3B and unpublished data) (19). 3J). Standard STRING-defined confidence values of 0.40 were used as cut-offs for putative interactions. NIK can phosphorylate IKKα. At 5 or 8 dpi, lungs were harvested and viral titers were determined by plaque assay. The difference in these reactivation curves is statistically significant (P = 0.003). Subsequently, cells were loaded with the β-lactamase substrate CCF2/AM (Invitrogen).
Furthermore, KSHV and RRV encode genes for viral IL-6, interferon regulatory factors (v-IRFs), and viral chemokines (v-MIP-I, -II, and -III) (12, 50, 92, 102, 104, 125-127). Based on the nuclear morphology, the positive staining was detected in lymphocytes as well as cells with monocytoid-like nuclei. If the latter is true, the discrepancy with the requirement of Y120 in vitro may be due to the absence of potential overlapping effector signaling molecules required by both the tyrosines in the in vitro settings, but substituted for or compensated by other factors in the in vivo setting. One hour after adsorption, inocula were removed and cells were treated with vehicle (DMSO) or ATM inhibitor KU-55933 (1 μM). The results of incubation with supernatant from the initial EBV-infected Sh-Sy5y cells and qPCR for EBV-DNA with primers to detect EBNA1 show that fresh Sh-Sy5y cells were positive for EBV genomes (Fig. The observation that γHV68 latent infection is regulated by different granzymes at early versus late time points postinfection may reflect qualitative differences between virus-specific immune effector cells at the two time points. Cells were harvested after 8 h, and the level of methionine incorporation was measured.
Results are presented as the means for triplicates with their standard deviations. J Virol79, 5059–5068. 4A) and presence (Fig. This conclusion was further confirmed by the loss of the interaction of the full-length M2 (RK/E) mutant with the cellular p32 protein in NIH 3T3 cells (Fig. (C) Multistep growth curve in WT and Caspase-1/11−/− MEFs upon infection with MHV68-eYFP at a low MOI (0.01 PFU/cell). Equivalent amounts of cell extracts were subjected to immunoprecipitation with anti-CDK2-agarose beads, and the precipitates were analyzed in an in vitro kinase assay utilizing GST-pRb as an exogenous substrate. The pellets were analyzed by Western blots using anti-gp150 (glycoprotein), ORF45 (tegument), ORF65 (small capsid protein), or anti-ORF49.
We have successfully generated a mutant virus that possesses these characteristics and have used it to show that MHV68 encodes at least one other protein that can disrupt (but not degrade) PML NBs, an action that is normally masked because ORF75c generally degrades the bulk of PML within a cell (unpublished results). KSHV is also known to cause B-cell primary effusion lymphoma (PEL), body cavity-based B-cell lymphoma (BCBL), and Multicentric Castleman’s disease (MCD) (Chang et al., 1994 Chang, Y., Cesarman, E., Pessin, M. For instance, the MHV68 M2 latency-associated gene alters B cell biology to induce the production of cytokines that promote B cell proliferation. Cells were washed, scraped into PBS, and pelleted at 18 hpi. This work was supported by NIH grant AI51663-01 (E.J.U.), American Cancer Society institutional research grant no. coli strain DH10B, which already contained the mutant BAC plasmid pHA6. This assay involves serially diluting harvested cells and plating them on MEF indicator monolayers (which are permissive for γHV68 replication).
R. Cells were then stained with a 1:100 dilution of all antibodies except for PE-conjugated antibodies and fluorescein isothiocyanate-GL7, which were used at 1:200, for 20 min on ice in the dark. coli GS111, which was used as the donor strain for allelic exchange with the recipient strain GS500 harboring the wild-type MHV-68 BAC. Env copy numbers were determined by quantitative PCR (qPCR) on a Light Cycler instrument (LC2.0 Roche). Overall, these findings provide insights into the role of M1-mediated regulation of MHV68 pathogenesis. Some of these EBV growth-transformed lymphoblasts are hypothesized to form germinal centers, with a concomitant downregulation of viral gene expression with expression of EBNA1, LMP1, and LMP2a (4, 5, 9). We have speculated that incoming viral genomes are subject to intrinsic gene silencing mechanisms regardless of whether the virus enters a productive infection or initiates nonproductive infection, as is the case for EBV.
ORF27 was found to encode a glycoprotein, gp48, which is expressed on the surfaces of infected cells and is present in purified virions. The activity of these exonucleases on eukaryotic mRNAs is kept in check by the presence of a 5′ 7-methylguanosine cap and a 3′ poly(A) tail, which prevent access to the message body. After intranasal (i.n.) infection of conventional mice, the virus spreads from the lung to the lymphoid tissue (29) and then persists in B lymphocytes (28) and in epithelial cells (27). Although ablation of STAT3 in B cells did not have a global effect on these assays of B cell function, it had long-term consequences for the viral load of the host, since virus latency was reduced at 6 to 8 weeks postinfection.