Human coronaviruses (CoVs) such as severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) cause epidemics of severe human respiratory disease. A conserved step of CoV replication is the translation and processing of replicase polyproteins containing 16 nonstructural protein domains (nsp's 1 to 16). The CoV nsp5 protease (3CLpro; Mpro) processes nsp's at 11 cleavage sites and is essential for virus replication. CoV nsp5 has a conserved 3-domain structure and catalytic residues. However, the intra-and intermolecular determinants of nsp5 activity and their conservation across divergent CoVs are unknown, in part due to challenges in cultivating many human and zoonotic CoVs. To test for conservation of nsp5 structure-function determinants, we engineered chimeric betacoronavirus murine hepatitis virus (MHV) genomes encoding nsp5 proteases of human and bat alphacoronaviruses and betacoronaviruses. Exchange of nsp5 proteases from HCoV-HKU1 and HCoV-OC43, which share the same genogroup, genogroup 2a, with MHV, allowed for immediate viral recovery with efficient replication albeit with impaired fitness in direct competition with wild-type MHV. Introduction of MHV nsp5 temperature-sensitive mutations into chimeric HKU1 and OC43 nsp5 proteases resulted in clear differences in viability and temperature-sensitive phenotypes compared with MHV nsp5. These data indicate tight genetic linkage and coevolution between nsp5 protease and the genomic background and identify differences in intramolecular networks regulating nsp5 function. Our results also provide evidence that chimeric viruses within coronavirus genogroups can be used to test nsp5 determinants of function and inhibition in common isogenic backgrounds and cell types. C oronaviruses (CoVs) are enveloped, positive-strand RNA viruses that infect a wide range of animal hosts. Human CoVs cause illnesses including the common cold and severe acute respiratory syndrome (SARS) as well as the recently identified Middle East respiratory syndrome (MERS) associated with infection of a novel coronavirus (1). Coronaviruses are members of the order Nidovirales, family Coronaviridae, and subfamily Coronavirinae. Among the viruses in Coronavirinae, four main genera have recently been designated (2): alphacoronaviruses, which contain human coronavirus 229E (HCoV-229E) and HCoV-NL63; betacoronaviruses, containing human coronaviruses SARS-CoV, HKU1, MERS-CoV, and OC43; and gammacoronaviruses and deltacoronaviruses, from which no current human coronaviruses have been identified. The betacoronavirus murine hepatitis virus (MHV) is a well-established model for the study of coronavirus replication and pathogenesis. The MHV genome is 32 kb in length and encodes 7 genes (Fig. 1A) (3-5). An essential step of CoV replication is the translation of the ORF1ab replicase polyproteins and the subsequent processing of up to 16 nonstructural proteins (nsp's 1 to 16), including the nsp12 RNA-dependent RNA polymerase (3,5,6). CoV nsp5 protease (3CLpro; Mpro) med...

A sparse-matrix screen for new crystallization conditions for the USP7 catalytic domain (USP7 CD ) led to the identification of a condition in which crystals grow reproducibly in 24-48 h. Variation of the halide metal, growth temperature and seed-stock concentration resulted in a shift in space group from P2 1 with two molecules in the asymmetric unit to C2 with one molecule in the asymmetric unit. Representative structures from each space group were determined to 2.2 Å resolution and these structures support previous findings that the catalytic triad and switching loop are likely to be in unproductive conformations in the absence of ubiquitin (Ub). Importantly, the new structures reveal previously unobserved electron density for blocking loop 1 (BL1) residues 410-419. The new structures indicate a distinct rearrangement of the USP7 BL1 compared with its position in the presence of bound Ub.

Polycystin 2-type cation channels PKD2 and PKD2L1 interact with polycystin 1-type proteins PKD1 and PKD1L3 respectively, to form receptor-cation-channel complexes. The PKD2L1-PKD1L3 complex perceives sour taste, whereas disruption of the PKD2-PKD1 complex, responsible for mechanosensation, leads to development of ADPKD (autosomal-dominant polycystic kidney disease). Besides modulating channel activity and related signalling events, the CRDs (C-terminal regulatory domains) of PKD2 and PKD2L1 play a central role in channel oligomerization. The present study investigates the aggregation state of purified full-length PKD2L1-CRD as well as truncations of CRDs from PKD2 channels. Far- and near-UV CD spectroscopy show that the full-length PKD2L1 CRD (PKD2L1-198) and the truncated PKD2 CRD (PKD2-244) are alpha-helical with no beta-sheet, the alpha-helix content agrees with sequence-based predictions, and some of its aromatic residues are in an asymmetric environment created at least by partially structured regions. Additionally, the CRD truncations exhibit an expected biochemical function by binding Ca2+ in a physiologically relevant range with Kd values of 2.8 muM for PKD2-244 and 0.51 muM for PKD2L1-198. Complimentary biophysical and biochemical techniques establish that truncations of the PKD2 and PKD2L1 CRDs are elongated molecules that assemble as trimers, and the trimeric aggregation state is independent of Ca2+ binding. Finally, we show that a common coiled-coil motif is sufficient and necessary to drive oligomerization of the PKD2 and PKD2L1 CRD truncations under study. Despite the moderate sequence identity (39%) between CRDs of PKD2 and PKD2L1, they both form trimers, implying that trimeric organization of CRDs may be true of all polycystin channels.

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Inorganic pyrophosphatases are potential targets for the development of novel antibacterial agents. A pyrophosphatase-coupled high-throughput screening assay intended to detect o-succinyl benzoic acid coenzyme A (OSB CoA) inhibitors led to the unexpected discovery of a new series of novel inorganic pyrophosphatase inhibitors. Lead optimization studies resulted in a series of 3-(3-aryl-pyrrolidin-1-yl)-5-aryl-1,2,4-triazine derivatives that were prepared by an efficient synthetic pathway. One of the tetracyclic triazine analogues 22h displayed promising antibiotic activity against a wide variety of drug-resistant Staphylococcus aureus strains, as well as activity vs. Mycobacterium tuberculosis and Bacillus anthracis, at a concentration that was not cytotoxic to mammalian cells.

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