Complete sequence and genomic analysis of murine gammaherpesvirus 68.

Murine gammaherpesvirus 68 (gammaHV68) infects mice, thus providing a tractable small-animal model for analysis of the acute and chronic pathogenesis of gammaherpesviruses. To facilitate molecular analysis of gammaHV68 pathogenesis, we have sequenced the gammaHV68 genome. The genome contains 118,237 bp of unique sequence flanked by multiple copies of a 1,213-bp terminal repeat. The GC content of the unique portion of the genome is 46%, while the GC content of the terminal repeat is 78%. The unique portion of the genome is estimated to encode at least 80 genes and is largely colinear with the genomes of Kaposi's sarcoma herpesvirus (KSHV; also known as human herpesvirus 8), herpesvirus saimiri (HVS), and Epstein-Barr virus (EBV). We detected 63 open reading frames (ORFs) homologous to HVS and KSHV ORFs and used the HVS/KSHV numbering system to designate these ORFs. gammaHV68 shares with HVS and KSHV ORFs homologous to a complement regulatory protein (ORF 4), a D-type cyclin (ORF 72), and a G-protein-coupled receptor with close homology to the interleukin-8 receptor (ORF 74). One ORF (K3) was identified in gammaHV68 as homologous to both ORFs K3 and K5 of KSHV and contains a domain found in a bovine herpesvirus 4 major immediate-early protein. We also detected 16 methionine-initiated ORFs predicted to encode proteins at least 100 amino acids in length that are unique to gammaHV68 (ORFs M1 to 14). ORF M1 has striking homology to poxvirus serpins, while ORF M11 encodes a potential homolog of Bcl-2-like molecules encoded by other gammaherpesviruses (gene 16 of HVS and KSHV and the BHRF1 gene of EBV). In addition, clustered at the left end of the unique region are eight sequences with significant homology to bacterial tRNAs. The unique region of the genome contains two internal repeats: a 40-bp repeat located between bp 26778 and 28191 in the genome and a 100-bp repeat located between bp 98981 and 101170. Analysis of the gammaHV68, HVS, EBV, and KSHV genomes demonstrated that each of these viruses have large colinear gene blocks interspersed by regions containing virus-specific ORFs. Interestingly, genes associated with EBV cell tropism, latency, and transformation are all contained within these regions encoding virus-specific genes. This finding suggests that pathogenesis-associated genes of gammaherpesviruses, including gammaHV68, may be contained in similarly positioned genome regions. The availability of the gammaHV68 genomic sequence will facilitate analysis of critical issues in gammaherpesvirus biology via integration of molecular and pathogenetic studies in a small-animal model.     

Cytokine gene expression in atopics: effect of IL-4 on IL-1 beta and IL-6 mRNA levels

Mononuclear cells from atopic blood donors showed increased IL-3 steady state mRNA levels. This finding complemented our earlier observations that cells from atopics also showed increased IL-4 but decreased IFN-gamma, IL-1 beta and IL-6 mRNA levels. Therefore, we investigated the effect of human recombinant IL-4 on cytokines mRNA levels in mononuclear cells from normals and atopics. In the presence of IL-4 steady state levels of IL-1 beta and IL-6 mRNA were decreased even if cells were co-stimulated with polyclonal activators such as PMA, PWM or PHA. No influence of IL-4 on granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3 or IFN-gamma mRNA levels was observed with the exception of a decreased IFN-gamma mRNA level in PWM stimulated cells.     

Superoxide-dependent nitroblue tetrazolium reduction and expression of cytochrome b-245 components by human tonsillar B lymphocytes and B cell lines

EBV-transformed B lymphocyte cell lines can generate superoxide, using an electron transport chain homologous, or even identical, to phagocytic NADPH-oxidase. We searched for normal, not virally transformed, B lymphocytes with analogous properties, using tonsils as the source of B cells. Unseparated tonsillar leukocytes contained cells capable of PMA-triggered superoxide dismutase-inhibitable reduction of nitroblue tetrazolium (NBT+ cells) well in excess of phagocytes (18.9 +/- 6.4% NBT+ cells with 1.3 +/- 0.9% granulocytes and 1.9 +/- 2.3% monocytes/macrophages, n = 8). NBT reduction was also inhibited by diphenylene iodonium, a selective inhibitor of phagocytic NADPH-oxidase. Cross-linking of surface Ig was equally effective as PMA in inducing NBT reduction among tonsillar leukocytes. NBT+ cells co-distributed with B cells on Percoll density gradients and were enriched among purified B cells obtained by SRBC rosetting twice and Sephadex G10 adherence (47.8 +/- 15.2% NBT+ cells among 90.5 +/- 5.5% B cells, 4.8 +/- 5.1% T cells, 1.2 +/- 0.77% monocytes/macrophages, and 0.73 +/- 0.6% granulocytes, n = 10). Further, mAb 7D5, directed against an extracellularly located epitope of the small subunit of cytochrome b-245 of phagocytes, stained the majority of tonsillar B cells (85 +/- 9.2% 7D5+ cells and 91.6 +/- 4.04% B cells, n = 3). Superoxide production, staining with 7D5 antibody, and expression of mRNA for the beta chain of cytochrome b-245 were further analyzed in cell lines. The EBV-BLCL F1 and the Burkitt lymphoma P3HR-1 both carried 7D5-detectable cytochrome b-245 Ag and expressed mRNA for the beta chain of the cytochrome b, both in similar amounts. However, only F1, not P3HR-1, was capable of PMA-triggered superoxide production. These data indicate that also normal nontransformed B lymphocytes possess the capacity to generate superoxide by a system apparently similar to phagocytic NADPH-oxidase, provisionally termed "B cell oxidase." Discrepancies observed in certain B cells and lines between expression of cytochrome b components and stimulus-induced superoxide production may be related to an absence or low level of other oxidase components or of the signal transduction mechanism. Conceivably, production of superoxide and derived reactive oxygen species by B cells may have cytotoxic, immunomodulatory, or mutagenic effects on the B cells themselves or on cells in their immediate vicinity.     

Macrophages are the major reservoir of latent murine gammaherpesvirus 68 in peritoneal cells

B cells have previously been identified as the major hematopoietic cell type harboring latent gammaherpesvirus 68 (gammaHV68) (N. P. Sunil-Chandra, S. Efstathiou, and A. A. Nash, J. Gen. Virol. 73:3275-3279, 1992). However, we have shown that gammaHV68 efficiently establishes latency in B-cell-deficient mice (K. E. Weck, M. L. Barkon, L. I. Yoo, S. H. Speck, and H. W. Virgin, J. Virol. 70:6775-6780, 1996), demonstrating that B cells are not required for gammaHV68 latency. To understand this dichotomy, we determined whether hematopoietic cell types, in addition to B cells, carry latent gammaHV68. We observed a high frequency of cells that reactivate latent gammaHV68 in peritoneal exudate cells (PECs) derived from both B-cell-deficient and normal C57BL/6 mice. PECs were composed primarily of macrophages in B-cell-deficient mice and of macrophages plus B cells in normal C57BL/6 mice. To determine which cells in PECs from C57BL/6 mice carry latent gammaHV68, we developed a limiting-dilution PCR assay to quantitate the frequency of cells carrying the gammaHV68 genome in fluorescence-activated cell sorter-purified cell populations. We also quantitated the contribution of individual cell populations to the total frequency of cells carrying latent gammaHV68. At early times after infection, the frequency of PECs that reactivated gammaHV68 correlated very closely with the frequency of PECs carrying the gammaHV68 genome, validating measurement of the frequency of viral-genome-positive cells as a measure of latency in this cell population. F4/80-positive macrophage-enriched, lymphocyte-depleted PECs harbored most of the gammaHV68 genome and efficiently reactivated gammaHV68, while CD19-positive, B-cell-enriched PECs harbored about a 10-fold lower frequency of gammaHV68 genome-positive cells. CD4-positive, T-cell-enriched PECs contained only a very low frequency of gammaHV68 genome-positive cells, consistent with previous analyses indicating that T cells are not a reservoir for gammaHV68 latency (N. P. Sunil-Chandra, S. Efstathiou, and A. A. Nash, J. Gen. Virol. 73:3275-3279, 1992). Since macrophages are bone marrow derived, we determined whether elicitation of a large inflammatory response in the peritoneum would recruit additional latent cells into the peritoneum. Thioglycolate inoculation increased the total number of PECs by about 20-fold but did not affect the frequency of cells that reactivate gammaHV68, consistent with a bone marrow reservoir for latent gammaHV68. These experiments demonstrate gammaHV68 latency in two different hematopoietic cell types, F4/80-positive macrophages and CD19-positive B cells, and argue for a bone marrow reservoir for latent gammaHV68.     

Chemiluminescence of mononuclear cells is enhanced during antigen recognition

Stimulation of phagocytes by several cytokines causes superoxide generation and consequently chemiluminescence. Since antigen-activated lymphocytes generate cytokines, we investigated whether antigen recognition by mononuclear cells, which contain both lymphocytes and monocytes, is accompanied by changes in lucigenin-dependent chemiluminescence. Mononulcear cells which underwent antigen-induced proliferation showed a delayed rise in lucigenin-dependent chemiluminescence in the absence of other stimuli. The common recall antigen Candida albicans increased spontaneous chemiluminescence of mononuclear cells from unselected donors up to 20-fold over control values after 48–72h of culture. With Rabies virus vaccine as specific antigenic stimulus, only mononuclear cells from rabies immunized individuals responded with enhanced delayed chemiluminescence. In contrast to opsonized zymosan and phorbol myristate acetate, antigens induced no oxidative burst within one hour after addition. Delayed mononuclear cel chemiluminescence was inhibited by the superoxide scavenger superoxide dismutase and by di-phenylene iodonium, a selective inhibitor of the phagocyte NADPH oxidase. A neutralizing monoclonal antibody against interferon-gamma completely abrogated antigen-induced chemiluminescence. Recombinant interferon-gamma by itself induced delayed mononuclear cell chemiluminescence. Thus, antigen-induced delayed mononuclear cell chemiluminescence represents activation of phagocyte NADPH oxidase by interferon-gamma generated by activated lymphocytes.     

Systematic identification of novel cancer genes through analysis of deep shRNA perturbation screens

Systematic perturbation screens provide comprehensive resources for the elucidation of cancer driver genes. The perturbation of many genes in relatively few cell lines in such functional screens necessitates the development of specialized computational tools with sufficient statistical power. Here we developed APSiC (Analysis of Perturbation Screens for identifying novel Cancer genes) to identify genetic drivers and effectors in perturbation screens even with few samples. Applying APSiC to the shRNA screen Project DRIVE, APSiC identified well-known and novel putative mutational and amplified cancer genes across all cancer types and in specific cancer types. Additionally, APSiC discovered tumor-promoting and tumor-suppressive effectors, respectively, for individual cancer types, including genes involved in cell cycle control, Wnt/β-catenin and hippo signalling pathways. We functionally demonstrated that LRRC4B, a putative novel tumor-suppressive effector, suppresses proliferation by delaying cell cycle and modulates apoptosis in breast cancer. We demonstrate APSiC is a robust statistical framework for discovery of novel cancer genes through analysis of large-scale perturbation screens. The analysis of DRIVE using APSiC is provided as a web portal and represents a valuable resource for the discovery of novel cancer genes.     

Plastidial alpha-glucan phosphorylase is not required for starch degradation in Arabidopsis leaves but has a role in the tolerance of abiotic stress

To study the role of the plastidial alpha-glucan phosphorylase in starch metabolism in the leaves of Arabidopsis, two independent mutant lines containing T-DNA insertions within the phosphorylase gene were identified. Both insertions eliminate the activity of the plastidial alpha-glucan phosphorylase. Measurement of other enzymes of starch metabolism reveals only minor changes compared with the wild type. The loss of plastidial alpha-glucan phosphorylase does not cause a significant change in the total accumulation of starch during the day or its remobilization at night. Starch structure and composition are unaltered. However, mutant plants display lesions on their leaves that are not seen on wild-type plants, and mesophyll cells bordering the lesions accumulate high levels of starch. Lesion formation is abolished by growing plants under 100% humidity in still air, but subsequent transfer to circulating air with lower humidity causes extensive wilting in the mutant leaves. Wilted sectors die, causing large lesions that are bordered by starch-accumulating cells. Similar lesions are caused by the application of acute salt stress to mature plants. We conclude that plastidial phosphorylase is not required for the degradation of starch, but that it plays a role in the capacity of the leaf lamina to endure a transient water deficit.     

Flow-cytometric analysis of mouse pritoneal macrophages

Flow-cytometric analysis of mouse peritoneal macrophages (MΦ) stained with acridine orange defined three populations with increasing RNA content. Resident MΦ displayed variable bimodal distributions of low and intermediate cellular RNA content with high RNA content only observed after in vitro stimulation with fetal calf serum or/and bacterial lipopolysaccharide (LPS). In contrast, only few resident macrophages from the LPS nonresponder strain C3H/HeJ increased in RNA content upon LPS stimulation. Macrophages with high RNA content developed transiently after in vivo stimulation with either thioglycollate broth or paraffin oil. Proteose peptone-elicited macrophages transformed only after additional in vitro stimulation with fetal calf serum and/or LPS. The magnitude of the in vitro response upon stimulation, assessed as increasing percentage of either intermediate or high RNA macrophages, was dependent on the composition of the MΦ population at the onset of in vitro culture. Effective increase in cellular RNA content was always paralleled by improved adherence of plated macrophages to the culture vessel. In conclusion, flow-cytometric measurement of macrophage populations might become a useful tool to quantify macrophage activation or stimulation.