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. 2018 Jul 3;9(4):e00823-18.
doi: 10.1128/mBio.00823-18.

RIG-I Detects Kaposi's Sarcoma-Associated Herpesvirus Transcripts in a RNA Polymerase III-Independent Manner

Yugen Zhang  1   2 , Dirk P Dittmer  1   2 , Piotr A Mieczkowski  3   4 , Kurtis M Host  1   2 , William G Fusco  5 , Joseph A Duncan  5 , Blossom Damania  6   2
Affiliations

RIG-I Detects Kaposi's Sarcoma-Associated Herpesvirus Transcripts in a RNA Polymerase III-Independent Manner

Yugen Zhang et al. mBio. .

Abstract

Retinoic acid-inducible gene I (RIG-I) is a cytosolic pathogen recognition receptor that initiates the innate immune response against many RNA viruses. We previously showed that RIG-I restricts Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation (J. A. West et al., J Virol 88:5778-5787, 2014, https://doi.org/10.1128/JVI.03226-13). In this study, we report that KSHV stimulates the RIG-I signaling pathway in a RNA polymerase (Pol) III-independent manner and subsequently induces type I interferon (IFN) responses. Knockdown or inhibition of RNA Pol III had no effect on beta interferon (IFN-β) induction by KSHV. By using high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) approach, we identified multiple KSHV regions that give rise to RNA fragments binding to RIG-I, such as ORF810420-10496, Repeat region (LIR1)119059-119204, and ORF2543561-43650 The sequence dissimilarity between these fragments suggests that RIG-I detects a particular structure rather than a specific sequence motif. Synthesized ORF810420-10496 RNA stimulated RIG-I-dependent but RNA Pol III-independent IFN-β signaling. In summary, several KSHV RNAs are sensed by RIG-I in a RNA Pol III-independent manner.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Innate immune responses against viral infections, especially the induction of type I interferon, are critical for limiting the replication of viruses. Retinoic acid-inducible gene I (RIG-I), a cytosolic RNA helicase sensor, plays a significant role in the induction of type I interferon responses following viral infection. Here, we identified multiple RNA regions in KSHV as potential virus ligands that bind to RIG-I and stimulate RIG-I-dependent but RNA Pol III-independent IFN-β signaling. Our results expand the role of RIG-I by providing an example of a DNA virus activating a canonical RNA-sensing pathway.

Keywords: Kaposi's sarcoma-associated herpesvirus; RIG-I; innate immunity.

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Figures

FIG 1
FIG 1
RNA Pol III is not required for IFN-β expression during KSHV primary infection or reactivation. (A and B) HEK293 cells in 24-well plates were treated with the indicated concentrations of the RNA Pol III inhibitor ML-60218. Eight hours after treatment, cells were transfected with poly(dA·dT) for 6 h (A) or infection with rKSHV.219 for 48 h (B), IFN-β mRNA level was measured by qRT-PCR. (C to F) HEK293 cells in 24-well plates were transfected with siRNAs targeting RNA Pol III or RIG-I. Twenty-four hours posttransfection, cells were infected with rKSHV.219 for 48 h. The relative expression levels of IFN-β (C), RNA Pol III (D), and RIG-I (E) normalized to β-actin were measured by qRT-PCR. (F) Western blotting showing efficient knockdown of RNA Pol III or RIG-I in HEK293 cells. (G to J) iSLK.219 cells latently infected with rKSHV.219 were transfected with nontargeting control, RNA Pol III, or RIG-I siRNA. Twenty-four hours after siRNA transfection, doxycycline was added to reactivate the iSLK.219 cells. At 72 h postreactivation, the relative expression levels of IFN-β (G), RNA Pol III (H), and RIG-I (I) normalized to β-actin were measured by qRT-PCR. (J) Western blots showing efficient knockdown of RNA Pol III or RIG-I in iSLK.219 cells. Data are presented as means plus standard deviations (SD). Error bars represent the variation range of duplicate experiments. The data are representative of three independent experiments. Values that are statistically significantly different are indicated by bars and asterisks as follows: **, P < 0.01; ***, P < 0.001.
FIG 2
FIG 2
Isolation and characterization of RIG-I/RNA complexes from latent and lytic iSLK cells. (A) Schematic representation of CLIP (cross-linking and endogenous RIG-I immunoprecipitation [IP]) procedure. CARDs, caspase activation and recruitment domains; CTD, C-terminal domain. (B) Western blot (WB) analysis from RIG-I pulldowns shows the efficiency and specificity of endogenous RIG-I IP. (C) Schematic representation of purified FLAG-RIG IP procedure. (D) FLAG-tagged RIG-I (FLAG-RIG-I) protein in an 8% polyacrylamide gel stained by an ultrasensitive Coomassie blue stain. (E) Western blot analysis from FLAG pulldowns shows the efficiency and specificity of purified FLAG-RIG IP. The Western blots are representative of three independent CLIPs.
FIG 3
FIG 3
Immunostimulatory activity of RNA from RIG-I and control (IgG) IPs in wild-type (WT) RIG-I and RIG-I knockout MEF cells. WT RIG-I and RIG-I knockout MEF cells in 24-well plates were transfected with four different immunoprecipitated RNA samples: (i) IgG antibody pulldown from latent iSLK.219 cells (IgG IP; No Dox); (ii) IgG antibody pulldown from lytic iSLK.219 cells reactivated with doxycycline (Dox) (IgG IP; Dox); (iii) RIG-I antibody pulldown from latent iSLK.219 cells (RIG-I; No Dox); and (iv) RIG-I antibody pulldown from lytic iSLK.219 cells (RIG-I IP; Dox). In parallel, cells were transfected with poly(I·C) or infected with VSV (positive controls). Twenty-four hours after RNA transfection or virus infection, total RNA was purified from transfected or infected cells and was treated with RNase A or not treated with RNase A, followed by qRT-PCR analysis for mouse IFN-β mRNA. (A) Enrichment of immunostimulatory RNA from RIG-I IP Dox compared with control (IgG) IP. (B) Enrichment of immunostimulatory RNA with RIG-I IP compared with total RNA (Input) from latent iSLK.219 cells (No Dox) or lytic iSLK.219 cells (Dox). (C) RNase A treatment of RIG-I-bound RNA as well as Input RNAs completely abolishes their immunostimulatory activity. (D) Mouse RIG-I mRNA in WT RIG-I and RIG-I−/− MEF by qRT-PCR analysis to confirm that there is no RIG-I in RIG-I−/− MEF. Data are presented as mean ± SD. Error bars represent the variation range of duplicate experiments. The data are representative of three independent experiments. **, P < 0.01; ***, P < 0.001.
FIG 4
FIG 4
KSHV RNAs are selectively enriched in RIG-I immunoprecipitates compared to control IgG immunoprecipitates by deep sequencing analysis. RNAs from RIG-I pulldown and control (IgG) pulldown from both latent and lytic iSLK.219 cells were subjected to Illumina deep sequencing analysis. Reads were aligned to KSHV JSC-1 isolated clones into BAC16 (GQ994935) and verified by whole-genome sequencing. Relative coverage is shown on the y axes. Further details are in Fig. S5 in the supplemental material.
FIG 5
FIG 5
qRT-PCR analysis of RIG-I-associated RNA from RIG-I and control (IgG) IPs. (A) Schematic of qRT-PCR procedure from endogenous RIG-I immunoprecipitation (IP) in latent or lytic iSLK cells. (B) qRT-PCR products of RNAs immunoprecipitated from endogenous RIG-I IP were run on an agarose gel, revealing KSHV-specific amplifications not present in the control IgG IP. The presence (+) or absence (−) of doxycycline (Dox) and reverse transcriptase (RT) are shown above the lanes. (C) Schematic of qRT-PCR procedure from purified recombinant FLAG-RIG-I IP in latent or lytic iSLK cells. (D) qRT-PCR products of RNAs immunoprecipitated from recombinant FLAG-RIG-I were run on an agarose gel, revealing KSHV-specific amplifications not present in the control IgG IP. (E) Schematic of qRT-PCR procedure from endogenous RIG-I IP in latent and lytic BCBL1 cells. (F) qRT-PCR products from RNAs immunoprecipitated from endogenous RIG-I IP of lytic BCBL1 cells were run on an agarose gel, revealing KSHV-specific gene bands not present in the control IgG IP. The data are representative of three independent qRT-PCR experiments.
FIG 6
FIG 6
KSHV ORF810420-10496 RNA induces IFN-β and RIG-I in RNA-transfected cells. (A to D) HEK293 cells in 24-well plates were transfected with 5′-ppp-dsRNA or poly(I⋅C) (positive controls), or with the indicated concentration (in nanograms or micrograms) of ORF810420-10496 RNA or not transfected (no-treatment control). At 24 h after transfection, cells were harvested for RNA extraction. The relative mRNA levels of IFN-β (A), RIG-I (B), and STING (C) normalized to β-actin were measured by qRT-PCR. The IFN-β protein level was measured by ELISA from the culture supernatants (D). (E to G) Uninfected iSLK cells in 24-well plates were transfected with 5′-ppp-dsRNA or poly(I·C) (positive controls) or with the indicated concentration of ORF810420-10496 RNA or not transfected (negative control). At 24 h after transfection, cells were harvested for RNA extraction. The relative mRNA levels of IFN-β (E), RIG-I (F), and STING (G) normalized to β-actin were measured by qRT-PCR. Data are presented as mean plus SD. Error bars represent the variation range of duplicate experiments. The data are representative of three independent experiments. **, P < 0.01; ***, P < 0.001.
FIG 7
FIG 7
IFN-β induction by KSHV ORF810420-10496 RNA is dependent on RIG-I but not on RNA Pol III. (A) HEK293 cells in 24-well plates were transfected with siRNAs targeting RIG-I or STING. At 48 h posttransfection, cells were transfected with 5′-ppp-dsRNA or poly(I·C) (positive controls), the indicated concentration (in micrograms) of ORF810,420-10,496 RNA, or not transfected (no-treatment control). At 24 h after ORF810420-10496 RNA transfection, IFN-β mRNA was measured by qRT-PCR. (B) WT RIG-I and RIG-I knockout MEF in 24-well plates were transfected with 5′-ppp-dsRNA (a positive control) or with the indicated concentration of ORF810420-10496 RNA or not transfected (no-treatment control). At 24 h after transfection, IFN-β mRNA was measured by qRT-PCR. (C) HEK293 cells in 24-well plates were transfected with siRNAs targeting RNA Pol III or RIG-I. At 48 h posttransfection, cells were transfected with poly(I·C) or poly(dA·dT) (positive controls) or with the indicated concentration of ORF810,420-10,496 RNA or not transfected (no-treatment control). At 24 h after ORF810420-10496 RNA transfection, IFN-β mRNA was measured by qRT-PCR. Data are presented as mean ± SD. Error bars represent the variation range of duplicate experiments. The data are representative of three independent experiments. **, P < 0.01; ***, P < 0.001.
FIG 8
FIG 8
KSHV ORF810420-10496 RNA activates both NF-κB and IRF3. HEK293 cells in 24-well plates were transfected with 5′-ppp-dsRNA (a positive control) or with the indicated concentration of ORF810420-10496 RNA or not transfected (no-treatment control). At 24 h after transfection, cellular protein lysates were analyzed by Western blotting for phosphorylated IκB (p-IκB) and IRF-3 (p-IRF3), total IκB and IRF-3, and actin. The data are representative of two independent experiments.
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