The lysate of the cells infected with either WT or M3FL was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the expression of FL was analyzed by Western blotting using anti-FL (sc-57603; Santa Cruz). Further, the animals had no other detected infections, as detailed in Materials and Methods. In addition to defining the specific phospho-tyrosine residues previously reported, we also identified numerous phosphorylation events on serine and threonine residues within ORF21/TK (Table 1). Upon inoculation of the upper respiratory tract infection, MHV68 first infects the neuroepithelium of the rostral nasal cavity (Frederico et al., 2012; Milho et al., 2012). To control for experimental variability, primers and probe sets specific for cellular small nucleolar RNA (snoRNA) 234 or snoRNA202 (see Table S2 in the supplemental material) were included in all experiments, as these small noncoding RNAs are generally stable during MHV68 infection (see Fig. 1, only MHV68 replicates productively in cell culture. We also considered the possibility that IFN-γ activates peritoneal cells ex vivo such that virus released during or after reactivation from latency is inactivated.
During hematopoiesis, common lymphoid progenitors in the bone marrow commit to the B cell lineage, after which they progress through multiple selection checkpoints at well-delineated stages. The STP of HVS subgroup A, STP-A, is less potent than STP-C in its transforming ability. Neutrophils (circled in panel B) and viral inclusions (circled in panel C) can be observed. Louis, MO) in PBS at room temperature for 20 min. (A–F) Primary B cells from either wild type C57BL6 (B6) mice or IL10−/− mice (as indicated) were transduced with retroviral vectors encoding either M2 or M2stop. Drugs and antibodies.Doxorubicin and phosphonoacetic acid (PAA) were purchased from Sigma-Aldrich and diluted in distilled water (dH2O). Cells were collected from all assays, and RNA was extracted for quantitative determination by RT amplification of viral genes.
Statistical analysis.For acute viral titers, data represent the mean ± standard error of the mean from at least two independent experiments, and statistical significance was determined using the Mann-Whitney test. Figure 3C shows that both the M-H/RTA and H-M/RTA chimeric proteins were able to activate the MHV-68 ORF57 promoter, which is consistent with the fact that both M/RTA and H/RTA are able to significantly activate MHV-68 p57luc. 2A). Despite the severe defect of v-cyclin-deficient viruses in reactivation from latency, the frequencies of viral genome-containing peritoneal cells were very similar between wt and v-cyclin-deficient viruses (wt γHV68, 1 in 11,200; v-cyclin.MR, 1 in 10,100; v-cyclin.Stop, 1 in 10,300; v-cyclin.LacZ, extrapolated to be ca. Quality was assessed on a denaturing agarose gel. Immunoblotting.Polyclonal rabbit antiserum (Cocalico, Reamstown, Pa.) was generated by using purified bacterially expressed v-cyclin protein as described previously (66). Symbols show mean ± SEM of triplicate cultures.
Wild-type MHV-68 virus and an STM BAC mutant harboring a transposon at the sequence of the BAC plasmid were also tested as a control. Isolation of five strains of herpesviruses from two species of free living small rodents. M., and Minson, A. The reporter plasmids were cotransfected into BHK-21, 293T, and NIH 3T3 cells with either pCMV-FLAG/RTA or pCMV-FLAG (Kodak). Protein purification and mass spectrometry.For the identification of M2-binding proteins, [35S]methionine- and [35S]cysteine-labeled mouse A20 B cells (107 cells) or 5 liters of A20 cells were resuspended in lysis buffer (0.15 M NaCl, 0.5% Nonidet P-40, and 50 mM HEPES buffer [pH 8.0]) containing protease inhibitors. However, latency amplification was unimpaired (Fig. National Institutes of Health (NIH) murine 3T12 fibroblasts were cultured at 37°C with 5% CO2 in DMEM containing 8% FBS, 2 μM l-glutamine, 50 U of penicillin/ml, and 50 μg of streptomycin/ml (8% cMEM).
Additional diagnostic cuts were performed to assess the introduction, and subsequent rescue, of specific mutations: HpaI for O72.Stop, NgoMIV for K104E, and SpeI for E133V. The uppercase letters indicate the inserted nonviral sequences, including PacI and PstI sites. 89004-308) that contained 0.2 ml of 1-mm-diameter zirconia-silica beads (BioSpec) and 1 ml of cold cMEM. Although γHV-68’s genome differs from EBV, it elicits an immune response in mice that shares many features with EBV. 2000. Spontaneous and maximum releases were measured by incubation with medium alone and 1% Triton X-100, respectively. (E) Frequency of splenocytes harboring viral genomes 43 to 45 dpi.
Given its importance in both acute and latent G2HV infection and its association with disease, understanding LANA function is an area of intense experimental focus, making LANA a prime target for novel treatments of KSHV-related malignancies (36, 37). 1A to D). Indeed, one such protein, encoded by MHV-68 ORF45, is essential for gene expression in the immediate-early phase of infection (21). However, one group has classified ORF24 as being an early gene (1). Then, two-independent bacterial artificial chromosome-derived ORF31-null MHV-68 mutants (31STOP) were generated and found to be defective in virus production in fibroblast cells. Early inhibition of lytic replication does not impact the progression of the latent infection, and latency is established in lymphoid tissues following infection with a replication-deficient mutant virus.