Xenbase: a genomic, epigenomic and transcriptomic model organism database.

Xenbase (www.xenbase.org) is an online resource for researchers utilizing Xenopus laevis and Xenopus tropicalis, and for biomedical scientists seeking access to data generated with these model systems. Content is aggregated from a variety of external resources and also generated by in-house curation of scientific literature and bioinformatic analyses. Over the past two years many new types of content have been added along with new tools and functionalities to reflect the impact of high-throughput sequencing. These include new genomes for both supported species (each with chromosome scale assemblies), new genome annotations, genome segmentation, dynamic and interactive visualization for RNA-Seq data, updated ChIP-Seq mapping, GO terms, protein interaction data, ORFeome support, and improved connectivity to other biomedical and bioinformatic resources.

The envelope glycoprotein of human endogenous retrovirus HERV-W induces cellular resistance to spleen necrosis virus.

Human endogenous retrovirus type W (HERV-W) envelope glycoprotein (Env) has recently been reported to induce fusion in cells expressing the RD-114 and type D retrovirus receptor (RDR) and to serve as a functional retroviral envelope protein. In this report, another biological function for HERV-W was demonstrated by testing its ability to protect cells against retroviral infection. Spleen necrosis virus (SNV), a gammaretrovirus was chosen for testing resistance because it uses RDR to enter cells. An HERV-W Env expression plasmid was transfected into canine osteosarcoma cells (D-17), which are permissive for SNV infection. Cell fusion assays were performed to demonstrate biological function of HERV-W Env in D-17 cells. The presence of HERV-W env sequences was confirmed in stably transfected cell clones by using polymerase chain reaction. Viral infectivity assays were performed with SNV and amphotropic Murine leukemia virus (MLV-A) pseudotyped vector viruses to measure titers in D-17 cells expressing HERV-W Env and in negative control cells. The HERV-W Env caused fusion of D-17 cells in culture and greatly reduced infection by SNV vector virus. A 1000- to 10,000-fold decrease in SNV infectivity was observed for D-17 cells expressing HERV-W Env as compared to D-17 cells that were not expressing HERV-W Env. In contrast, infection by MLV-A pseudotyped vector virus was not significantly reduced. Thus, HERV-W Env confers host cell resistance to infection by SNV. This is the first report of a human endogenous retrovirus gene product blocking infection by any exogenous retrovirus.

High fidelity of homologous retroviral recombination in cell culture.

Genetic variation continues to be a major obstacle in the development of therapies and vaccines against retroviral infections and contributes extensively to viral pathogenesis and persistence. Recombination is one mechanism that increases retroviral variation by shuffling mutations from different genomes. Recent studies suggest that recombination not only shuffles the mutations but also generates them at high rates during reverse transcription. In contrast to these recent studies, this investigation shows that recombination does not generate mutations during recombination. A spleen necrosis virus (SNV)-based homologous recombination system was used to test the hypothesis that retroviral recombination is a high-fidelity process during replication of the virus in cell culture. The system consisted of a pair of SNV vectors expressing two drug resistance genes. The vectors were constructed so that cells containing recombinant proviruses could be selected by a double drug-resistant phenotype. Restriction enzyme digestion and agarose gel electrophoresis were used to map the location of recombination within 182 proviruses. Sequencing and single-strand conformation polymorphism techniques were then used to check for mutations within the recombinant proviruses. Since no mutations were detected among the 182 recombinants that were analyzed, homologous recombination is a high-fidelity process for retroviruses in cell culture.

Differential modulation and subsequent blockade of mitogenic signaling and cell cycle progression by Pasteurella multocida toxin.

The intracellularly acting protein toxin of Pasteurella multocida (PMT) causes numerous effects in cells, including activation of inositol 1,4,5-trisphosphate (IP(3)) signaling, Ca(2+) mobilization, protein phosphorylation, morphological changes, and DNA synthesis. The direct intracellular target of PMT responsible for activation of the IP(3) pathway is the G(q/11)alpha-protein, which stimulates phospholipase C (PLC) beta1. The relationship between PMT-mediated activation of the G(q/11)-PLC-IP(3) pathway and its ability to promote mitogenesis and cellular proliferation is not clear. PMT stimulation of p42/p44 mitogen-activated protein kinase occurs upstream via G(q/11)-dependent transactivation of the epidermal growth factor receptor. We have further characterized the effects of PMT on the downstream mitogenic response and cell cycle progression in Swiss 3T3 and Vero cells. PMT treatment caused dramatic morphological changes in both cell lines. In Vero cells, limited multinucleation, nuclear fragmentation, and disruption of cytokinesis were also observed; however, a strong mitogenic response occurred only with Swiss 3T3 cells. Significantly, this mitogenic response was not sustained. Cell cycle analysis revealed that after the initial mitogenic response to PMT, both cell types subsequently arrested primarily in G(1) and became unresponsive to further PMT treatment. In Swiss 3T3 cells, PMT induced up-regulation of c-Myc; cyclins D1, D2, D3, and E; p21; PCNA; and the Rb proteins, p107 and p130. In Vero cells, PMT failed to up-regulate PCNA and cyclins D3 and E. We also found that the initial PMT-mediated up-regulation of several of these signaling proteins was not sustained, supporting the subsequent cell cycle arrest. The consequences of PMT entry thus depend on the differential regulation of signaling pathways within different cell types.

Localization of the intracellular activity domain of Pasteurella multocida toxin to the N terminus.

We have shown that Pasteurella multocida toxin (PMT) directly causes transient activation of Gqalpha protein that is coupled to phosphatidylinositol-specific phospholipase Cbeta1 in Xenopus oocytes (B. A. Wilson, X. Zhu, M. Ho, and L. Lu, J. Biol. Chem. 272:1268-1275, 1997). We found that antibodies directed against an N-terminal peptide of PMT inhibited the toxin-induced response in Xenopus oocytes, but antibodies against a C-terminal peptide did not. To test whether the intracellular activity domain of PMT is localized to the N terminus, we conducted a deletion mutational analysis of the PMT protein, using the Xenopus oocyte system as a means of screening for toxin activity. Using PCR and conventional cloning techniques, we cloned from a toxinogenic strain of P. multocida the entire toxA gene, encoding the 1,285-amino-acid PMT protein, and expressed the recombinant toxin as a His-tagged fusion protein in Escherichia coli. We subsequently generated a series of N-terminal and C-terminal deletion mutants and expressed the His-tagged PMT fragments in E. coli. These proteins were screened for cytotoxic activity on cultured Vero cells and for intracellular activity in the Xenopus oocyte system. Only the full-length protein without the His tag exhibited activity on Vero cells. The full-length PMT and N-terminal fragments containing the first 500 residues elicited responses in oocytes, but the C-terminal 780 amino acid fragment did not. Our results confirm that the intracellular activity domain of PMT is localized to the N-terminal 500 amino acids of the protein and that the C terminus is required for entry into cells.