BAC DNA from wild-type MHV-68 (WT) and M3FL was digested with EcoRI, HindIII, and SmaI and analyzed by agarose gel electrophoresis. In addition, each time the animals were infected experimentally, uninfected control mice were likewise analyzed and found to be MHV-68 negative by virus isolation, reactivation, PCR, and serology. Further, we recently demonstrated c-Jun phosphorylation and related AP-1 transcription factor activation during MHV68 replication . Maintenance of latency is typically evaluated 42 days post-infection. Sequences of several putative miRNAs were also included in the study: mghv-miR-M1-2-5p (TMER2), mghv-miR-M1-8-5p, mghv-miR-M1-8-3p and mghv-miR-M1-13-3p (TMER6), and mghv-miR-M1-15-3p (TMER8). Amounts of viral transactivators were increased 4-fold (pPAN-69Luc), 5-fold (BHLF1Luc), or 10-fold (pRpLuc and 57pLuc [++]) to restore activation in the presence of p65. We also determined whether the presence of IFN-γ affected the capacity of γHV68 to infect and induce cytopathic effect in these MEFs.
The presence of preformed infectious virus in bone marrow cells may be indicative of an exceedingly low level of acute replication in the bone marrow itself or may reflect the trafficking of lytically infected or reactivating cells through the bone marrow. The first ORF of the HVS genome codes for highly related STPs in all three subgroups (27, 28, 70, 99). We further hypothesize that altered cell tropism in IFN-γ−/− mice may be central to the requirement for these viral genes in acute pathogenesis. Louis, MO). Therefore, we observed only a modest increase in pSTAT5 levels upon GM-CSF treatment compared to unstimulated B cells. Resolved proteins were transferred to nitrocellulose membranes and probed with antibodies directed to the indicated proteins. NT-2 cells show properties of early embryo cells and can be used to study the early stages of human neurogenesis (59).
We did not detect any persistent lytic replication in either granzyme-deficient or wild-type mice in these experiments (data not shown). 3B). 1). 1A). The DNA arrays were then denatured (0.66 M NaCl, 0.5 M NaOH) and washed (double-distilled water and then 40 mM phosphate buffer, pH 7.3). Digests were electrophoresed on 1% agarose gels and transferred by alkaline transfer to Nytran nylon membranes (Turboblot; Schleicher & Schuell, Keene, N.H.) according to the manufacturer’s recommendations. A replicate experiment gave equivalent results.
Replication-competent mutants were reconstituted by transfecting their BAC DNAs into permissive cells (22). 74, 6964-6974. (1990a). The resistance to CHX and PAA treatment was quantitated by expressing GAPDH-normalized values from the treated array as a percentage of the GAPDH-normalized value in the untreated array. A PCR-amplified M2 fragment with BamHI sites at both ends was cloned into the BamHI cloning site of the pEBG vector to yield the pEBG/M2 construct. In contrast, there was a small but significant lytic replication deficit after intranasal infection of mice (Fig. Isoflurane was used for anesthesia to conduct infections and terminal harvests.
Recombinant BAC DNA was isolated and transfected into Vero-CRE cells, and viral supernatants were passaged through Vero-CRE cells a second time and then used to generate large viral stocks in NIH 3T12 cells. Particularly, a Myc-tagged ORF49 was generated using a modified version of the pCS3-MT plasmid as a destination vector containing the 6× Myc tag with additional sequences (a gift from Jin-Hyun Ahn, Sungkyunkwan University, Republic of Korea). Female mice between 6 and 8 weeks of age were anesthetized for a short period of time and inoculated intranasally with 100 PFU of MHV68 diluted in 20 μl cold cMEM, except for the 50% infective dose (ID50) experiment in which 1, 10, and 100 PFU of MHV68 were used (Table 1). Experimental autoimmune encephalomyelitis (EAE) is a well-studied and accepted model for the study of MS in rodents . London B 356:569–579). Seven days later the cultures were harvested and added to target cells at effector-to-target ratios ranging from 100 to 1 to 6 to 1. Limiting dilution viral genome PCR analysis was used to determine the frequency of latency in single-cell suspensions of bulk, nonsorted splenocytes and CD19+ B cells from mice infected with 100 PFU of M1Δ (bulk, ∼1/610; CD19+, ∼1/805; purity, 98.2% ± 0.8%) and M1.MR (bulk, ∼1/596; CD19+, ∼1/507; purity, 98.7% ± 0.3%).
Indeed, LANA expression is robust throughout both the KSHV and MHV68 lytic replication cycles (26, 28–32). Subsequently, the Tet resistance cassette was removed, resulting in a deletion between nucleotides 26,778 to 28,191 and leaving a small residual insert consisting of an FLP recombination target site and short vector sequences in the disrupted region. MHV-68 is a facile model for studying the gammaherpesvirus lytic phase (33, 41, 44, 51, 52), particularly virion structure, composition, and morphogenesis (6, 42, 48). ORF24 of MHV-68 is 39% and 26% identical to the Kaposi’s sarcoma-associated herpesvirus and Epstein-Barr virus homologs, respectively (23). We described here a primary characterization of this protein and its requirement for lytic replication. The virus exploits the life cycle of the B cell and latency is maintained long term in resting memory B cells.