Toll-like receptor signaling controls reactivation of KSHV from latency

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease. Like other herpesviruses, KSHV establishes life-long latency in the human host with intermittent periods of reactivation. Physiological triggers of herpesviral reactivation are poorly defined. Toll-like receptors (TLRs) recognize pathogens and are vital for the host innate immune response. We screened multiple TLR agonists for their ability to initiate KSHV replication in latently infected PEL. Agonists specific for TLR7/8 reactivated latent KSHV and induced viral lytic gene transcription and replication. Furthermore, vesicular stomatitis virus (VSV), a bonafide physiological activator of TLR7/8, also reactivated KSHV from latency. This demonstrates that secondary pathogen infection of latently infected cells can reactivate KSHV. Human herpesviruses establish life-long latency in the host, and it is plausible that a latently infected cell will encounter multiple pathogens during its lifetime and that these encounters lead to episodic reactivation. Our findings have broad implications for physiological triggers of latent viral infections, such as herpesviral reactivation and persistence in the host.

Fig. 1.

TLR stimulation and KSHV reactivation. (A) TLR expression in PEL cells. Gene expression of the ten TLRs was determined in BCBL-1 and BC-3 by quantitative RT-PCR using primers specific to each human TLR gene. 18S gene expression was used as a loading control. (B) KSHV reactivation in response to TLR agonists. BCBL-1 cells were treated with 10 μg/mL of the indicated TLR ligands and 20 ng/mL positive control (TPA) for 48 h. DNA was isolated and KSHV replication was determined by qPCR for KSHV ORF49 or ORF57 gene expression compared to vehicle control. (C) Time course of KSHV reactivation after TLR8 stimulation. BCBL-1 cells were treated with vehicle control, ssPoly-U, or a combination of TPA and Na-Butyrate. RNA was isolated at the indicated time points, and KSHV ORF49 and ORF57 gene expression was measured by qRT-PCR. (D) Single-stranded RNA reactivates KSHV from 4 different PEL. BCBL-1, BC-3, BCP-1, and VG-1 PEL cell lines were treated with vehicle control, 50 μg/mL ssPoly-U, or a combination of TPA and NaB for 48 h. Fold reactivation was calculated using real-time PCR values of the TLR agonist treated samples versus the vehicle control treated samples. All values were normalized to GAPDH.

Fig. 2.

TLR7/8, and not TLR4, reactivates latent KSHV. (A) TLR7 and TLR8 ligands reactivate KSHV. BCBL-1 cells were treated with increasing concentrations of CL-075, Imiquimod, CpG DNA, or a combination of TPA and NaB for 48 h. (B) LPS does not activate KSHV replication. BCBL-1, VG-1, BCP-1, and BC-3 cells were treated with either LPS for 48 h or a combination of TPA and NaB. ORF57 transcription was measured by qRT-PCR using GAPDH as an endogenous control.

Fig. 3.

Single-stranded RNA activates an innate immune response. (A) Single-stranded RNA activates the canonical NF-κB pathway. IkB-α protein expression was determined by western blot 5 h post-treatment with ssPoly-U. (B) Single-stranded RNA activates the NF-κB promoter in PEL. BCBL-1 cells were nucleofected with 200 ng NF-κB promoter-luciferase plasmid. After 48 h, cells were pooled and treated with ssPoly-U for an additional 16 h. Cells were then harvested and luciferase assays were performed. (C) TLR7/8 stimulation activates IFN and NF-κB transcription. BC-3 and BCBL-1 cells were treated with 50 μg/mL ssPoly-U for 48 h. IFN-α,β,γ, NF-kB1, and NF-kB2 transcription levels were determined by qRT-PCR. All values were normalized to GAPDH as the endogenous control. (D) IRF-7 expression is up-regulated by single-stranded RNA. Cells were treated with ssPoly-U for 24 h, lysed, and IRF-7 protein expression was analyzed by western blot. (E) Inhibition of IRF-7 function blocks ssPoly-U-induced KSHV reactivation. Flag-tagged IRF-7DN was analyzed by western blot 48 h after nucleofection of BCBL-1 cells (Left). pcDNA3-IRF-7DN or pcDNA3 control transfected cells were treated with 50 μg/mL ssPoly-U, equivalent amount of vehicle (mock sample), or 20 ng/mL TPA. Cells were analyzed for viral reactivation 24 h post-treatment by performing a western blot for vIL-6 protein expression (Right).

Fig. 4.

Single-stranded polyU RNA activates KSHV replication. (A) ssPoly-U activates the KSHV RTA/ORF50 promoter. BCBL-1 cells were nucleofected with 5 μg RTAp-Luciferase reporter plasmid and 48 h later cells were stimulated with ssPoly-U or vehicle control for 16 h. Luciferase activity of both samples was measured. (B) KSHV viral load analysis. KSHV viral DNA was isolated 48 h after treatment of BCBL-1 cells with ssPoly-U or vehicle control. QPCR was performed with vIL-6 primers to determine viral DNA loads. (C) Viral Reactivation. Western blot for KSHV vIL-6 and actin (loading control) after treatment of BCBL-1 cells with the indicated agonists.

Fig. 5.

Whole genome profiling of KSHV after ssPoly-U treatment. (A) ssPoly-U induces KSHV replication. KSHV viral arrays were performed after treatment of BCBL-1 and BC-3 with 50 μg/mL LPS, 50 μg/mL ssPoly-U, or a combination of TPA and NaB. Heat maps for each array are shown. (B) Time course of KSHV replication. Heat map of KSHV viral arrays after 0 h, 48 h, and 72 h treatment with 50 μg/mL ssPoly-U. Relative gene expression levels are represented by blue, white, and red, which indicate no detectable, intermediate, or high KSHV gene expression, respectively. (C) ssPoly-U reactivation of PEL generates infectious KSHV virions. BCBL-1 and BC-3 cells were treated with 50 μg/mL ssPoly-U, an equivalent amount of vehicle, or 20 ng/mL TPA. Forty-eight hours post-treatment, supernatants were harvested and used to infect Vero cells. Ninety-six hours postinfection of Vero cells, intracellular DNA was isolated and KSHV viral loads were quantified by qPCR. Values were normalized to GAPDH. Fold increase in KSHV viral loads from ssPoly-U treated PEL, compared to mock-treated PEL is shown.

Fig. 6.

TLR7/8 stimulation mediates KSHV reactivation in response to single-stranded RNA and VSV infection. (A) Knock-down of TLRs 7 and 8 in BCBL-1 cells. Western blot for TLR7 and TLR8 protein expression in BCBL-1 cells stably transfected with the psiRNA-luciferase control, psiRNA-TLR7, or psiRNA-TLR8 plasmids. Tubulin is shown as a loading control. (B) TLR7/8 mediate KSHV reactivation. ssPoly-U treatment of psiRNA-luciferase, psiRNA-TLR7, and psiRNA-TLR8 stable BCBL-1 cell lines was performed. Expression of the lytic protein, vIL6, was determined by western blot 24 h after ssPoly-U treatment of each cell line. Tubulin is shown as a loading control. (C) VSV infection of PEL mediates KSHV reactivation. BCBL-1 cells were infected with VSV at an MOI of 1. KSHV vIL-6 protein expression was determined by western blot analysis.