The multistep growth curve assay was performed to investigate the growth of M3FL and the parental wild-type MHV-68 during multiple rounds of replication in mouse fibroblast NIH 3T12 cell line at an MOI of 0.05. 1C) demonstrated that wood and BALB/c mice had equivalent loads. 3A), which is consistent with the presence of PML phosphopeptides in control, but not infected, MS analyses. MHV68 mutants lacking genes necessary for productive replication or DNA replication fail to establish WT levels of latency in the spleens of infected mice after intranasal inoculation (Coleman et al., 2003; Flano et al., 2005; Moser et al., 2006; Tibbetts et al., 2006; Gill et al., 2009, 2010; Milho et al., 2011; Minkah et al., 2015). Together these data demonstrated that the expression of both previously annotated and prospective MHV68 miRNAs varied widely and could be subdivided into at least 3 kinetic classes. Plaque assays performed on the supernatants from these cells also demonstrated a dose-dependent decrease in virus production with increasing expression of p65 (Fig. IFN-γ-treated cultures receiving H22 at days 1, 2, 3, and 5 reactivated with a frequency of 1/2,320, 1/2,905, 1/2,915, and 1/6,380, respectively; the cultures receiving PIP at each of these days were indistinguishable from the IFN-γ-only treated cultures (≤1/18,000).
S1 and S3 in the supplemental material). Furthermore, oncogenic v-ras can complement the HVS STP oncogene to induce lymphocyte transformation and does so more efficiently than normal c-ras (52). Original magnification, ×10. Several defects are attributed to the β2 microglobulin deficiency, including lack of classical CD8 T cells, hepatic iron overload, and abnormal IgG homeostasis due to the inefficient expression of neonatal Fc receptor (28, 42, 70, 73). Figure 3. For nutlin-3a treatments, mock-infected or infected cells were treated with nutlin-3a (10 μM) 4 h before harvest at 18 h postinfection. (B to E) Cells were harvested at 1, 2, 3, 5, 7, and 9 dpi for EBNA1, EBNA3C, BZLF1, and gp350 transcript analysis.
These mice also exhibited a small but statistically significant (P = 0.03) twofold increase in the frequency of cells harboring viral genome relative to that in wild-type mice (Fig. M-H/RTA was also the only chimeric construct able to induce lytic replication and processing of viral DNA (data not shown) and the production of infectious virions (Fig. IFN-γ did not alter transcript levels for immediate-early gene 73, which encodes the LANA protein (Fig. Statistically significant differences: A, P < 0.0001 for both v-cyclin.LacZ and v-cyclin.Stop compared to wt γHV68; B, P < 0.001 for v-cyclin.LacZ compared to wt γHV68. Hybridization signals were quantified with a phosphorimager (Packard). Growth of wild-type (wt) γHV68 in contact-inhibited MEFs was compared with that of v-cyclin.LacZ by infecting MEF monolayers with 1 PFU per well in 96-well plates. inoculation, which reaches the spleen directly . (C) Plaque morphology of the ORF49null and the wild-type virus. 9, 165-174. Med. Cloned array elements were spotted onto nylon membranes in quadruplicate. Cell extracts, immunoprecipitations, and immunoblots.For preparations of cell extracts for precipitation (6), transfected cells grown in 100-mm-diameter dishes were washed with ice-cold PBS and lysed with 1 ml of lysis buffer (150 mM NaCl, 1% Nonidet P-40, 50 mM Tris [pH 7.5], 0.2 mM EDTA) supplemented with a protease inhibitor cocktail (Roche). WT MHV68 and ORF50stop P2 stocks were concentrated to >108 PFU/ml by centrifugation at 4°C for 90 min at 35,000 × g in a Beckman-Coulter (Brea, CA) JA-20 rotor. Cells were infected in low-serum medium at a multiplicity of 10 for 1 h, washed twice with PBS, and replaced with low-serum medium.
Radiolabeled probes specific to ORF49 or RTA were generated by the random priming method using the ORF49 PCR products (nt 67012 to 67519) or the ORF50 PCR products (nt 68647 to 69178), respectively, as a template with [α-32P]dCTP. Ex vivo reactivation assay.Spleens and peritoneal exudate cells (PECs) from four infected mice per group were harvested and pooled in cold cMEM. Interestingly, γHV-68 MOG CFA mice (Figure 1C, blue line) developed milder paralysis than γHV-68 EAE mice, yet still displayed a disease course similar to EAE alone (Figure 1C, black line). J. After 5 days of incubation at 37°C the assays were fixed with methanol and stained with Giemsa stain, and then the plaques were enumerated microscopically. The B cell compartment is the primary reservoir of MHV68 latency upon dissemination to the secondary lymphoid tissues. In addition, “light” and “heavy” media were supplemented with 10% dialyzed FBS (Thermo Scientific Pierce), 100 U penicillin/ml, 100 μg/ml of streptomycin, and 2 mM l-glutamine.
The single deletion mutants A, B, and C showed a similar growth pattern to that of the parental virus, although mutant A did so with slightly delayed kinetics. PCR, cloning, and plasmids.An enhanced green fluorescent protein (EGFP) fusion expression plasmid containing full-length MHV-68 ORF52 was generated by PCR amplification from genomic herpesvirus DNA and cloning in frame into pEGFPC1 (Clontech, Mountain View, CA) with primers 1 and 2 (Table 1).