Y RNAs are conserved endogenous RIG-I ligands across RNA virus infection and are targeted by HIV-1

Abstract

Pattern recognition receptors (PRRs) protect against microbial invasion by detecting specific molecular patterns found in pathogens and initiating an immune response. Although microbial-derived PRR ligands have been extensively characterized, the contribution and relevance of endogenous ligands to PRR activation remains overlooked. Here, we characterize the landscape of endogenous ligands that engage RIG-I-like receptors (RLRs) upon infection by different RNA viruses. In each infection, several RNAs transcribed by RNA polymerase III (Pol3) specifically engaged RLRs, particularly the family of Y RNAs. Sensing of Y RNAs was dependent on their mimicking of viral secondary structure and their 5'-triphosphate extremity. Further, we found that HIV-1 triggered a VPR-dependent downregulation of RNA triphosphatase DUSP11 in vitro and in vivo, inducing a transcriptome-wide change of cellular RNA 5'-triphosphorylation that licenses Y RNA immunogenicity. Overall, our work uncovers the contribution of endogenous RNAs to antiviral immunity and demonstrates the importance of this pathway in HIV-1 infection.

Keywords: Biological sciences; Immunology; Transcriptomics.

Graphical abstract

Figure 1

A differential affinity screen identifies Y RNAs and other POL3 RNAs as RIG-I ligands mobilizable during RNA virus infection (A) Promoter IFN-β-luciferase reporter activity in WT or MAVS^−/− (left) 293 cells infected with measles virus (MV) or dengue virus 4 (DV-4) at MOI of 1 and 0.5, respectively, or (right) 293-4x4 co-cultivated with HIV-1-infected MT4C5 at a ratio of MT4C5:293-4x4 of 1:1. 5′-PPP is a short in vitro transcribed RNA RIG-I agonist transfected at a concentration of 10 ng/mL. (B) Twenty-four hours post-infection with MV, DV-4, after co-culture with HIV-1-infected MT4 or in noninfected (NI) control, sequencing reads were mapped to human genome Hg38. Differential analyses were performed between RIG-I/RNA and Cherry/RNA samples. Genes are represented following their normalized count in cellular RNA (x axis) and their fold enrichment (log2) to RIG-I compared with Cherry control (y axis) (average of three independent replicates). Genes that showed a log2(FC) > 2 and adj-pval<0.05 are represented with larger dot size. Among these, Y RNAs are labeled with red dots vtRNA in brown and other Pol3 transcripts are shown in orange. Canonical Y RNAs and Pol3 transcripts that show enrichment in more than two conditions (virus or NI) are specifically annotated. (C) Venn diagram representing individual transcripts specifically enriched to RIG-I compared with Cherry in any of MV, DV-4, or HIV-1-infected conditions but absent in NI condition. (D) Families of RNA repeats that show specific affinity to RIG-I compared with Cherry in at least one infected or NI condition, computed according to their relative enrichment compared with NI. The name of the repeat (e.g. Zombi_B) and the subfamily to which it belongs (e.g. Mariner) are indicated. (A) Data representative of n = 3 independent experiments. Bars show mean ± SEM of technical triplicates. Student’s t test; ∗p < 0.05; ∗∗∗p < 0.001. (B–D) Enrichment calculated from the mean of n = 3 infection/RLR-purification/sequencing experiments.

Figure 2

RNY4 RIG-I agonist activity is conferred by RNA 5′-PPP moieties and viral-mimicking specific secondary structure (A) Promoter ISRE-luciferase reporter activity in HAP1 cells control (CTL) or knockout (ko) for each individual RLR or downstream adaptor MAVS, transfected with 30 ng/mL of IVT Y RNA (RNY1, RNY3-5), 10 ng/mL poly(I:C) low- or high-molecular-weight (p(IC) LMW/HMW), 30 ng/mL of total eukaryotic RNA, or treated with 100 U/mL recombinant IFN-β. (B) Promoter ISRE-luciferase reporter activity in HAP1 cells CTL or RIG-I ko transfected with 30 ng/mL IVT RNY4 full length or lacking specific substructure (Figure S2C). RNY4 dephos: RNY4 was additionally pretreated with alkaline phosphatase to remove 5′- triphosphate extremity. (C) RNY1, RNY4, and IFN-β RNA levels were measured by qPCR after transfection of WT or MAVS^−/− 293T with plasmids coding for RNY4 sequence and supplemented with a plasmid coding for RIG-I. U6-RNY4: p2RZ plasmid encoding full-length RNY4 downstream of Pol3 U6 promoter with a ribozyme sequence placed directly in 3’. CMV-RNY4: same plasmid with Pol2 CMV promoter instead of U6 (Table S7). NT: empty plasmid. (D) Probability of thermodynamically stable sequence folding along RNY4 secondary structure in the 5′ end of each transcript, for dataset of human Y RNA families, (+)ssRNA viruses genomes (Flaviviridae or non-Flaviviridae), or human noncoding RNA (ncRNA), mRNAs, and Pol3 transcripts, compared with average probability of the same sequences randomly scrambled. (A–B) Data representative of n = 3 independent experiments. Bars show mean ± SEM of technical duplicates. (C) Bars show mean ± SEM of n = 3 independent experiments. Student’s t test ∗p < 0.05.

Figure 3

HIV-1-dependent downregulation of DUSP11 licenses endogenous 5′-PPP RNAs immunogenicity in infected cells (A) Ratio of 5′-PPP and 5′-P-bearing RNY1 and RNY4 in Jurkat cells 48 h postinfection with HIV-GFP or in noninfected (NI) Jurkat cells. Relative 5′-PPP/5′-P RNA levels were determined through differential enzyme digestion followed by qPCR analysis relative to β-actin mRNA. RN7SL1 and U17b are 5′-PPP and 5′-P RNA controls, respectively. (B) DUSP11 protein levels measured at different times points after Jurkat T cell infection with HIV-GFP. (C) DUSP11 protein levels measured in NI or HIV-GFP-infected productively (GFP+) or nonproductively (GFP−) CD4 primary cells from five different donors 48 h postinfection. CD4 T cells were beads-sorted from total PBMC and activated with phytohemagglutinin-L (PHA) for 72 h prior to infection with HIV-GFP. Forty-eight hours postinfection, productively infected cells were FACS sorted according to GFP expression. DUSP11 protein levels are quantified relative to GAPDH (see also Figure S3C). (D and E) 5’-PPP RNA sequencing of Jurkat CTL or DUSP11^−/− cells. Individual genes (D) and repeats (E) are plotted according to the average percentage of their 5′-PPP subsets in three Jurkat control clones (x axis) or DUSP11^−/− clones (y axis). Positions of RN7SL1 and U17b genes are indicated, representing 5′-PPP and 5′-P RNA controls, respectively. (F) RNY4 RNA enrichment on RIG-I in DUSP11^−/− cells. RNY4 RNA level were measured by qPCR from total RNA, RIG-I-bound, and Cherry-bound fractions in 293 expressing ST-RIG-I or ST-Cherry and either WT, deficient for DUSP11 or after infection with MV. RIG-I binding is computed by measuring the abundance of RNY4 bound to RIG-I compared with protein control Cherry, after normalization to the total cellular RNA abundance in cells overexpressing RIG-I or Cherry, relative to GAPDH mRNA levels. Data are represented in Log2 FC compared with WT NI condition. (G) Heatmap of qPCR values measuring expression level of a panel of IFN-I stimulated genes in Jurkat control, DUSP11^−/−, or control treated overnight with recombinant IFN-β. Expression levels are normalized to β-actin mRNA levels and to Jurkat control. Genes with significant enrichments in DUSP11^−/− cells compared with control are indicated with stars. (A) Bars show mean ± SEM of n = 3 independent experiments. Student’s t test; ∗p < 0.05. (B) Western blot representative of n = 3 independent experiments. (C) Bars show mean ± SEM of n = 5 donors. (F) Bars show mean ± SEM of n = 5 independent experiments. (G) Heatmap shows mean of three control and three DUSP11^−/− Jurkat clones. (H) Bars show mean ± SEM of n = 3 experimental replicates. (G) Student’s paired t test; ∗p < 0.05, ∗∗p < 0.01.

Figure 4

HIV-1 VPR induces DUSP11 downregulation and subsequent increase of endogenous 5’-PPP RNAs (A) DUSP11 protein levels measured at 6 and 48 h after Jurkat T cell infection with WT NL4.3 HIV-1 or the same clone deleted for VPR protein. (B) DUSP11 protein levels in FACS-sorted Jurkat cells 72 h following transduction with lentiviruses coding for ovalbumin (OVA) used as a control, HIV-1 VPR WT, or an HIV-1 VPR(Q65R) defective mutant. UNG2 serves as control of a VPR target downregulated by Q65R mutant (Langevin et al., 2009). UNG1 is a product detected by the same antibody that is not targeted by VPR. (C) DUSP11 protein levels 48 h after HIV-1 infection in Jurkat cells treated with antiretroviral inhibitor or nontreated (NT). T-20: fusion inhibitor enfuvirtide (5 uM); AZT: reverse transcriptase inhibitor zidovudine (1 mM); Ral: integrase inhibitor raltegravir (10 uM). (D) 5’-PPP RNA sequencing of FACS-sorted Jurkat 48 h after infection with HIV-GFP WT or HIV-GFP ΔVPR. Repeats are plotted according to their increase in 5′ triphosphorylation between infected and noninfected cells. Repeats with significant changes in 5′-PPP status upon HIV WT infection are plotted together with corresponding values upon HIV-ΔVPR infection (average of three infection replicates each). (E) Promoter IFN-β-luciferase reporter activity in 293-4x4 after co-culture with HIV-GFPWT- or HIV-GFPdVPR-infected MT4C5. 293-4x4 without co-culture (NT) or co-cultured with noninfected (NI) MT4 serve as negative control, 293-4x4 transfected with 10 ng/ul of poly(I:C) low molecular weight (LMW) serve as positive control. The induction of IFN-β-luciferase is shown as fold change to NT control. (F) Relative quantification of DUSP11 protein level in CD4 T cells from HIV + patients prior to and after antiretroviral treatment. Paired t test; ∗p < 0.05. See also Figure S4C. (A and C) Numbers at the bottom indicate semi-quantification of relative DUSP11/GAPDH levels normalized to NI conditions. (A, B, and C) Western blot representative of n = 2 independent experiments. (E) Mean of three independent experiments.