Identification and characterization of host proteins bound to dengue virus 3' UTR reveal an antiviral role for quaking proteins

Abstract

The four dengue viruses (DENV1-4) are rapidly reemerging infectious RNA viruses. These positive-strand viral genomes contain structured 3' untranslated regions (UTRs) that interact with various host RNA binding proteins (RBPs). These RBPs are functionally important in viral replication, pathogenesis, and defense against host immune mechanisms. Here, we combined RNA chromatography and quantitative mass spectrometry to identify proteins interacting with DENV1-4 3' UTRs. As expected, RBPs displayed distinct binding specificity. Among them, we focused on quaking (QKI) because of its preference for the DENV4 3' UTR (DENV-4/SG/06K2270DK1/2005). RNA immunoprecipitation experiments demonstrated that QKI interacted with DENV4 genomes in infected cells. Moreover, QKI depletion enhanced infectious particle production of DENV4. On the contrary, QKI did not interact with DENV2 3' UTR, and DENV2 replication was not affected consistently by QKI depletion. Next, we mapped the QKI interaction site and identified a QKI response element (QRE) in DENV4 3' UTR. Interestingly, removal of QRE from DENV4 3' UTR abolished this interaction and increased DENV4 viral particle production. Introduction of the QRE to DENV2 3' UTR led to QKI binding and reduced DENV2 infectious particle production. Finally, reporter assays suggest that QKI reduced translation efficiency of viral RNA. Our work describes a novel function of QKI in restricting viral replication.

Keywords: QKI; RNA elements; dengue virus; host factors; translation.

FIGURE 1.

Identification of DENV1-4 3′ UTR interacting proteins. (A,B) Schematic presentation of SILAC-based quantitative mass spectrometry following RNA affinity chromatography. Mass spectrometry data of selected proteins are shown. (C) Representative western blotting results, showing RBPs that interact with indicated DENV RNAs. In vitro transcribed RNA molecules containing tobramycin aptamer sequences and indicated DENV sequences were coupled with tobramycin conjugated sepharose beads. RNA bound beads were mixed with HuH7 cell lysates. After incubation, beads were washed and eluted. Resultant eluates were probed for indicated proteins using immunoblotting. (ND) Not detected.

FIGURE 2.

DENV4 RNA interacts with QKI in infected cells. (A) Input samples and immunoprecipitated materials were analyzed by immunoblotting using antibodies against FLAG tag, showing immunoprecipitated FLAG-tagged QKI-5 and QKI-6, respectively. (B,C) Total RNA was isolated from immunoprecipitated materials and was analyzed by RT-qPCR using primers specific for indicated RNAs. Each experiment, in biological triplicates, was repeated three times. Statistical significance was determined using a two-tailed t-test: (*) P = 0.05; (**) P = 0.01; (***) P = 0.001. Error bars indicate SEM.

FIGURE 3.

QKI depletion enhances viral particle production of DENV4. (A) Representative western blotting results, showing knockdown of QKI in HuH7 cells. (B–E) HuH7 were transfected with nontargeting control siRNA (NTC) or siRNAs targeting QKI (siQKI-1 and siQKI-2) and, two days later, cells were infected with DENV4 at MOI = 0.5 or with DENV2 at MOI = 10. Twenty-four hours post-DENV4 infection and 48 h post-DENV2 infection, supernatants were collected and viral titer was determined by focus formation assay. Cells were harvested for total RNA extraction, and levels of viral RNA accumulation were measured by RT-qPCR using primers that amplify a region in the middle of the viral coding sequence (gRNA). Each experiment, in biological triplicates, was repeated at least three times. Statistical significance was determined using a two-tailed t-test: (**) P = 0.01; (***) P = 0.001. Error bars indicate SEM.

FIGURE 4.

A QRE is important for QKI interaction in DENV 3′ UTR. (A–C) Schematic presentation of DENV 3′ UTR sequences used in RNA chromatography experiments. (D–F) Representative western blotting results showing interaction between indicated RNAs and QKI or DDX6. Indicated RNAs were immobilized on tobramycin sepharose beads, and RNA bound beads were mixed with HuH7 cell lysates. After incubation, beads were washed and eluted with excessive amount of tobramycin. Eluates were analyzed by immunoblotting.

FIGURE 5.

Functional characterization of QRE in DENV 3′ UTR. (A,B) HuH7 cells were infected with wild-type DENV4 viruses (DENV4-WT) or mutant DENV4 viruses (DENV4-mut8) at MOI = 0.5. Twenty-four hours post-infection, supernatants were collected and titer was measured by focus formation assay. Total RNA was isolated from infected cells and analyzed by RT-qPCR using primers that amplify a region in the middle of the viral coding sequence (gRNA). (C,D) HuH7 cells were infected with wild-type DENV2 viruses (DENV2-WT) or mutant DENV2 viruses (DENV2-mut1) at MOI = 10. Two days post-infection, samples were collected and analyzed as described above. (E) HuH7 cells were infected with DENV2 WT or mut1 viruses at MOI = 10. Supernatants were harvested at 1, 48, and 96 h post-infection and titer was measured by focus formation assay. Each experiment, in biological triplicates, was repeated at least two times. Statistical significance was determined using a two-tailed t-test: (**) P = 0.01; (***) P = 0.001. Error bars indicate SEM.