KSHV and the toll of innate immune activation

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi’s sarcoma (KS), the most common cancer afflicting HIV-infected individuals.¹ KSHV is also associated with B cell lymphoproliferative disorders primary effusion lymphoma (PEL) and multicentric Castlesman’s disease (MCD).² As with all herpes-viruses, the KSHV lifecycle is comprised of both latent and lytic phases. Minimal viral gene expression is observed during latency, which facilitates maintenance of viral genomes and evasion of host immune detection. Reactivation of virus from latency results in lytic replication, which is necessary for life-long viral persistence and transmission. Lytic replication is also highly correlated with the development and severity of KS pathogenesis. A fundamental question in KSHV and herpesvirus biology involves determining physiological activators of viral reactivation.

KSHV reactivation is dependent on the lytic switch protein, replication and transcription activator (RTA). RTA regulates lytic gene expression directly by binding RTA-responsive elements in lytic gene promoters or via indirect mechanisms through interactions with cellular transcription factors. The RTA promoter contains multiple, different binding sites for transcription factors that are activated by chemical and physiological triggers of KSHV reactivation. For example, phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA/PMA) and histone deacetylase (HDAC) inhibitors activate the RTA promoter and provide a valuable tool in vitro to study KSHV reactivation. TPA activates protein kinase C and mitogen-activated protein kinase (MAPK) pathways resulting in activator protein-1 (AP-1) complex formation. Activated AP-1 binds the RTA promoter to drive RTA expression. In addition, an SP1 transcription factor binding site in the RTA promoter is normally masked by nucleosomes in latently infected cells. Upon treatment with HDAC inhibitors butyrate or trichostatin A, chromatin-remodeling proteins Ini1/Snf5 and histone acetyltransferease CBP enable SP1 binding to the RTA promoter.³ The hormones epinephrine/norepinephrine have been reported to reactivate KSHV from latency, indicating that cellular stress leads to KSHV reactivation. Epinephrine/norepinephrine-dependent RTA expression occurs through activation of β-adrenergic receptors and the protein kinase A (PKA) pathway.⁴ KSHV reactivation has also been observed with cytokines such as interferon-γ, oncostatin M, and hepatocyte growth factor, while interferon-α inhibits reactivation.⁵ Several hypoxia response elements are present in the RTA promoter, and it was shown that hypoxia induced transcription factors HIF-1α and HIF-2α resulted in activation of RTA.⁶ Finally, several lines of evidence suggest that super-infection with KSHV itself, or coinfection of KSHV latently infected cells with other herpesviruses, or HIV, induces KSHV reactivation.^7,8 Recently, we demonstrated that the mechanism of virus-induced reactivation is regulated by activation of specific innate immune receptors in KSHV latently infected cells.⁹

Toll-like receptors (TLRs) are a key component of the innate immune response to invading pathogens.¹⁰ TLRs are transmembrane proteins that reside either on the cell surface or within endosomes and contain 21 to 25 leucine-rich repeat regions that are specific to conserved pathogen associated molecular patterns (PAMPs). Of the 10 identified human TLRs, cell surface TLRs 1, 2, 4, 5 and 6 detect bacterial PAMPs. Viral nucleic acid that is released in the endosome can be detected by TLR 3 (double-stranded RNA), TLR 7/8 (single-stranded RNA) and TLR 9 (hypomethylated DNA). In addition, TLR 7 appears to detect group B streptococcus mRNA as well. The activation of TLRs by specific agonists stimulates the activation of NFκB and interferon regulatory factor (IRF) transcription factors, and the establishment of the cellular antiviral state. NFκB and IRFs translocate to the nucleus to drive expression of proinflammatory cytokines and type I interferon. Until recently, the influence of TLRs on the KSHV lifecycle was unknown. Our group demonstrated that primary infection of monocytes with KSHV results in TLR 3-dependent induction of proinflammatory chemokines and interferon, most notably CXCL10 and IFNβ.¹¹ This response is downregulated at later times in infection as the virus establishes latency. Although traditionally associated with the detection of bacteria, KSHV infection of endothelial cells resulted in TLR 4 downregulation, and it was shown that two viral proteins, the KSHV encoded G-protein coupled receptor (vGPCR) and viral IRF-1 (vIRF-1) partially inhibited TLR4 expression.¹²

In light of increasing evidence suggesting a role for TLRs in mediating KSHV infection, we sought to determine whether TLR agonists could induce viral reactivation from KSHV-infected PEL, which harbor latent virus. TLR profiling revealed that PEL cells express multiple TLRs, including TLRs 7 and 8. We screened TLR activation with TLR-specific agonists and found that stimulation of TLR 7 or 8 with synthetic single-stranded RNA resulted in KSHV reactivation (see Fig. 1). Interestingly, TLRs 7 and 8 arose from a common ancestral gene, are often functionally redundant due to their high sequence homology, and can be activated by the same ligands.^13,14 Reactivation was demonstrated by expression of KSHV lytic proteins, whole viral genome transcription as well as production of progeny virions. shRNA knockdown experiments indicated that reactivation was mostly dependent on TLR 8. Additional transcription factors likely play a role in reactivation since inhibition of IRF-7 by overexpression of a dominant-negative mutant reduced, but did not abolish, reactivation (Fig. 1). Furthermore, infection of PEL cells with vesicular stomatitis virus, an RNA virus and a known activator of the TLR 7/8 pathway, also reactivated KSHV.

Figure 1.

TLRs 7 and 8 control reactivation of KSHV from latency. Stimulation of TLRs 7 and 8 by single-stranded RNA activates NFκB and IRF-7 transcription factors leading to the production of proinflammatory cytokines and type I interferon, activation of the KSHV lytic transactivator, RTA. RTA drives reactivation of KSHV from latency and enables the virus to enter the lytic cycle. Lytic replication results in the production of progeny virions, leading to viral spread and persistence of KSHV.

The discovery that TLR 7/8 stimulation reactivates KSHV from latency may have therapeutic implications for the treatment of KSHV-associated malignancies. Recent interest in using viruses to treat cancer has focused on the ability of certain viruses such as VSV to selectively infect and lyse tumor cells. Our results suggest that this may be applicable to KSHV-infected lymphomas. However, KSHV malignancies typically occur in immunosuppressed individuals, in which case treatment with VSV could exacerbate disease due to KSHV reactivation resulting in viral replication and spread. Therefore, combination treatment with viral replication inhibitors may inhibit spread while still enabling VSV-mediated tumor lysis. In addition, in certain instances it may be beneficial to block viral reactivation and spread by administering TLR 7/8 antagonists. Although this approach would not eradicate KSHV infected cells, it could prevent reactivation and cell-to-cell transmission by maintaining latency. In summary, we report a novel link between host innate immunity and herpesviral reactivation. Our study suggests that KSHV has evolved to sense host “danger” signals to facilitate its survival and propagation by escaping a cell that is fated to die.