The KSHV capsid at 24 Å resolution. The hemoglobin level of the Matrigel plugs treated as in (G) was determined with hemoglobin content calculated based on the standard curve. MHV68 infection induces IRF-1 expression in a type I IFN-dependent but viral DNA damage response-independent manner.IRF-1 is a short-lived transcription factor that is constitutively expressed at low levels in a variety of tissues, including spleen (39). 71, 4278−4283 (1997). Cells from each sample group were loaded into individual agarose plugs and run on a horizontal agarose gel. Viral titer data were statistically analyzed using the Mann-Whitney nonparametric two-tailed t test. The DNA was extracted with phenol-chloroform and precipitated with ethanol, along with 1 μg of glycogen carrier (Glycoblue; Ambion).

In general, we did observe a close correlation between overall sequence conservation and conservation of predicted pre-miRNA candidates. Therefore, we determined kinetics of IFNβand IRF2 expression during MHV68 infection in the spleen of wildtype, IFNAR1-/-, and IRF2-/- mice. 2A). The infected cells were overlaid with 1% methylcellulose-containing growth medium. Finally, immunoblotting with an anti-Bo10-c15 rabbit polyserum confirmed that only the Bo10 MuDir mutant virions lacked gp180, encoded by the spliced Bo10 product (Figure 2D) while content of other proteins such as gB appeared to be normal (Figure S1). (Fig. mLANA was then transferred into pDEST24 (Life Technologies) using Gateway LR Clonase II enzyme mix (Life Technologies).

Few eGFP+ cells were evident in SCLNs 3days after i.n. Based on these studies, the authors also proposed that in EBV+ cancers characterized by type II latency (i.e., lacking EBNA2), STAT3 may drive LMP1 expression (Chen et al., 2001, 2003). cDNA was generated from sorted GFP+ cells independently from RNA-seq libraries. To examine whether latent infection of MHV-68 in persistently infected SH-SY5Y cells can be further reactivated into productive lytic replication, the cells infected with MHV-68/EGFP or WT were treated with TPA and sodium butyrate (NaB), which are well-known lytic inducers, in the presence and the absence of ganciclovir (GCV; a potent antiviral drug) for 36 h. After transfection, cells were either starved overnight in Earle’s balanced salt solution (starvation medium) or grown in nutrient-rich media with the addition of 2× essential amino acids and 2× nonessential amino acids. Both productive and latent replications of MHV-68/EGFP in prolonged culture of infected SH-SY5Y cells. (A, B) Primary macrophages were infected as indicated, and chromatin was collected at 16 hpi and subjected to ChIP using HDAC1-specific (A) or HDAC2-specific (B) antibodies or a nonspecific …

M1 function suppresses reactivation from latency in accordance with Vβ4+ CD8+ T cell activation. Immunofluorescence with the anti-p43 serum showed ORF11-specific cytoplasmic staining (Fig. γHV68 infection is associated with an increased incidence of lymphoproliferative diseases in BALB-β2m-deficient mice. The latently infected B lymphoma cell line, S11, was derived from long-term MHV68-infected mice that spontaneously developed lymphoproliferative disease (Usherwood et al., 1996b). The detection limit for this assay was 5 PFU. Successful Flp recombination was established by replica plating of bacteria onto chloramphenicol or chloramphenicol plus kanamycin and confirmed by restriction enzyme mapping of BAC DNA. A 60-bp probe was designed to contain the complementary sequence (nucleotides [nt] 71370 to 71424) upstream of the translational start site of ORF52 (nt 71364), as well as 5 nt of the noncomplementary sequence at the 3′ end (5′-AGG GAG AAT AAC AAC AAG TTG AGC AAG GGT GCA GGT TGT CTC CAG GGC AC TGC TTG GGC G-3′).

C). Virus stocks were prepared using BHK-21 cells, and viral titers were determined by a plaque assay on BHK-21 cells as described previously (65). Recombinant BACs were selected and confirmed by PCR and restriction fragment length polymorphism analysis. C57BL/6J (Harlan U.K.) and BAFF-R−/− mice (26) (kindly provided by Andrew Sage and Lauren Baker, Division of Cardiovascular Medicine, Cambridge University Medical School) were maintained at the Cambridge University Department of Pathology animal unit and infected with MuHV-4 when 6 to 12 weeks old, either intranasally (i.n.) in 30 μl of Dulbecco’s modified Eagle’s medium (DMEM) under isoflurane anesthesia (104 PFU) or intraperitoneally (i.p.) in 100 μ1 of DMEM (105 PFU). The bacteria were then transformed with a temperature-sensitive Flp recombinase expression plasmid, pCP20, to excise the kanamycin resistance gene. The genomic sequence of γHV68 is available and confirms its close relationship with other gammaherpesviruses (33). The activation domains of the proteins were swapped as indicated in the diagram, …

In this study, we show that vaccination with a reactivation-deficient virus reduced long-term latent infection of wild-type challenge virus to an undetectable level. After intranasal inoculation, the virus replicates productively in the lungs, then establishes a latent infection in lymphoid tissue, predominantly the spleen 4, although other cells can also maintain a latent infection 5678. With regard to persistent EC survival of infection, the number of surviving cells is greatly reduced in the absence of the viral cyclin (v-cyclin), while the amount of virus produced per cell is unaffected (63).