150 dpi) (A) or 50 mg/kg of cyclosporine A (at ca. 5A). A number of motifs present in control peptides were characterized by acidic residues downstream and an Arg upstream of the phospho-acceptor, including an AKT-related RXXS* motif (Fig. During the course of infection, the gHVs traverse numerous cell types before gaining access to germinal center B cells, and ultimately memory B cells. MHV68 miRNAs contribute to efficient infection of memory B cells in vivo. It is possible that IFN-γ is required for latently infected cells to transition to a transcriptionally quiescent state from which reactivation is inefficient. We thank David Allman for helpful discussions.
The primers selected for the PCR analysis of the IgH locus are capable of amplifying only a select number of Vh families and are unable to detect heavy chain rearrangements utilizing Jh4. Antibodies used for surface staining of primary B cells include Thy1.1-APC (eBioscience) and CD19-FITC. Doxorubicin responsiveness was completely abolished in infected cells by 14 h postinfection. 7A and B). Louis, MO 63110. ORF M3 transcripts could not be detected in the presence of CX (Fig. H.
To examine whether the M3 promoter is responsive to RTA, we constructed two reporter plasmids by inserting two regions of the M3 promoter (1,200 and 600 bp upstream of the M3 TATA box) into the pGL3-Basic plasmid (Promega) containing the firefly luciferase coding sequence. The results represent average values from three individual experiments, and the error bars indicate standard deviations. We confirmed that BMDMs responded to the foreign DNA ligands CpG and poly(dA-dT) (Fig. (A) Direct binding of ORF49 to PARP-1. J., Stewart, J. Infected cells were used as the comparative control to account for virus-induced changes in host protein quantities and/or phosphorylation status (50). Thus, the recombination plasmid pHA2 containing a 1.5-kbp fragment homologous to the left end of the MHV-68 genome and the BAC vector including thegpt and gfp genes was constructed (Fig.
sIgD is a marker for mature, naive B cells but is downregulated sometime during the germinal-center reaction as naive B cells differentiate into either plasma or memory B cells. Cullen, B. Based on Poisson distribution, the frequencies of reactivation and viral genome-positive cells were obtained from a nonlinear regression fit of the data where the regression line intersects at 63.2%. − RT, no reverse transcriptase was added to the reaction mixture. In the DR4 mouse model, activation of an endogenous retrovirus contributes to the development of lymphoid tumors (Raffegerst et al., manuscript in preparation). Thus, this strongly argues that M1 gene expression is largely limited to the infected plasma cell population. The contribution to reactivation from preformed infectious virus was determined by parallel plating of mechanically disrupted cells (latent virus cannot reactivate from killed cells).
After 24 h the cells were treated with MG132 (10 μM) or lactacystin (10 μM) for 45 min, followed by virus infection for another 4 h. Insoluble debris was removed by centrifugation (13,000 × g, 15 min). We further confirmed that Xrn1 is involved in SOX-induced RNA degradation in an siRNA-independent manner by use of a flaviviral structural element (SLII) that can block 5′-3′ RNA degradation by Xrn1 . Ig production by B cells after exposure to MHV-68 in vitro. Validated BAC clones were transfected into NIH 3T3 cells, and recombinant γHV68 was further amplified in NIH 3T3 cells. (A) Diagram of the cysteine-rich region in ORF75c aligned with ORF75a and ORF75b. In addition to regulating transcription of viral lytic genes, KSHV LANA inhibits the activity of several host cell cycle regulatory proteins.
The Rta protein functions as a transcriptional activator. Murine gammaherpesvirus 68 (MHV-68 or γHV-68) is genetically and biologically related to EBV and KSHV and is considered an important experimental system to study virus-host interactions and viral pathogenesis (29–32). 2) than those with the characteristics of naive (CD62Lhi) or resting (CD44lo) cells. Notably, M2 is only transiently expressed in wild-type (wt) mice, during the peak of viral latency (day 14 postinfection) (42). Acute-phase splenic latency is also affected, with peak latency reduced ∼50-fold with respect to MHV-68. Upon inoculation of inbred strains of mice, γHV68 undergoes acute-phase replication in mutiple organs, including the lungs and spleen. A distinctive characteristic of herpesviruses is the capacity to establish lifelong infections where the virus persists in healthy carriers by hiding in a cellular reservoir that expresses only few latency-associated viral genes.
Thus, these data demonstrate that TLR stimulation can drive MHV68 reactivation from latency and suggests that periodic pathogen exposure may contribute to the homeostatic maintenance of chronic gammaherpesvirus infection through stimulating virus reactivation and reseeding latency reservoirs. SOX and muSOX localize to both the nucleus and cytoplasm of infected cells. Infection of the AC-RTA virus elicited both cellular immune responses and virus-specific IgG at a level comparable to that elicited by infection of the wild-type virus. PU-H71 is a purine-scaffold Hsp90 inhibitor that, in contrast to other Hsp90 inhibitors, displays unique selectivity for binding the fraction of Hsp90 that is preferentially associated with oncogenic client proteins and enriched in tumor cells (teHsp90).