Quantification of viral genome and transcript.The total genomic DNA from the infected lungs or spleen tissue was prepared using the DNeasy kit (Qiagen) and then subjected to quantitative real-time PCR as previously described (40). The cycling parameters were as follows: hot start at 95°C for 10 min, denaturation at 94°C for 10 s, annealing at 60°C for 20 s, and extension at 72°C for 15 s. 2B). 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). MHV68 miRNA and TMER nomenclature.Several previous reports identified mature virus-encoded miRNAs (see Table S1 in the supplemental material) expressed in cells infected with MHV68 in vitro (11, 37, 38, 41, 43). Reactivation assay.A total of 107 KS-1 cells (KSHV-positive, EBV-negative primary effusion B-cell lymphoma; gift from J. We used 10 μg/ml to fully neutralize 10 U of IFN-γ/ml in the ex vivo neutralization experiments.
At various times postinoculation, femurs and tibias were harvested and flushed with 10 ml Dulbecco modified Eagle medium containing 10% fetal calf serum. Recently, a herpesvirus called RRV was isolated that is related to but distinct from KSHV (30). In normal BALB/c mice, cycKO virus replication was also decreased relative to WT virus replication, but to a lesser extent than that in BALB/c IFN-γ−/− mice. Therefore, chronic inflammation associated with persistent EBV and KSHV infections might contribute to gammaherpesvirus lymphomagenesis. However, the requirement of these individual tyrosine residues in some essential functions of M2, namely establishment of latency, reactivation from latency coupled with plasma cell differentiation, and IL-10 production is unknown. Consistent with these findings, a phosphoproteomic analysis of lytic MHV68 infection in fibroblasts also identified phosphorylated H2AX and a number of additional ATM targets (22). The mechanism of reactivation in both B and epithelial cells is not specifically understood.
Recent comparisons of CTLs from GzmB-cluster−/− and GzmB−/− mice revealed a more severe defect in cytotoxicity in GzmB-cluster−/− effector cells compared to GzmB−/− cells (P. Based on the reporter assay data, we determined whether H/RTA could reactivate MHV-68 from latency. 1B). We have performed these analyses with both CD8 T-cell-deficient (CD8−/−) and B-cell-deficient (MuMT) mice. The original stock of MHV-68 (strain g2.4) was kindly provided by S. The KSHV and HVS v-cyclins, in conjunction with cdk’s, have been demonstrated to phosphorylate the retinoblastoma protein (pRb) and promote cell cycle progression, like their mammalian homologs (2,8). However epithelial-derived virions still infect epithelial cells better than myeloid cells, and myeloid-derived virions still infect epithelial and myeloid cells better than B cells.
The inserted ORF49 DNA fragment was amplified via PCR from the MHV-68 genome (pMHV-68), using the primers ORF49F (5′-aga gaattc AACAGAAAACATG GGGGATG-3′) and ORF49R (5′-cgc ggatcc CTACGAGGGAATTT CTGC-3′). In regions that are endemic for Plasmodium falciparum transmission, mathematical modeling data suggests that immunity to severe non-cerebral malaria requiring hospitalization in children may be attained after 1–2 infections . Using this technique, we want to elucidate the role of host and viral genes in the virus-host interaction. At early times after infection, the frequency of PECs that reactivated gammaHV68 correlated very closely with the frequency of PECs carrying the gammaHV68 genome, validating measurement of the frequency of viral-genome-positive cells as a measure of latency in this cell population. Immunity 20, 305–317. We have constructed custom membrane arrays representing nearly all of the known and predicted MHV-68 ORFs to explore the patterns of viral gene expression. Thus, γHV68 infections of mice can provide a genetically tractable animal model for the study of gammaherpesvirus pathogenesis and for the development of therapeutic strategies against human herpesviruses (46, 56).
Moreover, despite genetic divergence in latency genes among MHV-68, KSHV, and EBV, many lytic cycle genes show a high level of conservation among all three viruses. IMPORTANCE Gammaherpesviruses persist for the lifetime of the host. 334-339. Cyclin A expression allows for the continued activation of CDK2 and subsequently binds and activates CDK1 as S phase progresses and the G2/M boundary is reached, at which stage cyclin B levels are maximal for the regulation of mitosis in conjunction with CDK1. In this review, I will summarize the role of the GMPs and highlight the available data describing the immune-evasion properties of these proteins. Moreover, ORF49 directly bound to RTA and its negative cellular regulator, poly(ADP-ribose) polymerase-1 (PARP-1), and disrupted the interactions of RTA and PARP-1. We also detected a higher frequency of persistent infection in PML−/− peritoneal cells.
The v-cyclin encoded by murine gammaherpesvirus 68 (gammaHV68) induces cell cycle progression and is an oncogene (L. We utilized murine gamma herpesvirus 68 (γHV-68), the murine homolog to EBV, to examine how infection by a virus like EBV could enhance CNS autoimmunity. The partial glycoprotein B sequence up to the DNA polymerase gene showed 96.6 % nucleotide sequence identity to the Rupicapra rupicapra gammaherpesvirus 1 and 81.5 % to ovine herpesvirus 2.