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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 [59]. 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 [6], and ongoing stimulation by B cells is critical for maintenance of the TFH population [27], [28]. 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).

To confirm that the signals were due to viral reactivation, we examined the expression of viral latent and lytic transcripts as well as the viral genomes in the spleen. Although there appeared to be a lower level of both infectious centers and viral DNA in wood mice at later times postinfection, up to 40 days p.i. Sequence logos generated for each data set using the ICE-LOGO resource (http://iomics.ugent.be/icelogoserver/logo.html), a weighted representational analysis, demonstrate general differences in phosphorylated sequences between control and infected systems (Figs. The next step of B cell maturation occurs upon detection of a cognate antigen for the BCR, accompanied by CD40 co-signaling, leading to the formation of the germinal center (Goetz and Baldwin, 2008). mLANA is critical for the maintenance of the viral episome during latency and is therefore expressed in a large fraction of latently infected B cells, including developing, naive, germinal-center, and memory B cells (47, 50). Studies in which only infected cells are either IFN-γ responsive or IFN-γ unresponsive will be required to definitively address this question. This concept is supported by studies of the EBV latency protein LMP2A, whose expression in transgenic mice is associated with the bypass of normal B cell developmental checkpoints in the bone marrow (6).

In this assay, rearranged, but not germ line, heavy chain sequences are amplified using forward primers homologous to conserved framework region 3 of three murine Vh gene families (Fig. After confirming the presence of required mutation by sequencing, the PCR products were electroporated into the M2/galK intermediate and recombinants were selected on minimal plates containing glycerol and 2-deoxy-D-galactose. If p53 functions are specifically inhibited by the virus, we hypothesized that infected cells would become resistant to treatment with exogenous p53-activating stimuli as a consequence. The results showed that supernatants collected at 2 dpi from ACV-treated cells had similar reductions in GFP-EBV genome copy numbers whether they were generated from Sh-Sy5y or LCL1 cells, with a drop of approximately 40 to 50% (Fig. We thank members of the labs of H.W.V. Northern blot analysis.The array data presented here are reflected in previous work using alternate methods of examining transcription (14, 21, 29, 35, 44). (2005a).

At 12 hpi, >57% of the ORFs examined were expressed in the C-RTA virus at fivefold-higher levels than in the parental virus (data not shown). 5C). Despite infection levels reaching ca. 4B). 312:71–100). The fraction of light isotopic peptides containing [6-12C]arginine and [6-12C]lysine was calculated by dividing the peak area of the light peptide spectrum of a given protein by the sum of the peak areas for light and heavy peptide spectra for the same protein (ratio light/[light plus heavy]). Propagation of the mutant virus in fibroblasts expressing recombinase Cre results in deletion of the BAC vector sequences.

An examination of the frequency of viral-genome-positive cells within the sorted populations revealed that both B-cell and non-B-cell fractions harbor virus at day 16 postinfection. 89, 2456–2458 (2015). Generation of MyD88-mixed-bone-marrow chimeric mice.B6.SJL-PtprcaPepb/BoyJ wild-type (Ly5.1) mice (The Jackson Laboratory) were lethally irradiated (950 rad) with two doses of 475 rad in a 20-h interval and reconstituted with bone marrow from both MyD88+/+ (Ly5.1) and MyD88−/− (Ly5.2) mice. Firefly luciferase activity was normalized with Renilla luciferase activity. C) Analysis of hyposplenism over time after infection. The other major cell population in the spleen that is infected by MHV68 are plasma cells (CD138hi, B220low) [23], [27]. The resultant purified cell populations were resuspended in cMEM supplemented with 10% dimethyl sulfoxide and stored at −80°C for limiting-dilution PCR assays or at 4°C in cMEM for limited-dilution ex vivo reactivation assays as described below.

Captured images were imported and processed in Adobe Photoshop. Southern blotting.DNAs were extracted from virus stocks by alkaline lysis (19), digested with restriction endonucleases, electrophoresed in 0.8% agarose gels, and transferred to positively charged nylon membranes (Roche Diagnostics). RNA was Northern blotted with probes to the 3′ end of the GAPDH coding region, or to 18S. Furthermore, the absence of this cytokine has no obvious effect on the pathogenesis of infection (23). Generation of recombinant γHV68.The bacterial artificial chromosome (BAC) system was used to generate recombinant γHV68 as previously described (10, 11). Preparation of virus stocks, infection of mice, and analysis of acute and latent infection was carried out as described previously (22). Of the viral transcripts expressed during latent infections, mLANA (a homologue of the Kaposi’s sarcoma-associated herpesvirus [KSHV] latency-associated nuclear antigen [LANA]) is critically required, as mLANA-null viruses are unable to establish and maintain latent infections following intranasal inoculation of mice (26, 48).

Cell lines derived from KSHV- or EBV-associated lymphomas are latently infected with virus. TBK1 and IKKε can be activated by engagement of PAMPs by the PRRs, including Toll-like receptors (TLRs), cytoplasmic RIG-1-like receptors (RLRs), or cytosolic DNA sensors (15–22). Using cells obtained after a 6-day pulse, gates were set around the population of choice to obtain the percentage of BrdU incorporation (Fig.