No abstract

No abstract

Hadwiger and co-workers (5,7,14) found that pisatin, a phytoalexin produced in pea plants, could be induced by various chemicals which were presumed to affect the conformation of DNA; based on this and inhibitor experiments they (7, 14) suggested that pisatin production depended upon both de novo RNA and protein synthesis. To date, however, no systematic study that would link de novo RNA and protein synthesis, phytoalexin production, and disease resistance has been conducted. Plant and Fungus. The soybean cultivar Harosoy 63 (resistant to race 1 of P. megasperma var. sojae) was grown as described previously (22) and used throughout the experiments unless otherwise specified. Race 1 of P. megasperma var. sojae was maintained on V-8 juice agar. For inoculum production, the fungus was grown for 3 to 5 days in a pea broth medium (10) and rinsed with deionized H20 prio. to inoculation.Seven-to 8-day-old plants were cut at the base of the hypocotyls and the cut ends placed in deionized H20 or aqueous solutions of either blasticidin S, a protein synthesis inhibitor (4)

In order to understand and possibly treat B-cell malignancies associated with latent gammaherpesvirus infection, it is vital to understand the factors that control the balance between the two transcriptional states of gammaherpesviruses: latency and lytic replication. We used murine gammaherpesvirus 68 (MHV 68) as a model system to investigate how engagement of endosomal Toll-like receptors (TLRs) impacts reactivation from latency in vitro and establishment of latent infection in vivo. We found that treatment with TLR7 ligand R848 or TLR9 ligand CpG oligodeoxynucleotide (ODN) suppresses reactivation of MHV 68 in vitro. These suppressive effects correlated with the ability to activate cellular transcription factor NF-B. Downregulation of TLR9 by RNA interference in vitro led to a reduction of nuclear levels of NF-B p65 and consequently to an increase of spontaneous reactivation in cells latently infected with MHV 68, indicating that the TLR9 pathway suppresses spontaneous reactivation events. In vivo, sustained stimulation of TLR7 by repeated R848 treatment led to an increased frequency of infected splenocytes compared to mock-treated control results. Frequencies of infected splenic B cells in tlr7 ؊/؊ or tlr9 ؊/؊ mice after establishment of latency did not differ from those seen with their wild-type counterparts. Nevertheless, MHV 68-infected B cells from tlr9 ؊/؊ mice showed a higher frequency of reactivation than B cells from wild-type or tlr7 ؊/؊ mice in ex vivo reactivation assays. Thus, we show a suppressive effect of TLR7 or TLR9 triggering on MHV 68 reactivation that correlates with NF-B activation and that the mere presence of a functional TLR9 signaling pathway contributes to dampen lytic gammaherpesvirus reactivation in infected cells. IMPORTANCEA hallmark of gammaherpesviruses is their establishment of latency in B cells that is reversible through lytic reactivation. Latency can result in B-cell malignancies. Activation of the innate immune system is thought to contribute to controlling the switch between the transcriptional states of latency and reactivation. Nevertheless, the mechanisms involved are not clear. Here, we show that engagement of Toll-like receptor 7 (TLR7) and TLR9 suppresses reactivation of murine gammaherpesvirus MHV 68 in vitro and that stimulation of TLR7 in vivo increases the frequency of infected cells. TLR7 and TLR9 are innate immunity sensors of nucleic acids localized in endosomes. Additionally, we demonstrate that impairment of TLR9 signaling in latently infected B cells leads to increased reactivation. Thus, activated endosomal TLR7 and TLR9 pathways play an important role in promoting establishment of latent gammaherpesvirus infection. Counteracting signaling of these pathways allows reactivation and could represent treatment targets in gammaherpesvirus-associated malignancies.