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. 2018 Nov 19;9(1):4841.
doi: 10.1038/s41467-018-07314-7.

RIG-I like receptor sensing of host RNAs facilitates the cell-intrinsic immune response to KSHV infection

Yang Zhao  1 , Xiang Ye  1 , William Dunker  1 , Yu Song  1   2 , John Karijolich  3   4   5
Affiliations

RIG-I like receptor sensing of host RNAs facilitates the cell-intrinsic immune response to KSHV infection

Yang Zhao et al. Nat Commun. .

Abstract

The RIG-I like receptors (RLRs) RIG-I and MDA5 are cytosolic RNA helicases best characterized as restriction factors for RNA viruses. However, evidence suggests RLRs participate in innate immune recognition of other pathogens, including DNA viruses. Kaposi's sarcoma-associated herpesvirus (KSHV) is a human gammaherpesvirus and the etiological agent of Kaposi's sarcoma and primary effusion lymphoma (PEL). Here, we demonstrate that RLRs restrict KSHV lytic reactivation and we demonstrate that restriction is facilitated by the recognition of host-derived RNAs. Misprocessed noncoding RNAs represent an abundant class of RIG-I substrates, and biochemical characterizations reveal that an infection-dependent reduction in the cellular triphosphatase DUSP11 results in an accumulation of select triphosphorylated noncoding RNAs, enabling their recognition by RIG-I. These findings reveal an intricate relationship between RNA processing and innate immunity, and demonstrate that an antiviral innate immune response can be elicited by the sensing of misprocessed cellular RNAs.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Knockdown of RLRs and MAVS enhances KSHV lytic reactivation in iSLK.219 cells. a Schematic of RLR-MAVS signaling pathway. b iSLK.219 cells were transfected with indicated siRNAs for 48 h and then treated with Dox for 72 h. GFP and RFP were imaged 48 h post-Dox treatment. Bar indicates 750 μm. c RFP positive cells were quantified by flow cytometry 48 h post-Dox treatment. d RNA extracted from iSLK.219 24 h post-reactivation and expression of the indicated genes was quantified by RT-qPCR. e Western blot analysis of cell lysate from latent and 48 h post-Dox treatment iSLK.219 cells described in (b). f HEK293T cells were infected with supernatants of reactivated iSLK.219. GFP images were captured 48 h postinfection. Bar indicates 300 μm. g Quantification of GFP positive cells in (f). h RNA was extracted from HEK293T cells in (f) and KSHV LANA gene expression was monitored by RT-qPCR. i Western blot analysis of cell lysates from latent and 24 h post-Dox treatment iSLK.219 cells in (b). Error bars in all panels represent mean ± SD from three independent experiments. p Values were determined by the Student’s t test, *p < 0.05, **p < 0.01
Fig. 2
Fig. 2
Overexpression of MDA5 or RIG-I restricts KSHV lytic reactivation in iSLK.219 cells. a iSLK.219 F-RIG-I, F-MDA5, and Control (Con) cells were reactivated for 72 h at which time GFP and RFP images were captured. Bar indicates 750 μm. b RNA was extracted from cells in (a) and expression of the indicated viral genes was quantified by RT-qPCR. c Western blot analysis of cell lysates prepared from cells described in (a). d HEK293T cells were infected with supernatants from cells described in (a). GFP images were taken 48 h postinfection. Bar indicates 300 μm. e Quantification of GFP-positive cells in (d). f RNA was extracted from HEK293T cells in (d). Expression of KSHV LANA was quantified by RT-qPCR. Error bars in all panels represent mean ± SD from three independent experiments. p Values were determined by the Student’s t test, *p < 0.05, **p < 0.01
Fig. 3
Fig. 3
RLRs-MAVS signaling pathway restricts KSHV lytic reactivation in BC-3 cells. a RNA was extracted from BC-3 cells expressing the indicated shRNAs and the target mRNA level was quantified by RT-qPCR. b Expression of KSHV viral genes in cells described in (a) was quantified by RT- qPCR. c BC-3 F-RIG-I, F-MDA5, and Control (Con) cells were reactivated for 48 h. Cell lysates from latent and lytic cells were analyzed by immunoblotting for the indicated proteins. d RNA was extracted from BC-3 cells described in (c) and expression of the indicated viral genes was quantified by RT-qPCR. Error bars in all panels represent mean ± SD from three independent experiments. p-values were determined by the Student’s t test, *p < 0.05, **p < 0.01
Fig. 4
Fig. 4
RIG-I and MDA5 bind host RNAs during lytic reactivation in PEL. a HCT116 dual cells were transfected with RNA purified from latent and lytic BC-3 cells either by IgG or J2 antibody. Cells were harvested 24 h posttransfection and subjected to luciferase assay. Mock indicates no antibody. b Pie chart representation of gene biotypes identified by F-RIG-I and F-MDA5 fRIP-seq. c PCA analysis. d, e The gene ontology overrepresentation analysis of enriched RNAs from F-MDA5 (d) and F-RIG-I (e) fRIP-seq. f Distribution of MDA5 fRIP-seq reads on the NOP14 locus. g fRIP-qPCR analysis of NOP14 and GINS. h Distribution RIG-I fRIP-seq reads at the vtRNA loci. i fRIP-qPCR analysis of vtRNAs. Error bars in all panels represent mean ± SD from three independent experiments. p Values were determined by the Student’s t test, *p < 0.05, **p < 0.01
Fig. 5
Fig. 5
Accumulation of immunostimulatory 5′-ppp-vtRNAs during lytic reactivation. a Predicted secondary structure of vtRNAs generated by RNAfold. b SYBR-Gold staining of in vitro transcribed vtRNAs with or without CIP treatment. c HCT116 ISG54-luciferase reporter cells were transfected with 100 ng in vitro transcribed vtRNAs with or without CIP treatment. Cells were harvested 24 h posttransfection and subjected to luciferase assay. Mock indicated cells without RNA transfection and was set as 1. d BC-3 cells were reactivated for 3 days and expression of DUSP11 was quantified by RT-qPCR. L latency, D1–D3 lytic reactivation for 1 day to 3 days. The DUSP11 expression was normalized to the level of 18S rRNA and L was set as 1. e Cell lysates were prepared from BC-3 cells described in (d) and DUSP11 protein levels were monitored by Western blot. GAPDH was run as a loading control. f Latent and lytic BC-3 cells were subjected to RNAP II ChIP-qPCR analysis. Signals were normalized to input. g Total RNA, extracted from latent or 72 h postreactivation BC-3 cells, was subjected to splint-ligation to quantify 5′-monophosphorylated vtRNAs. * denotes a product of adapter-adapter ligation (see Supplementary Fig. 7). h HCT116 ISG54-luciferase reporter cells were transfected with vtRNA or U1 RNA isolated by antisense oligonucleotide affinity selection from either latent or lytic BC-3 cells. Cells were harvested 12 h posttransfection and subjected to luciferase assay. Mock indicated cells without RNA transfection and was set as 1. Error bars in all panels represent mean ± SD from three independent experiments. p Values were determined by the Student’s t test, *p < 0.05, **p < 0.01
Fig. 6
Fig. 6
5′-triphosphate containing vtRNAs block KSHV lytic reactivation. a iSLK.219 cells were mock transfected, or transfected with 100 ng in vitro transcribed vtRNAs with or without CIP treatment, or a RIG-I ligand RNA (3pRNA) and reactivated by adding Dox 4 h posttransfection. GFP and RFP images were captured 48 h postreactivation. Bar indicates 300 μm. b Quantification of RFP positive cells in (a). c Expression of the indicated viral genes was determined in latent and 48 h post-Dox treatment cells by RT-qPCR. d Expression of the indicated genes was quantified in latent and 24 h post-Dox treatment cells by RT-qPCR. c, d The gene expression was normalized to the level of 18S rRNA and Mock in latent cells was set as 1. Error bars in all panels represent mean ± SD from three independent experiments. p Values were determined by the Student’s t test, *p < 0.05, **p < 0.01. e The model depicting how RLRs-MAVS pathway is activated and restricts KSHV lytic reactivation. In latency, DUSP11 removes 5′ triphosphates of vtRNAs, thus preventing their recognition by RIG-I. During lytic reactivation, 5′-end processing of vtRNAs is attenuated due a reduction in DUSP11 expression. 5′-ppp-vtRNAs and long dsRNAs are sensed by RIG-I and MDA5, respectively. RLRs elicit an antiviral gene expression program through MAVS and downstream phosphorylation of IRF3
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