To confirm the sites of infection and to further identify other tissues or organs which were infected but may be difficult to detect by noninvasive imaging due to its limit of sensitivity, ex vivo imaging of the individual organs/tissues was performed after euthanizing the M3FL-infected mice. 3A), which were predominantly T cells (not shown). The STRING algorithm links genes or proteins into networks based on published functional or informatics-predicted interactions . This phenomenon is strain-dependent, as C57BL/6 mice depleted of CD8+ T cells suffer delayed clearance from the lung accompanied by increased viral latency in the spleen (Stevenson et al., 1999b). (C) Expression of viral coding genes surrounding miRNA mutations. The concentration of IFN-γ used was 100 U/ml. For each sample group in each experiment (n = 3 experiments), splenocytes from 5 mice were pooled.
The cytoplasmic domain of K15 is constitutively tyrosine phosphorylated, and the tyrosine residue within the putative SH2 binding motif is a major site of phosphorylation by cellular tyrosine kinases (22). 3A to C, insets). Despite variable numbers of YFP+ B cells observed with the different viruses, there was little differences noted in the total number of germinal center (GC) B cells (CD3−CD4−CD8−/B220+, GL7+CD95+) and plasma cells (CD3−CD4−CD8−/B220l°CD138hi) upon infection via either IN or IP route (Figures 5C–5F). These data therefore agree with observations of cultured cells and provide in vivo relevance confirming that p53 is induced during lytic MHV68 infection. This undoubtedly indicated that EBV infection was successful. Peritoneal cells from infected caspase 3−/− and B6 mice were analyzed for the frequency of cells which reactivated virus ex vivo and which harbored viral genome at 16 (A) or 42 (B) dpi. For instance, the level of expression of some caspase genes is reduced in Stat1−/− cells, and expression can be rescued with a Stat1 mutant that cannot be phosphorylated or form canonical Stat1 homodimers (18, 23).
For other genes, like ORF 52, with no designated role, it is possible to make functional predictions by comparing their expression profiles to those of better-characterized genes. 4C to E). J. Liang, C., Feng, P., Ku, B., Dotan, I., Canaani, D., Oh, B. Sensitivity of MHV-68 transcription to inhibition of protein synthesis during de novo infection.Herpesvirus immediate-early or alpha genes are defined as those that are expressed in the presence of CHX, a de novo protein synthesis inhibitor (35). 3A). RNA was DNase treated (Ambion Turbo DNA-Free kit), RNA was reverse transcribed into cDNA using SuperScript III (Invitrogen), and the cDNA was subjected to quantitative PCR using ABsolute Blue QPCR SYBR with Low ROX (Thermo Scientific) in an ABI 7500 real-time PCR system (Applied Biosystems) according to the manufacturer’s instructions.
Panels labeled BamHI and EcoRI represent general diagnostic Southern analyses for all recombinants, while those labeled NgoMIV, HpaI, and SpeI are diagnostic Southern blots for the specific mutants v-cyc[K104E], v-cyc[STOP], and v-cyc[E133V], respectively. BHK-21 cells were infected with MHV-68 at an MOI of 5 and harvested at the indicated time points. To distinguish between preformed infectious virus present in PECs and reactivating virus, cells were also mechanically disrupted before setting up the ex vivo reactivation assays. Blocking buffer (PBS with 1% BSA; 0.05% Tween and 10% FBS) was added in each well for 1 h. A. This work was supported by NIH grants AI37597 (D.L.W.) and AI42927 (M.A.B.), the Trudeau Institute, the Cancer Research Campaign (United Kingdom), and The Royal Society. However, there was a striking, high level of infectious virus detected in the lungs of 6 out of 10 M1Δ-infected animals even at high dilutions of lung lysates (50% of the wells exhibited CPE at a 1:250 dilution).
Briefly, protein samples were harvested by disrupting cells with either radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 20 mM Tris, 2 mM EDTA, 1% NP-40, and 0.25% deoxycholate supplemented with phosphatase and protease inhibitors) or IP lysis buffer. Data shown are from a representative experiment which was repeated once with similar results. This differs from other herpesviral proteins, such as herpes simplex virus type 1 ICP22 and human cytomegalovirus IE2 40, which have been demonstrated to be involved in late gene expression at the level of mRNA stabilization and protein translation, respectively (15, 24). Therefore, we infected BALB/c mice with MHV-68, and at various times postinfection we removed the spleens and bronchoalveolar lavage (BAL) and used an IFN-γ ELISPOT assay to measure the frequency of cells responding to this epitope. For penetration, prewarmed medium was added for a 2-h period of incubation at 37°C. Data were analyzed by using FloJo software (TreeStar, Inc., San Carlos, Calif.). Viral MicroRNA Targetome of KSHV-Infected Primary Effusion Lymphoma Cell Lines.
Formation of stable conjugates between CD4 T cells and B cells is SAP dependent , and ongoing stimulation by B cells is critical for maintenance of the TFH population , . Signaling through TLRs has been shown to play a role in class switching (28, 36) as well as to be necessary for maintenance of neutralizing and long-term antibody responses against infections (20, 57).