Dengue subgenomic RNA binds TRIM25 to inhibit interferon expression for epidemiological fitness

Abstract

The global spread of dengue virus (DENV) infections has increased viral genetic diversity, some of which appears associated with greater epidemic potential. The mechanisms governing viral fitness in epidemiological settings, however, remain poorly defined. We identified a determinant of fitness in a foreign dominant (PR-2B) DENV serotype 2 (DENV-2) clade, which emerged during the 1994 epidemic in Puerto Rico and replaced an endemic (PR-1) DENV-2 clade. The PR-2B DENV-2 produced increased levels of subgenomic flavivirus RNA (sfRNA) relative to genomic RNA during replication. PR-2B sfRNA showed sequence-dependent binding to and prevention of tripartite motif 25 (TRIM25) deubiquitylation, which is critical for sustained and amplified retinoic acid-inducible gene 1 (RIG-I)-induced type I interferon expression. Our findings demonstrate a distinctive viral RNA-host protein interaction to evade the innate immune response for increased epidemiological fitness.

Fig. 1. sfRNA is a critical determinant of the epidemic potential of DENV-2

(A) Plaque titers of PR-1 and PR-2B DENV-2 viruses at 24 hpi in HuH-7 cells infected at a multiplicity of infection (MOI) of 0.1. PFU, plaque-forming units. (B) PR-1 and PR-2B gRNA in infected HuH-7 cells (MOI = 0.1), 24 hpi. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C) PR-1 and PR-2B sfRNA in infected HuH-7 cells (MOI = 0.1), 24 hpi. (D) Ratio of sfRNA:gRNA in HuH-7 cells infected with PR-1 and PR-2B viruses at MOI 0.1, 24 hpi. (E) Quantification of sfRNA:gRNA ratios in HuH-7 cells at 48 hpi with clinical isolates from Nicaragua. Numbers below the bars refer to the names of the viral isolates. Data are expressed as mean ± SD from three independent experiments. **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, as determined by t test.

Fig. 2. PR-2B DENV-2 replication is associated with reduced expression of IFN-β relative to PR-1 DENV-2

(A) Plaque titers after infection of HuH-7 cells with selected viruses from each PR clade at 24 and 96 hpi. (B) DENV-2 gRNA levels in infected HuH-7 cells after infection (MOI = 0.5) with selected viruses from each clade at various time points postinfection. (C to F) Expression levels of IFN-β in infected HuH-7 cells at 24 hpi (C), 48 hpi (D), 72 hpi (E), and 96 hpi (F). PR-1: blue bars; PR-2B: red bars. (G) Plaque titers of supernatant quantified at 24 hpi with representative viruses from each clade, following knockdown of IRF-3 in HuH-7 cells by small interfering RNA (siRNA). siC: control RNA; siIRF-3: siRNA against IRF3. (H) Quantification of DENV-2 gRNA in infected primary monocytes at 24 hpi. Infection was performed with selected PR-1 and PR-2B DENV-2 viruses opsonized with enhancing concentrations of humanized 3H5 monoclonal antibody against DENV-2 E protein. (I) Quantification of sfRNA:gRNA ratios in primary monocytes, 24 hpi. (J) IFN-β expression in primary monocytes at 24 hpi. (K) Plaque titers from supernatant of infected primary monocytes at 72 hpi. Data are expressed as mean ± SD from three independent experiments. n.s.: not significant; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, as determined by t test.

Fig. 3. sfRNA attenuates type I interferon antiviral response, resulting in increased viral replication

(A) Quantification of gRNA levels and sfRNA: gRNA ratios in HuH-7 cells, 72 hours after electroporation with replicons bearing nucleotide residues representative of PR-1 or PR-2B DENV-2. Bars show the gRNA levels (left axis), and lines show sfRNA:gRNA ratios (right axis). (B) Luciferase reporter activity for the original replicon (pDENrep-FH) and mutated replicons at various time points after electroporation. Determination of firefly luciferase (FLuc) levels served as internal normalization controls. (C) Quantification of IFN-β and NF-κB expression using quantitative reverse transcription–polymerase chain reaction (QRT-PCR) at 96 hours after electroporation and (D) luciferase reporter activity following co-electroporation of pDENrep-FH with control RNA or sfRNA from PR1 or PR2B. FLuc levels served as internal normalization controls. (E) Quantification of IFN-β expression in HuH-7 cells transfected with polyIC only, polyIC and size-matched control RNA, or sfRNA from PR-1 or PR-2B, by QRT-PCR at 24 hours after transfection. Data are expressed as mean ± SD from three independent experiments. n.s.: not significant; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, as determined by t test.

Fig. 4. sfRNA binds TRIM25 to inhibit TRIM25 deubiquitylation

(A) RNA-immunoprecipitation (RIP) was performed on HuH-7 cells at 24 hpi with one virus from each clade (MOI = 5). Infected cells were subjected to TRIM25 or IgG control pull-down followed by immunoblotting with TRIM25 to confirm efficacy of pull-down. Input: cell lysate before RIP; Sup: supernatant after incubation of antibody-coated beads with cell lysate; Pellet: final product after washing. Bar graphs show fold enrichment of sfRNA and gRNA upon pull-down with TRIM25, as measured by QRT-PCR. (B) EMSA of synthetic PR-1 and PR-2B sfRNA (30 nM) incubated with increasing concentrations of TRIM25. Arrow indicates the position of the sfRNA-protein (ribonucleoprotein) complex. Amount of ribonucleoprotein complex was quantified and expressed as a percentage of total RNA signal. (C) Lysates from HuH-7 cells infected with one virus from each clade (MOI = 5) were subjected to TRIM25 or IgG control pull-down followed by immunoblotting with TRIM25 and ubiquitin (UB). Arrow indicates ubiquitylated bands. (D) Bar chart depicts percentage of ubiquitylated TRIM25 over total TRIM25. (E) After transfection with PR-1 or PR-2B sfRNA, HuH-7 cell lysates were subjected to RIG-I pull-down followed by immunoblotting with RIG-I, TRIM25, and UB. (F) PR-1 and PR-2B DENV-2 replication at 24 hpi in TRIM25-silenced HuH-7 cells infected with one virus from each clade. siC: control RNA; siTRIM25: siRNA against TRIM25. Differences in gel electrophoresis conditions could have led to slight variations in mobility of prestained protein markers. Data are expressed as mean ± SD from three independent experiments. n.s.: not significant; *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001, as determined by t test.