In order to understand and possibly treat B-cell malignancies associated with latent gamma-herpesvirus infection, it is vital to understand the factors that control the balance between the two transcriptional states of gamma-herpesviruses: latency and lytic replication. We used murine gamma-herpesvirus (MHV)-68 as a model system to investigate how engagement of endosomal Toll-like receptors (TLRs) impacts on reactivation from latency in vitro and on establishment of latent infection in vivo. We found that treatment with TLR7 ligand R848 or TLR9 ligand CpG ODN suppresses reactivation of MHV-68 in vitro. These suppressive effects correlated with the ability to activate cellular transcription factor NFκB. Downregulation of TLR9 by RNA interference in vitro led to a reduction of nuclear levels of NFκB p65 and consequently to an increase of spontaneous reactivation in MHV-68 latently infected cells, indicating that the TLR9 pathway contributes to limiting 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. Frequencies of infected splenic B-cells in tlr7(-/-) or tlr9(-/-) mice after establishment of latency did not differ from their wild-type counterpart. Nevertheless, MHV-68-infected B-cells from tlr9(-/-) mice showed a higher frequency of reactivation compared to 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 NFκB activation and that the mere presence of functional TLR9 signaling pathway contributes to dampen lytic gamma-herpesvirus reactivation in infected cells.
IMPORTANCE A hallmark of gamma-herpesviruses 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 receptors (TLRs)7 and 9 suppress reactivation of murine gamma-herpesvirus MHV-68 in vitro and that stimulation of TLR7 in vivo increases the frequency of infected cells. TLRs 7 and 9 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 gamma-herpesvirus infection. Counteracting signaling of these pathways allows for reactivation and could represent treatment targets in gamma-herpesvirus-associated malignancies.