as a model to study the role of the immune system in virus-induced ventral nervous system demyelination. To date, the pathology of demyelination is still not clear; thus, further insights into the replication and transcription of the model are required to better use this model for further study of demyelination, replication, and transcription of other coronaviruses.MHV-A59 is a positive-strand RNA coronavirus with a genome of 31 kb in length, of which about 22 kb is encompassed by the replicase gene containing two large overlapping open reading frames (ORF), termed ORF1a and ORF1b. The replicase gene expresses two large polyproteins, pp1a (495 kDa) and pp1ab (803 kDa), where expression of pp1ab involves a Ϫ1 ribosomal frameshift just upstream of the ORF1a translation termination codon (31). Neither pp1a nor pp1ab is detected intact in MHV-infected cells, since they are cotranslationally and posttranslationally processed by three proteases (two papain-like proteases, PLP1 and PLP2, and a main protease, M pro ) into at least 14 mature nonstructural proteins (1,23,29). These nonstructural p...

Structural and Functional Analyses of the Severe Acute Respiratory Syndrome Coronavirus Endoribonuclease Nsp15

“…When arranged into a hexamer, the six active sites are exposed at the surface of the hexameric structure. Furthermore, the active site of Nsp15 can be modeled according to the residues of RNase A that share a common mechanism of catalysis (21,22). In this structure, the C-terminal tail of Nsp15 folds back on the active site, suggesting that it may play a role in specific recognition of the cognate uridylate.…”

The Coronavirus Endoribonuclease Nsp15 Interacts with Retinoblastoma Tumor Suppressor Protein

“…Furthermore, cleavage occurs through the formation of a 2=-3= cyclic phosphodiester product in a mechanism identical to that of RNase A (4). Crystal structures of the SARS-CoV and the mouse hepatitis virus (MHV) Nsp15 have been reported, and both proteins form hexamers in solution (3,36,42).Mutations in the active site of Nsp15 that apparently abolish endoribonuclease activity in vitro reduce viral infectivity by up to 2 logs (20, 35). However, some mutations outside of the active site are reported to have a larger effect on virus viability, suggesting that Nsp15 has role(s) in coronavirus infection apart from its function as an endoribonuclease (18,20).…”

Crystallization and preliminary X-ray crystallographic analysis of a nonstructural protein 15 mutant fromHuman coronavirus 229E

“…4). For SARS-CoV and MHV, two size-exclusion chromatography peaks indicate that their nsp15 proteins form hexamers as well as monomers (Ricagno et al, 2006;Xu et al, 2006). Although several mutations were made to attempt to destroy the trimertrimer interactions of nsp15 in SARS-CoV, only monomers could be observed instead of trimers (Bhardwaj et al, 2008).…”

“…In previous investigations on nsp15s, even though several sitedirected mutations were made, trimers only existed in the transition state between hexamers and monomers. Stable trimeric nsp15 has not been obtained before; thus, its structure and enzymatic activity have not yet been reported (Ricagno et al, 2006;Joseph et al, 2007;Xu et al, 2006). In this work, trimeric nsp15 from HCoV-229E was successfully obtained by mutating Ile26 and Asn52 to alanine, and the expression, purification, crystallization and preliminary X-ray diffraction studies of this trimeric nsp15 are described.…”

“…The crystal structures of these two homologous nsp15s have been determined previously (Xu et al, 2006;Ricagno et al, 2006), demonstrating that nsp15 is a hexamer formed by a dimer of trimers (PDB entries 2gth and 2h85). In addition, the structure of the monomeric nsp15 in SARS-CoV was determined through an N-terminal domain truncation (PDB entry 2ozk; Joseph et al, 2007).…”