Discovery of a viral NLR homolog that inhibits the inflammasome

Abstract

The NLR (nucleotide binding and oligomerization, leucine-rich repeat) family of proteins senses microbial infections and activates the inflammasome, a multiprotein complex that promotes microbial clearance. Kaposi's sarcoma-associated herpesvirus (KSHV) is linked to several human malignancies. We found that KSHV Orf63 is a viral homolog of human NLRP1. Orf63 blocked NLRP1-dependent innate immune responses, including caspase-1 activation and processing of interleukins IL-1β and IL-18. KSHV Orf63 interacted with NLRP1, NLRP3, and NOD2. Inhibition of Orf63 expression resulted in increased expression of IL-1β during the KSHV life cycle. Furthermore, inhibition of NLRP1 was necessary for efficient reactivation and generation of progeny virus. The viral homolog subverts the function of cellular NLRs, which suggests that modulation of NLR-mediated innate immunity is important for the lifelong persistence of herpesviruses.

Fig. 1. Orf63 is a viral homolog and inhibitor of NLRP1

(A–D) Stable Orf63 expression in THP-1 cells is detected by immunoblot (top panel in A). THP-1-Control and THP-1-Orf63 cells were either treated with vehicle control or primed with 5ng/ml LPS for 1 hour, followed by stimulation with 10μg/ml MDP for 6 hours. Supernatants were harvested and analyzed for IL-1β by enzyme linked immunosorbent assay (ELISA) and values were normalized to extracellular lactate dehydrogenase (LDH) released (A) or analyzed for pro-IL-1β and cleaved IL-1β by immunoblot (B). Supernatants were also analyzed for IL-18 by ELISA and normalized to extracellular LDH (C) and by immunoblot (D). (E) THP-1-Control or THP-1-Orf63 cells were treated as described above and extracellular LDH was calculated relative to positive control. (F) Procaspase-1, pro-IL-1β, ASC (together denoted as CIA) and NLRP1 expression plasmids were transfected into 293T cells with Orf63 or vector control. Cell extracts and supernatants were harvested 24 hours later and subjected to an IL-1β ELISA and immunoblot for cleaved IL-1β (G) 293T cells were transfected with the indicated expression constructs followed by immunoblot for IL-1β expression. (H) NLRP1 and procaspase-1 were transfected into 293T cells with Orf63 or vector control for 24 hours. Caspase-1 enzymatic activity was determined by incubating lysates with caspase-1 substrate, WEHD-7-amino-4-trifluoromethyl coumarin (AFC). Fluorescence emission was measured every 15 minutes for 2 hours. (I) Caspase-1 was immunoprecipitated from 293T cells transfected with the indicated expression plasmids followed by immunoblot. ** indicates statistical significance P ≤ 0.05 by two-tailed student's t-test. Data are representative of a minimum of three experimental replicates.

Fig. 2. Orf63 interacts with NLRP1

(A) 293T cells were transfected with expression plasmids for NLRP1, Orf63 or both plasmids and 48 hours later, NLRP1 was immunoprecipitated followed by immunoblotting for Orf63. (B) A reverse immunoprecipitation was performed. 293T cells were transfected with expression plasmids for NLRP1, Orf63 or both plasmids and 48 hours later, Orf63 was immunoprecipitated followed by immunoblotting for NLRP1. (C) Co-immunoprecipitation of purified Orf63 and NLRP1-GST proteins followed by immunoblotting for Orf63-FLAG and GST-NLRP1. (D) 293T cells were transfected with Orf63 and full-length or mutant NLRP1 expression plasmids followed by immunoprecipitation of Orf63 and immunoblotting for NLRP1. (E) 293T cells were transfected with Orf63-N and Orf63ΔN mutants and full-length NLRP1 expression plasmids for 48 hours. Immunoprecipitations were performed for NLRP1 followed by immunoblots for Orf63-N and Orf63ΔN mutants. Asterisk indicates non-specific band in input lanes. Data are representative of a minimum of three experimental replicates.

Fig. 3. Orf63 inhibits NLRP1 inflammasome formation and is necessary for IL-1β inhibition during viral infection

(A) Indicated expression plasmids were transfected into 293T cells. NLRP1 was immunoprecipitated 48 hours later and procaspase-1 interactions with NLRP1 were determined by immunoblot. (B) 293T cells were transfected with indicated expression plasmids for 48 hours. NLRP1-myc was immunoprecipitated with anti-myc-antibody and interactions with NLRP1-V5 detected by immunoblot. (C) 293T cells were transfected with indicated expression plasmids followed by immunoprecipitation of either Orf63 or NOD2 48 hours later. Interaction of Orf63 with NOD2 was detected by immunoblot (D) KSHV-infected primary human monocytes were transfected with siRNAs against Orf63 or non-targeting controls. 48 hours later, Orf63 and GAPDH transcription was analyzed by PCR. (E) Control and Orf63 siRNA monocytes were analyzed for IL-1β expression by ELISA 48 hours after transfection with Orf63 siRNA or non-targeting control siRNA. (F) Transcription of lytic viral genes, Orf49, Orf50 and Orf57, in KSHV-infected monocytes was analyzed by qPCR 48 hours after knock-down of Orf63. (G) BCBL-1 PEL cells were nucleofected with siRNAs against Orf63 or a non-targeting control. 24 hours later, the cells were treated with 25ng/ml TPA. 96 hours post-TPA treatment, Orf49, Orf57 and Orf63 transcription was analyzed by qPCR. (H) BCBL-1 PEL cells were treated as in panel G, and 96 hours later, supernatants were used to infect naïve Vero cells. 72 hours later, KSHV viral load in the infected Vero cells was determined by qPCR for Orf49. ** indicates statistical significance P ≤ 0.05 by two-tailed student's t-test. Data are representative of a minimum of three experimental replicates.

Fig. 4. Orf63 inhibits the NLRP3 inflammasome

(A–D) THP-1-control or THP-1-Orf63 cells were mock treated or primed with 5ng/ml LPS for 1 hour followed by stimulation with 2.5mM ATP for 6 hours. Supernatants were harvested and analyzed by ELISA for IL-1β and normalized to extracellular LDH (A) or for pro-IL-1β and cleaved IL-1β by immunoblot (B). ATP-stimulated cells were also examined for IL-18 expression by ELISA and values were normalized to extracellular LDH (C) and pro-IL-18 and cleaved IL-18 by immunoblot (D). (E) THP-1-control or THP-1-Orf63 cells were treated with ATP as described above and subjected to an LDH release assay. (F) 293T cells were transfected with NLRP3 and Orf63 expression plasmids for 48 hours. NLRP3 was immunoprecipitated followed by immunoblotting for Orf63. (G) GST and GST-Orf63 was purified and subjected to SDS-PAGE and Coomassie staining. (H) GST or GST-Orf63 protein was incubated with glutathione beads and ³⁵S-methionine-labeled NLRP1, NLRP3 or luciferase protein as previously described (29). Complexes were washed several times and subjected to SDS-PAGE. The gel was dried and the bound radiolabeled proteins were detected by a phosphorimager. The first three lanes represent 20% of the input of each of the three labeled proteins added to the GST and GST-Orf63 binding reactions. ** indicates statistical significance P ≤ 0.05 by two-tailed student's t-test. Data are representative of a minimum of three experimental replicates.