Murine gammaherpesvirus-68 (MHV-68), a natural pathogen of mice, is being evaluated as a model of Epstein Barr Virus (EBV) infection for use in investigation of the effects of immunomodulatory therapy on herpesvirus pathogenesis in humans. Here we characterize Rta activation of γHV68 gene 57, which is abundantly transcribed during the early phase of virus replication. Here, we demonstrate that MHV68 lytic infection induces p53 phosphorylation and stabilization in a manner that is dependent on the DNA damage response (DDR) kinase ataxia telangiectasia mutated (ATM). The virus persists indefinitely in myeloma cells, without any apparent cytopathic effect, but with the production of infectious virus. In the present study, we utilized an animal model of an Epstein Barr Virus (EBV)-like infection, murine gammaherpesvirus 68 (HV-68), to question whether such a virus could modulate the expression of CRYAB by antigen presenting cells. Our data show that γHV68 alters sleep, activity, and temperature in a manner suggestive of fatigue. Treatment of cells with ACV results in a significant reduction of linear genomes, but has no effect on the level of circular DNA molecules.
Moreover, RT-PCR analysis showed that there was no significant increase in expression of viral genes associated with lytic infection. Taken together, these data suggest that the resident CNS cell types, astrocytes and microglia, are not significant sources of proinflammatory IL-12p70 in response to gammaherpesvirus infection. A number of genes have been identified and probes for the lytic and latent cycle are being developed. Nucleic acid was extracted 2X with saturated phenol/chloroform/isoamyl alcohol (25:24:1), precipitated with 2 volumes of EtOH, and resuspended in PBS with 25 μg/ml RNase A (Sigma-Aldrich). After incubation for 30 min at 37oC, DNA was extracted 1X with phenol/chloroform/isoamyl alcohol, precipitated with 2 volumes ethanol, microfuged for 10 min at 16,000 × g and washed with 75% EtOH. To accomplish this, they must evade recognition and clearance by the immune system. APOBEC4 (A4) was discovered by computational searches for genes homologous to A1 and, like A2, has no ascribed function.
Total RNA was isolated using Trizol (Invitrogen; Carlsbad, CA), as previously described[15, 16, 18]. To date, the mode of gammaherpesvirus persistence in the host has been addressed only with EBV. RNA concentrations were determined with a Gene Spec III spectrophotometer (Naka Instruments, Japan) using a 10 μl cuvette. For cDNA synthesis, one μg of RNA was reverse-transcribed in the presence of random hexamers (50 ng/μl), 10 mM dNTPs, 2.5 mM MgCl2 using ImProm-II reverse transcriptase (Promega) in the buffer supplied by the manufacturer. cDNA was precipitated with one-tenth volume of 3 M sodium acetate (pH 5.2) and 3 volumes of EtOH, and resuspended in 50 μl of nuclease-free H2O. Genomic DNA and mRNA transcript levels were examined by PCR. For semiquantitative PCR, 100 ng of DNA or cDNA was combined with 2.5 U of Taq polymerase (Promega), 0.2 mM each dNTP, 25 pmol of each primer and PCR buffer containing 2.5 mM MgCl2 as provided by the manufacturer.
Samples were cycled using 95° denaturation for 35 seconds, 60°C annealing for 75 seconds and 72°C extension for 90 seconds, with the first three cycles using extended denaturation, annealing and extension times. Antibody is clearly capable of neutralizing mucosal virions in other infections . While most viruses make do with a single cell-binding glycoprotein, herpesviruses typically employ at least four. Forty percent of each amplified PCR product was electrophoresed on an ethidium bromide-stained 2% agarose gel and photographed under UV illumination. The BALB/c-3T3 cells (ATCC CCL163) were trypsinized, washed once, and infected with MHV-68 at a multiplicity of infection of 10 for 1 to 2 h in Dulbecco modified Eagle medium containing 10% fetal calf serum (FCS) (HyClone, Logan, Utah) at 37°C or left uninfected as controls. This polyclonal B cell activation can lead to the emergence of auto-antibodies but MuHV-4 infection is usually not associated with the development of auto-immune diseases or lymphomas in immune competent mice . Louis, MO 63110.
Phone: (314) 362-9223 (H.W.V.) or (314) 362-0367 (S.H.S.). It has been reported that EBV and KSHV can be reactivated from latency by DNA methylation inhibitor reagents such as 5-AzaC , . The paradigm of EBV latency suggests that some genes are expressed exclusively during the latent phase of replication (13). Therefore, we hypothesized that it may have different biological characteristics and thus yield additional insights into gammaherpesvirus pathogenesis. Processing of snoRNA-derived hairpins gives rise to Dicer dependent but Drosha-/Dgcr8-independent miRNAs. At the indicated times post infection, mice were euthanized and individual sera were taken. Spleens and thymuses were disrupted in a tissue homogenizer, using a protocol that preserves cell viability, and filtered over Nitex to remove splenic stroma (1).
60-amino-acid motifs with four conserved cysteine residues (disulfide bonded together in the manner, 1-3, 2-4) (39, 53), which are critical for binding C3b and C4b (2, 49). Speck, and H. The activity of arginase 1 was determined as follows: Cells were lysed in 100 μl of 0.1% Triton X-100 for 30 min at 37°C, and microfuged to remove debris. K3 may therefore be important for the survival of lytically infected DCs. 100 μl of 0.5 M L-arginine (pH 9.5) was added and incubate at 37°C for 30 min. Although the role of chronic inflammation in the development of gammaherpesvirus-associated malignancies has not been examined, other chronic inflammatory stimuli, such as Helicobacter pylori infection, are major contributors to the development of cancer (12). v-cyc[K104E] and v-cyc[E133V] mutants were generated by allelic exchange essentially as previously described (60).
The absorbance was read at 570 nm. Some of these EBV growth-transformed lymphoblasts are hypothesized to form germinal centers, with a concomitant downregulation of viral gene expression with expression of EBNA1, LMP1, and LMP2a (4, 5, 9). At 15 days post infection, mice were euthanized and spleens were removed. We thus hypothesized that, like KSHV RTA, MHV-68 RTA might recognize and bind to a promoter in the left oriLyt region and transactivate the expression of a downstream viral gene(s) (60, 61). Louis, MO). Reactivation data for 6 weeks in panel F represent a single experiment with spleen cells pooled from four to seven mice per group. Whether these responses are similarly dependent on CD4 T cell help during infection is unknown.
To isolate T lymphocytes, spleens were removed from normal, uninfected mice, and splenic leukocytes obtained as described above. Furthermore, the dominant-negative mutants were able to suppress viral lytic replication following transfection of virion DNA or infection with virus. The labeled oligonucleotide was hybridized to the arrays for 15 h at 50°C in 10 ml of hybridization buffer. Additionally, in contrast to the apparent lack of a role for XBP-1 in MHV68 reactivation, we show that another host transcription factor, IRF4—which is essential for plasma cell differentiation—plays a critical role in MHV68 reactivation from B cell latency.