KSHV hexons and pentons have the same height (∼160 Å), as measured from the part of the floor next to the channel, to their outer tip. The H-M/RTA construct was able to activate the MHV-68 p57 promoter but was unable to reactivate MHV-68 or complement the MHV-68 RTA-deficient virus. Membranes were probed with polyclonal serum recognizing multiple MHV68 lytic antigens (upper panel) and actin MAb (lower panel). ). Viral titers in BALB/c IFN-γ−/− mice that were sacrificed on day 5, 8, 11, or 14 postinfection (A) or succumbed to WT infection on the indicated days (B) and those in BALB/c mice sacrificed on days 5, 8, and 15 postinfection (C) are shown. 3D).
This introduced a premature stop codon into M7, thereby terminating gp150 translation after 62 amino acids. Although functional domains of MHV-68 Rta have not been defined, based on the amino acid sequence homology to EBV and HHV-8 Rta, it is likely that the N terminus of MHV-68 Rta contains DNA binding and dimerization domains while the activation domain is located at the C terminus. Follicular hyperplasia, as defined by the presence of a prominent germinal center reaction with otherwise normal histology, was occasionally observed and was not scored as lymphoproliferative disease. Limiting dilution viral genome PCR analysis was utilized to determine the frequency of latency from CD19+ IgD− splenocytes from mice 6 months postinfection with M1Δ and M1.MR (∼1/183 and ∼1/955, respectively; P = 0.08). MHV68 infection significantly reduced the proportion of B cells in spleens from infected wild-type and PML−/− mice probably because of significant increases in non-T, non-B, or nonmacrophage cell types, which we classified as “other” (Fig. The indirect immunofluorescence assay was performed as described previously (22).
(A) BHK-21 or FT-24 cells were infected with 24S at an MOI of 2 with (+) or without (−) PAA. Jacoby, M. Briefly, bulk splenocytes or sorted cells were thawed, counted, and resuspended in isotonic buffer. Strikingly, when assessing YFP marking of the splenic plasma cell population by the M1pYFP virus, the vast majority of YFP+ cells exhibited a plasma cell phenotype (on average >75% of YFP+ cells) (Figure 4). For quantitation of reactivation, a limiting-dilution reactivation assay was performed as previously described (31). 2C), suggesting that only a small proportion of MHV68-infected cells in the lung supported persistent viral replication.
After blocking of the membrane with 3% milk, immunoblot detection was performed with the corresponding primary antibodies by incubation at 4°C overnight and with secondary peroxidase-conjugated antibody for 60 min at room temperature. For the frequency of cells which reactivated virus ex vivo and which harbored viral genome, data represent the mean ± the standard error of the mean from at least three independent experiments and were analyzed by nonlinear regression (sigmoidal dose curve with nonvariable slope) using GraphPad Prism (GraphPad, San Diego, Calif.). After 20 h, the cells were harvested, washed with ice-cold phosphate-buffered saline (PBS), pelleted, resuspended in 50 mM NaCl, and sonicated for 45 s on ice. Every PCR plate contained control reactions (uninfected cells and 10 copies, 1 copy, and 0.1 copy of plasmid DNA in a background of 104 cells). A [32P]dCTP-labeled probe (APBiotech, Little Chalfont, United Kingdom) was generated by random primer extension (Nonaprimer kit; Qbiogene, Bingham, United Kingdom) according to the manufacturer’s instructions. The RNA was then treated with tobacco acid pyrophosphatase (TAP) to remove the cap structure from full-length mRNA, leaving a 5′-monophosphate.
Limiting dilution PCR assays to detect either challenge virus or vaccine virus. The mice were generated as previously described (38) and were bred in-house. The recombinant MHV-68 BAC plasmids of 11ST and 11ST/MR were generated by the two-step allelic exchange method (41, 42). Briefly, bulk splenocyte or FACS-sorted populations were obtained from mice at days 16 and 42 postinfection as described above. Viruses, cells, and plaque assays.MHV-68 was originally obtained from the American Type Culture Collection (VR1465), and the recombinant virus tw25 was constructed by homologous recombination to contain the enhanced green fluorescence protein (EGFP) expression cassette inserted at nucleotide (nt) 1839 without disrupting any known ORF. These results indicate that the drug concentrations used in this experiment fall within an appropriate range and the inhibition of ORF 45 transcripts by cycloheximide and phosphonoacetic acid was not due to nonspecific toxicity of the inhibitors.