The Gammaherpesviruses Kaposi's Sarcoma-Associated Herpesvirus and Murine Gammaherpesvirus 68 Modulate the Toll-Like Receptor-Induced Proinflammatory Cytokine Response

Abstract

The human pathogen Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease, establishes lifelong latency upon infection. Murine gammaherpesvirus 68 (MHV68) is a well-established model for KSHV. Toll-like receptors (TLRs) play a crucial role for the innate immune response to pathogens. Although KSHV and MHV68 are detected by TLRs, studies suggest they modulate TLR4 and TLR9 signaling, respectively. In this study, we show that in bone marrow-derived macrophages (BMDMs), MHV68 did not induce a detectable proinflammatory cytokine response. Furthermore, MHV68 abrogated the response to TLR2, -4, -7, and -9 agonists in BMDMs. Similarly to observations with MHV68, infection with KSHV efficiently inhibited TLR2 signaling in THP-1 monocytes. Using a KSHV open reading frame (ORF) library, we found that K4.2, ORF21, ORF31, and the replication and transcription activator protein (RTA)/ORF50 inhibited TLR2-dependent nuclear factor kappa B (NF-B) activation in HEK293 TLR2-yellow fluorescent protein (YFP)-and Flag-TLR2-transfected HEK293T cells. Of the identified ORFs, RTA/ORF50 strongly downregulated TLR2 and TLR4 signaling by reducing TLR2 and TLR4 protein expression. Confocal microscopy revealed that TLR2 and TLR4 were no longer localized to the plasma membrane in cells expressing RTA/ORF50. In this study, we have shown that the gammaherpesviruses MHV68 and KSHV efficiently downmodulate TLR signaling in macrophages and have identified a novel function of RTA/ORF50 in modulation of the innate immune response. IMPORTANCEThe Toll-like receptors (TLRs) are an important class of pattern recognition receptors of the innate immune system. They induce a potent proinflammatory cytokine response upon detection of a variety of pathogens. In this study, we found that the gammaherpesviruses murine gammaherpesvirus 68 (MHV68) and Kaposi's sarcoma-associated herpesvirus (KSHV) efficiently inhibit the TLR-mediated innate immune response. We further identified the KSHV-encoded replication and transcription activator protein (RTA) as a novel modulator of TLR signaling. Our data suggest that the gammaherpesviruses MHV68 and KSHV prevent activation of the innate immune response by targeting TLR signaling.

Murine Gammaherpesvirus 68 Pathogenesis Is Independent of Caspase-1 and Caspase-11 in Mice and Impairs Interleukin-1β Production upon Extrinsic Stimulation in Culture

“…6). The lytic transactivator RTA has been reported to inhibit multiple cell signaling pathways, including the TLR, MAVS, and NF-B signaling pathways (63)(64)(65). However, the reduction in IL-1␤ at 9 hpi did not require RTA or RTA-dependent viral genes.…”

“…Mutations in MHV68 orf50 severely limit viral gene expression and block virus replication (30,62). RTA has additional functions in host signaling pathways, including the degradation of NF-B signaling proteins, impairment of cytokine production, and interference with TLR signaling (63)(64)(65). To examine whether RTA or RTA-dependent gene expression drives suppression of IL-1␤, we infected BMDMs with a recombinant MHV68-ORF50stop virus (30) prior to LPS and ATP treatment and evaluated the intracellular and secreted levels of IL-1␤ at 9 hpi.…”

Multi-step regulation of innate immune signaling by Kaposi's sarcoma-associated herpesvirus

“…RTA, viral E3 ubiquitin ligase, effectively inhibits TLR2 and TLR4 signaling in THP-1 monocytes by causing their degradation (40). In addition, RTA indirectly degrades TRIF (41).…”

Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen: Replicating and Shielding Viral DNA during Viral Persistence

“…2C) (106). The NF-B pathway plays an important role in suppressing the lytic replication of KSHV and MHV68 and promoting latency (107)(108)(109). Several latent KSHV proteins, including full-length LANA and vFLIP, are potent activators of NF-B (109)(110)(111)(112)(113).…”