The infection in the salivary gland area started to be detected at D10 and continued until D18, while the signals in the thymus area could be seen only at D10. (C) Viral antigen (PAP method) is expressed by type I (arrow) and type II (arrowhead) pneumocytes. Because our MS analyses focused on timepoints associated with robust infection-induced alterations in total phosphoprotein profiles, we next sought to correlate infection-associated phosphoproteomic changes with specific stages in the viral replication cycle. Normal B cell maturation is dependent on limited survival signals and deletion of self-reactive cells. with 104 PFU MHV68. The Δ2 and Δ3 mutants lack the entire C-terminal activation domain or DNA binding/dimerization domain, respectively, and are essentially inactive for transactivation. These data corroborate previous results that IFN-γ acts during cell reactivation and not on infectious virus (Fig.
While the approximate frequency of infection of pro-pre-B cells was very low at 15 days, it could not be accounted for solely by contamination from immature or mature B cells. LMP2a contains 12 transmembrane domains linked by loops and a short stretch of amino-terminal and carboxy-terminal domains (Fig.3). The incidence of lymphoproliferative lesions was significantly increased in γHV68-infected males. Figure 4. Induction of ATM-p53 signaling during MHV68 lytic replication.MHV68 induces ATM and H2AX phosphorylation, and infected cells exhibit additional DDR-related phosphorylation events during lytic replication in fibroblasts (20, 22, 43–45). Fluorescence microscopy was carried out at 1, 3, 6, and 9 dpi to monitor for GFP expression. GzmBcluster−/− mice also had a small, but statistically significant, increase in the frequency of cells harboring viral genome compared to wild-type (P = 0.05) or GzmAXB−/− (P = 0.02) mice (Fig.
(A) A BAC clone containing a recombinant MHV-68 RTA-deficient virus was cotransfected with pFLAG (V), pFLAG-H/RTA (H), pFLAG-M/RTA (M), pFLAG-E/RTA (E), pFLAG-M-H/RTA (M-H), pFLAG-H-M/RTA (H-M), pFLAG-E-M/RTA (E-M), or pFLAG-E-H/RTA (E-H) into BHK-21 (shown) or 293T cells. Luciferase activity was normalized to β-galactosidase expression, and the value from cells transfected with empty pGL2 vector was subtracted for each condition. (A) Background-subtracted data from two repeated arrays (time point, 5 h p.i.) were plotted against each other. Samples from the same mice were examined for the presence of fibrosis or atrophy of the spleen as previously described (21). 2D). 11. Antibody to a lytic cycle viral protein decreases gammaherpesvirus latency in B-cell-deficient mice.
RNA expression levels are shown in a tabular form (Fig. The central proline and positively charged amino acids of M2 contribute to its nuclear localization.Despite its small coding sequence, M2 contains distinct regions, namely, four proline-rich regions and a central positively charged region. The supernatant was removed, and then cells were lysed in 500 μl of fresh BMM-Low by freezing. Briefly, PECs and splenocytes were harvested from infected mice at the times indicated, and single-cell suspensions were generated. The lysates were cleared by low-speed centrifugation to remove cell debris, and then the virus titers of the supernatants were analyzed by plaque assays. Hence, for this study, the mice were inoculated intranasally with 100 PFU of MHV68. For intracellular staining, CNS mononuclear cells, splenocytes and lymph nodes cells were stimulated for 4 hours in IMDM (Gibco) containing 10% FBS (Gibco), GolgiPlug (BD Biosciences), 10 ng/ml PMA and 500 ng/ml ionomycin; or for 24 hours with MOG peptide (100 µM) with addition of GolgiPlug during the last 5 hours of incubation.
P., and Davison, A. Data are representative of two experiments. 2B and D), while the frequency of viral-genome-positive IgD+ B cells was below the limit of accurate quantitation (Fig. Mascot Distiller was used to determine the fraction of peptides containing light isotopes of arginine and lysine. “P” indicates the probes used for Southern blot analysis, corresponding to nucleotides 25,097 to 25,833 (P1), 101,141 to 102,045 (P2), and 101,443 to 102,699 (P3). This defect in late antigen expression was also seen when ORF65 expression was examined. We did not observe any difference in the latent virus burden between the two groups at 28 days postinfection.
However, because the frequency of homologous recombination in eukaryotic cells is low and selection against nonrecombinant wild-type (WT) virus is necessary, this technique is often ineffective, laborious, and time-consuming. At early and intermediate times postinfection the virus was predominantly found within the B-cell compartment and resides within both naive and more mature B cells lacking surface immunoglobulin D (sIgD), which exhibit features representative of memory B cells. & Izaurralde, E. Here we show in a mouse model of gammaherpesvirus infection that infected B cells require signals from CD4 T cells for proliferation. Phone: 44-1223-336921. Analysis of wild-type × MyD88−/− mixed-bone-marrow chimeric mice demonstrated that there is a selective failure of MyD88−/− B cells to participate in germinal-center reactions as well as to become activated and undergo class switching. We explored the interaction between ZAP and murine gammaherpesvirus 68 (MHV-68), whose life cycle has latent and lytic phases.