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

Figures

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  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…

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  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…

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  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…

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  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…