An Important Role for Mitochondrial Antiviral Signaling Protein in the Kaposi's Sarcoma-Associated Herpesvirus Life Cycle
- R. M. Longnecker, Editor
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FIG 1Western blot confirming knockdown of MAVS and RIG-I in 293 cells. (A) MAVS depletion compared to the scrambled control. (B) RIG-I depletion compared to the scrambled control. Actin was used as a loading control.
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FIG 2KSHV infection of control and knockdown cell lines was monitored for GFP expression by fluorescence microscopy for 48 or 72 h. Control cells and MAVS-depleted and RIG-I-depleted stable knockdown 293 cells were infected for 48 h (A) or 72 h (B). GFP expression was significantly increased in both MAVS-depleted and RIG-I-depleted cells compared to the scrambled control. (C) Quantitation of viral genomes at 2 h postinfection (30 min postspinoculation). There was no significant difference in viral genomes detected between the shMAVS and shRIG-I cells compared to shSCR cells at this time point.
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FIG 3Global viral gene transcription was assessed using a viral qPCR array. Poly(A) RNA was isolated from scrambled control, MAVS-depleted, or RIG-I-depleted 293 cells infected with wild-type KSHV at 0 h and 48 h postinfection. Global viral gene transcription was increased in both MAVS- and RIG-I-depleted cells compared to the control. Shown is a heat map representation of relative expression levels of significantly changed mRNAs. Red indicates the presence and blue the absence of a particular mRNA. The names of the primers, indicating the gene name and primer position on the genome, are shown below the heat map. Primers were clustered using centroid clustering and Manhattan distance as metrics. A dendrogram of the result is shown above the heat map.
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FIG 4IFN-β transcription was monitored following infection of control, MAVS-depleted, or RIG-I-depleted 293 cells at 24 h postinfection with either wild-type KSHV or UV-inactivated KSHV. Both MAVS-depleted and RIG-I-depleted 293 cells showed decreased amounts of IFN-β transcripts compared to that in the control at 24 (A) and 48 (B) h postinfection. MAVS or RIG-I knockdown in 293 cells infected with UV-inactivated KSHV resulted in no induction of IFN-β transcription, indicating a requirement for infectious virus particles for stimulation of type I IFN.
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FIG 5Effect of MAVS and RIG-I depletion on KSHV reactivation. iSLK cells latently infected with KSHV.r219 were transfected with scrambled control, MAVS, or RIG-I shRNA constructs for 48 h. (A) Knockdown was confirmed by Western blotting. (B) At 48 h postreactivation viral transcript levels were monitored by qPCR. MAVS depletion resulted in a 7-fold increase and RIG-I depletion resulted in a 3.5-fold increase in viral genomes. (C) Reactivation was monitored by RFP expression using fluorescence microscopy. Both MAVS-depleted and RIG-I-depleted cells showed increased RFP compared to that in the scrambled control cells at 48 h postreactivation. (D) Mean RFP intensity was quantified using NIS Elements (Nikon imaging software) on the Nikon Eclipse Ti. Data are shown as the average mean intensity over three independent experiments. Both shMAVS and shRIG-I cells showed increased RFP intensity over that in shSCR cells following reactivation.
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FIG 6Infectious virus production following reactivation of latent KSHV from scrambled control and MAVS-depleted or RIG-I-depleted cells. (A) GFP-positive cells were quantitated by counting multiple fields. Naive 293 cells infected with supernatants from reactivated cells depleted for either MAVS or RIG-I showed a significant increase in the number of GFP-positive cells compared to control cells. (B) Microscopy images of infected cells described for panel A. (C) qPCR confirms that 293 cells infected with supernatant from reactivated iSLK cells, depleted for MAVS or RIG-I, show increased viral transcript levels compared to 293 cells infected with supernatants from the scrambled control.
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FIG 7Detection of dsRNA following reactivation of iSLK cells latently infected with KSHV. iSLK cells latently infected with KSHV-BAC16 were reactivated for 48 h with doxycycline. Immunofluorescence assay for dsRNA was performed. dsRNA, indicated by red fluorescence visible in the cytoplasm, was detected in the cytoplasm of reactivated cells (+Dox) but not in unreactivated (−Dox) cells.
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FIG 8Latently infected B cells (BCBL1) were transduced with scrambled control (shSCR) or shMAVS lentiviruses. BCBL1 cells were incubated for 72 or 96 h with the lentiviruses, and expression of ORF57 mRNA transcript was measured. MAVS-depleted cells showed increased ORF57 viral gene transcription at both 72 (A) and 96 (B) h compared to the scrambled control. MAVS depletion was confirmed via qPCR. Fold changes were normalized to the housekeeping β-actin gene and calculated using the 2-ΔΔCT method where ΔΔCT = CT for target gene − CT for β-actin.
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