Coronavirus replication and transcription machinery involves multiple virus-encoded nonstructural proteins (nsp). We report the crystal structure of the hexadecameric nsp7-nsp8 supercomplex from the severe acute respiratory syndrome coronavirus at 2.4-angstroms resolution. nsp8 has a novel 'golf-club' fold with two conformations. The supercomplex is a unique hollow, cylinder-like structure assembled from eight copies of nsp8 and held tightly together by eight copies of nsp7. With an internal diameter of approximately 30 angstroms, the central channel has dimensions and positive electrostatic properties favorable for nucleic acid binding, implying that its role is to confer processivity on RNA-dependent RNA polymerase.

New domains were progressively added to cytoplasmic aminoacyl transfer RNA (tRNA) synthetases during evolution. One example is the UNE-S domain, appended to seryl-tRNA synthetase (SerRS) in species that developed closed circulatory systems. Here we show using solution and crystal structure analyses and in vitro and in vivo functional studies that UNE-S harbours a robust nuclear localization signal (NLS) directing SerRS to the nucleus where it attenuates vascular endothelial growth factor A expression. We also show that SerRS mutants previously linked to vasculature abnormalities either deleted the NLS or have the NLS sequestered in an alternative conformation. A structure-based second-site mutation, designed to release the sequestered NLS, restored normal vasculature. Thus, the essential function of SerRS in vascular development depends on UNE-S. These results are the first to show an essential role for a tRNA synthetase-associated appended domain at the organism level, and suggest that acquisition of UNE-S has a role in the establishment of the closed circulatory systems of vertebrates.

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...

Recent studies suggested an essential role for seryl-tRNA synthetase (SerRS) in vascular development. This role is specific to SerRS among all tRNA synthetases and is independent of its well-known aminoacylation function in protein synthesis. A unique nucleus-directing domain, added at the invertebrate-to-vertebrate transition, confers this novel non-translational activity of SerRS. Previous studies showed that SerRS, in some unknown way, controls VEGFA expression to prevent vascular over-expansion. Using in vitro, cell and animal experiments, we show here that SerRS intervenes by antagonizing c-Myc, the major transcription factor promoting VEGFA expression, through a tandem mechanism. First, by direct head-to-head competition, nuclear-localized SerRS blocks c-Myc from binding to the VEGFA promoter. Second, DNA-bound SerRS recruits the SIRT2 histone deacetylase to erase prior c-Myc-promoted histone acetylation. Thus, vertebrate SerRS and c-Myc is a pair of ‘Yin-Yang’ transcriptional regulator for proper development of a functional vasculature. Our results also discover an anti-angiogenic activity for SIRT2.DOI: http://dx.doi.org/10.7554/eLife.02349.001

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The development of drugs with rapid distribution in the kidney and long-term retention in the renal tubule is a breakthrough for enhanced treatment of acute kidney injury (AKI). Here, l-serine–modified chitosan (SC) was synthesized as a potential AKI kidney–targeting agent due to the native cationic property of chitosan and specific interaction between kidney injury molecule–1 (Kim-1) and serine. Results indicated that SC was rapidly accumulated and long-term retained in ischemia-reperfusion–induced AKI kidneys, especially in renal tubules, which was possibly due to the specific interactions between SC and Kim-1. SC-TK-SS31 was then prepared by conjugating SS31, a mitochondria-targeted antioxidant, to SC via reactive oxygen species (ROS)–sensitive thioketal linker. Because of the effective renal distribution combined with ROS-responsive drug release behavior, the administration of SC-TK-SS31 led to an enhanced therapeutic effect of SS31 by protecting mitochondria from damage and reducing the oxidative stress, inflammation, and cell apoptosis.

XX males and XY females have a sex reversal disorder which can be caused by an abnormal interchange between the X and the Y chromosomes. We have isolated and characterized a novel gene on the Y chromosome, PRKY. This gene is highly homologous to a previously isolated gene from Xp22.3, PRKX, and represents a member of the cAMP-dependent serine threonine protein kinase gene family. Abnormal interchange can occur anywhere on Xp/Yp proximal to SRY. We can show that abnormal interchange happens particularly frequently between PRKX and PRKY. In a collection of 26 XX males and four XY females, between 27 and 35% of the interchanges take place between PRK homologues but at different sites within the gene. PRKY and PRKX are located far from the pseudoautosomal region where XY exchange normally takes place. The unprecedented high sequence identity and identical orientation of PRKY to its homologous partner on the X chromosome, PRKX, explains the high frequency of abnormal pairing and subsequent ectopic recombination, leading to XX males and XY females and to the highest rate of recombination outside the pseudoautosomal region.

Angiogenesis is recognized as an important hallmark of cancer. Although telomerase is thought to be involved in tumor angiogenesis, the evidence and underlying mechanism remain elusive. Here, we demonstrate that human telomerase reverse transcriptase (hTERT) activates vascular epithelial growth factor (VEGF) gene expression through interactions with the VEGF promoter and the transcription factor Sp1. hTERT binds to Sp1 in vitro and in vivo and stimulates angiogenesis in a manner dependent on Sp1. Deletion of the mTert gene in the first generation of Tert null mice compromised tumor growth, with reduced VEGF expression. In addition, we show that hTERT expression levels are positively correlated with those of VEGF in human gastric tumor samples. Together, our results demonstrate that hTERT facilitates tumor angiogenesis by up-regulating VEGF expression through direct interactions with the VEGF gene and the Sp1 transcription factor. These results provide novel insights into hTERT function in tumor progression in addition to its role in telomere maintenance.