HSC90 is required for nascent hepatitis C virus core protein stability in yeast cells

Hepatitis C virus core protein (Core) contributes to HCV pathogenicity. Here, we demonstrate that Core impairs growth in budding yeast. We identify HSP90 inhibitors as compounds that reduce intracellular Core protein level and restore yeast growth. Our results suggest that HSC90 (Hsc82) may function in the protection of the nascent Core polypeptide against degradation in yeast and the C-terminal region of Core corresponding to the organelle-interaction domain was responsible for Hsc82-dependent stability. The yeast system may be utilized to select compounds that can direct the C-terminal region to reduce the stability of Core protein.     

Large-scale development of expressed sequence tag-derived simple sequence repeat markers and diversity analysis in Arachis spp.

Large-scale development of expressed sequence tag simple sequence repeat (EST-SSR) markers was performed in peanut (Arachis hypogaea L.) to obtain more informative genetic markers. A total of 10,102 potential non-redundant EST sequences, including 3,445 contigs and 6,657 singletons, were generated from cDNA libraries of the gynophore, roots, leaves and seedlings. A total of 3,187 primer pairs were designed on flanking regions of SSRs, some of which allowed one and two base mismatches. Among the 3,187 markers generated, 2,540 (80%) were trinucleotide repeats, 302 (9%) were dinucleotide repeats, and 345 (11%) were tetranucleotide repeats. Pre-polymorphic analyses of 24 Arachis accessions were performed using 10% polyacrylamide gels. A total of 1,571 EST-SSR markers showing clear polymorphisms were selected for further polymorphic analysis with a Fluoro-fragment Analyzer. The 16 Arachis accessions examined included cultivated peanut varieties as well as diploid species with the A or B genome. Altogether 1,281 (81.5%) of the 1,571 markers were polymorphic among the 16 accessions, and 366 (23.3%) were polymorphic among the 12 cultivated varieties. Diversity analysis was performed and the genotypes of all 16 Arachis accessions showed similarity coefficients ranging from 0.37 to 0.97. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-011-9604-8) contains supplementary material, which is available to authorized users.     

Self-Enhancement of Hepatitis C Virus Replication by Promotion of Specific Sphingolipid Biosynthesis

Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.     

Prior immunization with severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) nucleocapsid protein causes severe pneumonia in mice infected with SARS-CoV

The details of the mechanism by which severe acute respiratory syndrome-associated coronavirus (SARS-CoV) causes severe pneumonia are unclear. We investigated the immune responses and pathologies of SARS-CoV-infected BALB/c mice that were immunized intradermally with recombinant vaccinia virus (VV) that expressed either the SARS-CoV spike (S) protein (LC16m8rVV-S) or simultaneously all the structural proteins, including the nucleocapsid (N), membrane (M), envelope (E), and S proteins (LC16m8rVV-NMES) 7-8 wk before intranasal SARS-CoV infection. The LC16m8rVV-NMES-immunized group exhibited as severe pneumonia as the control groups, although LC16m8rVV-NMES significantly decreased the pulmonary SARS-CoV titer to the same extent as LC16m8rVV-S. To identify the cause of the exacerbated pneumonia, BALB/c mice were immunized with recombinant VV that expressed the individual structural proteins of SARS-CoV (LC16mOrVV-N, -M, -E, -S) with or without LC16mOrVV-S (i.e., LC16mOrVV-N, LC16mOrVV-M, LC16mOrVV-E, or LC16mOrVV-S alone or LC16mOrVV-N + LC16mOrVV-S, LC16mOrVV-M + LC16mOrVV-S, or LC16mOrVV-E + LC16mOrVV-S), and infected with SARS-CoV more than 4 wk later. Both LC16mOrVV-N-immunized mice and LC16mOrVV-N + LC16mOrVV-S-immunized mice exhibited severe pneumonia. Furthermore, LC16mOrVV-N-immunized mice upon infection exhibited significant up-regulation of both Th1 (IFN-gamma, IL-2) and Th2 (IL-4, IL-5) cytokines and down-regulation of anti-inflammatory cytokines (IL-10, TGF-beta), resulting in robust infiltration of neutrophils, eosinophils, and lymphocytes into the lung, as well as thickening of the alveolar epithelium. These results suggest that an excessive host immune response against the nucleocapsid protein of SARS-CoV is involved in severe pneumonia caused by SARS-CoV infection. These findings increase our understanding of the pathogenesis of SARS.     

Effect of Bcl11b genotypes and gamma-radiation on the development of mouse thymic lymphomas

Bcl11b is a haploinsufficient tumor suppressor gene and expressed in many tissues such as thymus, brain and skin. Irradiated Bcl11b^+/− heterozygous mice mostly develop thymic lymphomas, but the preference of Bcl11b inactivation for thymic lymphomas remains to be addressed. We produced Bcl11b^+/− heterozygous and Bcl11b wild-type mice of p53^+/− background and compared their incidence of γ-ray induced thymic lymphomas. Majority of the tumors in p53^+/− mice were skin tumors, and only 5 (36%) of the 14 tumors were thymic lymphomas. In contrast, Bcl11b^+/−p53^+/− doubly heterozygous mice developed thymic lymphomas at the frequency of 27 (79%) of the 34 tumors developed (P = 0.008). This indicates the preference of Bcl11b impairment for thymic lymphoma development. We also analyzed loss of the wild-type alleles in the 27 lymphomas, a predicted consequence given by γ-irradiation. However, the loss frequency was low, only six (22%) for Bcl11b and five (19%) for p53. The frequencies did not differ from those of spontaneously developed thymic lymphomas in the doubly heterozygous mice, though the latency of lymphoma development markedly differed between them. This suggests that the main contribution of irradiation at least in those mice is not for the tumor initiation by inducing allelic losses but probably for the promotion of thymic lymphoma development.     

Update of MAGEST: Maboya Gene Expression patterns and Sequence Tags

MAGEST is a database for maternal gene expression information for an ascidian, Halocynthia roretzi. The ascidian has become an animal model in developmental biological research because it shows a simple developmental process, and belongs to one of the chordate groups. Various data are deposited into the MAGEST database, e.g. the 3′- and 5′-tag sequences from the fertilized egg cDNA library, the results of similarity searches against GenBank and the expression data from whole mount in situ hybridization. Over the last 2 years, the data retrieval systems have been improved in several aspects, and the tag sequence entries have increased to over 20 000 clones. Additionally, we constructed a database, translated MAGEST, for the amino acid fragment sequences predicted from the EST data sets. Using this information comprehensively, we should obtain new information on gene functions. The MAGEST database is accessible via the Internet at http://www.genome.ad.jp/magest/.     

Complete genomic sequence of nitrogen-fixing symbiotic bacterium Bradyrhizobium japonicum USDA110.

The complete nucleotide sequence of the genome of a symbiotic bacterium Bradyrhizobium japonicum USDA110 was determined. The genome of B. japonicum was a single circular chromosome 9,105,828 bp in length with an average GC content of 64.1%. No plasmid was detected. The chromosome comprises 8317 potential protein-coding genes, one set of rRNA genes and 50 tRNA genes. Fifty-two percent of the potential protein genes showed sequence similarity to genes of known function and 30% to hypothetical genes. The remaining 18% had no apparent similarity to reported genes. Thirty-four percent of the B. japonicum genes showed significant sequence similarity to those of both Mesorhizobium loti and Sinorhizobium meliloti, while 23% were unique to this species. A presumptive symbiosis island 681 kb in length, which includes a 410-kb symbiotic region previously reported by G?ttfert et al., was identified. Six hundred fifty-five putative protein-coding genes were assigned in this region, and the functions of 301 genes, including those related to symbiotic nitrogen fixation and DNA transmission, were deduced. A total of 167 genes for transposases/104 copies of insertion sequences were identified in the genome. It was remarkable that 100 out of 167 transposase genes are located in the presumptive symbiotic island. DNA segments of 4 to 97 kb inserted into tRNA genes were found at 14 locations in the genome, which generates partial duplication of the target tRNA genes. These observations suggest plasticity of the B. japonicum genome, which is probably due to complex genome rearrangements such as horizontal transfer and insertion of various DNA elements, and to homologous recombination.     

A water channel of the nematode C.elegans and its implications for channel selectivity of MIP proteins

A genome project focusingon the nematode Caenorhabditis elegans has demonstrated thepresence of eight cDNAs belonging to the major intrinsic proteinsuperfamily. We functionally characterized one of these cDNAs namedC01G6.1. Injection of C01G6.1 cRNA increased the osmotic waterpermeability (P_f) of Xenopusoocytes 11-fold and the urea permeability 4.5-fold but failed toincrease the glycerol permeability. It has been speculated that the MIPfamily may be separated into two large subfamilies based on thepresence or absence of two segments of extra amino acid residues (~15amino acids) at the second and third extracellular loops. BecauseC01G6.1 (designated AQP-CE1), AQP3, and glycerol facilitator (GlpF) all have these two segments, we replaced the segments of AQP-CE1 with thoseof AQP3 and GlpF to identify their roles. The functional characteristics of these mutants were principally similar to that ofwild-type AQP-CE1, although the values of P_f andurea permeability were decreased by 39-74% and 28-65%,respectively. These results suggest that the two segments of extraamino acid residues may not contribute to channel selectivity orformation of the route for small solutes.