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. 2022 Apr 13;96(7):e0196221.
doi: 10.1128/jvi.01962-21. Epub 2022 Mar 10.

RACK1 Associates with RNA-Binding Proteins Vigilin and SERBP1 to Facilitate Dengue Virus Replication

Alexis Brugier #  1 , Mohamed Lamine Hafirrassou #  1 , Marie Pourcelot  1 , Morgane Baldaccini  2 , Vasiliya Kril  1 , Laurine Couture  1 , Beate M Kümmerer  3 , Sarah Gallois-Montbrun  4 , Lucie Bonnet-Madin  1 , Pierre-Olivier Vidalain  5 , Constance Delaugerre  1   6 , Sébastien Pfeffer  2 , Laurent Meertens  1 , Ali Amara  1
Affiliations

RACK1 Associates with RNA-Binding Proteins Vigilin and SERBP1 to Facilitate Dengue Virus Replication

Alexis Brugier et al. J Virol. .

Abstract

Dengue virus (DENV) is a mosquito-borne flavivirus responsible for dengue disease, a major human health concern for which no effective treatment is available. DENV relies heavily on the host cellular machinery for productive infection. Here, we show that the scaffold protein RACK1, which is part of the DENV replication complex, mediates infection by binding to the 40S ribosomal subunit. Mass spectrometry analysis of RACK1 partners coupled to an RNA interference screen-identified Vigilin and SERBP1 as DENV host-dependency factors. Both are RNA-binding proteins that interact with the DENV genome. Genetic ablation of Vigilin or SERBP1 rendered cells poorly susceptible to DENV, as well as related flaviviruses, by hampering the translation and replication steps. Finally, we established that a Vigilin or SERBP1 mutant lacking RACK1 binding but still interacting with the viral RNA is unable to mediate DENV infection. We propose that RACK1 recruits Vigilin and SERBP1, linking the DENV genome to the translation machinery for efficient infection. IMPORTANCE We recently identified the scaffolding RACK1 protein as an important host-dependency factor for dengue virus (DENV), a positive-stranded RNA virus responsible for the most prevalent mosquito-borne viral disease worldwide. Here, we have performed the first RACK1 interactome in human cells and identified Vigilin and SERBP1 as DENV host-dependency factors. Both are RNA-binding proteins that interact with the DENV RNA to regulate viral replication. Importantly, Vigilin and SERBP1 interact with RACK1 and the DENV viral RNA (vRNA) to mediate viral replication. Overall, our results suggest that RACK1 acts as a binding platform at the surface of the 40S ribosomal subunit to recruit Vigilin and SERBP1, which may therefore function as linkers between the viral RNA and the translation machinery to facilitate infection.

Keywords: RACK1; RNA-binding proteins; SERBP1; Vigilin; dengue virus; host factors.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
The interaction between RACK1 and the 40S ribosome is required for dengue virus (DENV) infection. (A) Western blot analysis of RACK1 expression in control, RACK1KO, and RACK1KO HAP1 cells transcomplemented with a hemagglutinin (HA)-RACK1 cDNA. Cell lysates were probed with the indicated antibodies. Shown is a representative Western blot of n = 3 technically independent experiments. (B) Role of RACK1 in DENV infection. Control, RACK1KO, or RACK1KO cells transcomplemented with a cDNA encoding wild-type (WT) HA-RACK1 were infected at different multiplicities of infection (m.o.i) with DENV2-16681. Levels of infection were determined by flow cytometry using the 2H2 prM monoclonal antibody (MAb) at 48 h postinfection (hpi). The data shown are the means ± stardard error of the mean (SEM) of four independent experiments performed in duplicate. Significance was calculated using two-way analysis of variance (ANOVA) with Dunnett’s multiple-comparison test. (C) Western blot analysis of RACK1 expression in RACK1KO HAP1 transcomplemented with cDNA encoding WT HA-RACK1 or the HA-RACK1 D/E mutant (HA-RACK1 DE cDNA). Cell lysates were probed with the indicated antibodies. Shown is a representative Western blot of three independent experiments. (D) Impact of RACK1 association to the 40S subunit of the ribosome in DENV infection. Control, RACK1KO, and RACK1KO HAP1 cells transcomplemented with cDNA encoding WT HA-RACK1 or the HA-RACK1 DE mutant were infected at MOI 1 with DENV2-16681 and harvested at 48 hpi. Levels of infection were determined by flow cytometry as described above. The data shown are the means ± SEM of three independent experiments performed in duplicate. Significance was calculated using one-way ANOVA with Dunnett’s multiple-comparison test. ****, P < 0.0001; n.s, not significant; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; NI, not infected.
FIG 2
FIG 2
Global map of the RACK1 interactome in human cells. (A) Experimental scheme of our RACK1 immunoprecipitation approach. 293T cells expressing RACK1 or HA-RACK1 were lysed, and extracts were purified with anti-HA-coated beads before SDS-PAGE and mass spectrometry (MS) analysis. (B) Histogram indicating statistical enrichment for specific biological processes and cellular components, determined by Gene Ontology (GO) analysis. (C) Interaction network of RACK1-associated proteins identified by MS in 293T cells. Proteins were clustered into functional modules using enriched GO terms as a guideline and manual mining of literature. This panel is a representative network of n = 3 independent experiments showing similar results. LC-MS/MS, liquid chromatography-tandem MS.
FIG 3
FIG 3
RNA interference (RNAi) screen-identified Vigilin, SERBP1, and ZNF598 are DENV host-dependency factors. (A) Host-dependency factors (HDFs) found in our RNAi screen. The data shown are representative of three independent experiments. Host-dependency factors are marked in green. The positive control (small interfering RNA [siRNA] pool targeting RACK1) is highlighted in blue. (B) Validation of the interaction between RACK1 and endogenous Vigilin or SERBP1 in 293T cells by immunoprecipitation. Cell extracts from 293T cells expressing RACK1 or HA-RACK1 were subjected to affinity purification using anti-HA beads, and interacting proteins were revealed by Western blotting. The data shown are representative of three independent experiments. (C) Human primary fibroblasts were transfected with the indicated siRNA pools. RACK1, Vigilin, SERBP1, and ZNF598 expression in siRNA transfected cells was assessed by Western blot analysis 48 h posttransfection. (D) The viability of siRNA transfected fibroblasts described in B was monitored by cell titer glow analysis. The data shown are the means ± SEM of three independent experiments performed in triplicate. Significance was calculated using two-way ANOVA with Dunnett’s multiple-comparison test. (E) siRNA transfected fibroblasts described for panel B were challenged with DENV2-16681 at MOI 1. At 48 h posttransfection, the levels of infection were determined by flow cytometry using 2H2 MAb at 48 hpi. The data shown are the means ± SEM of three independent experiments performed in duplicate. Significance was calculated using one-way ANOVA with Dunnett’s multiple-comparison test. RLU, relative light units; siNT, nontargeting siRNA.
FIG 4
FIG 4
Impact of RACK1, Vigilin, SERBP1, and ZNF598 gene editing on infection by DENV and other enveloped viruses. (A) Sanger sequencing of VIGILIN, SERBP1, and ZNF598 in control and VigilinKO, SERBP1KO, or ZNF598KO HAP1 cells, respectively. (B) Validation of Vigilin, SERBP1, and ZNF598 gene editing by Western blot analysis. Shown is a representative Western blot of three independent experiments. (C) Impact of RACK1, Vigilin, SERBP1, and ZNF598 gene editing on cell viability in HAP1 cells by cell titer glow analysis. The data shown are the means ± SEM of three independent experiments performed in duplicate. Significance was calculated using two-way ANOVA with Dunnett’s multiple-comparison test. (D to G) Impact of RACK1/Vigilin/SERBP1/ZNF598 gene editing on DENV infectious cycle. The indicated cells were infected for 48 h at MOI 1 with DENV2-16681. (D) Supernatants from infected cells were harvested, and then the titer was determined by flow cytometry on Vero cells and expressed as fluorescence-activated cell sorter infectious unit (FIU)/mL. (E) Infection was assessed by immunoblot using anti-NS3, anti-prM, and anti-E DENV MAbs. The data shown are representative of three independent experiments. (F) Levels of infection were assessed by quantification of DENV viral RNA (vRNA) by quantitative reverse transcription-PCR using NS3 primers. The data shown are the means ± SEM of three independent experiments performed in duplicate. Significance was calculated using one-way ANOVA. (G) The indicated cells were infected with Zika virus (ZIKV) HD78 at MOI 2 (left), chikungunya virus (CHIKV) 21 at MOI 2 (middle), and vesicular stomatitis virus G protein-pseudotyped human immunodeficiency virus (VSV-pp) at MOI 2 (right). Levels of infection were determined by flow cytometry at 48 hpi. The data shown are the means ± SEM of at least two independent experiments performed in duplicate. Significance was calculated using one-way ANOVA with Dunnett’s multiple-comparison test. n.s, not significant; ****, P < 0.0001.
FIG 5
FIG 5
Vigilin and SERBP1 regulate DENV translation and replication. (A) The indicated cells were infected at MOI 1 with DENV-Luc. At the indicated time points, Renilla luciferase activity reflecting RNA translation (1 to 8 hpi) and replication (12 to 72 hpi) was measured. The data shown are the means ± SEM of three independent experiments performed in triplicate. Significance was calculated using two-way ANOVA with Dunnett’s multiple-comparison test. (B) The indicated cells were infected at MOI 1 with CHIKV-Luc. Gaussia luciferase activity was monitored at the indicated time points. The data shown are the means ± SEM of three independent experiments performed in triplicate. Significance was calculated using two-way ANOVA with Dunnett’s multiple-comparison test. (C) Impact of RACK1/Vigilin/SERBP1 KO on DENV life cycle in HAP1 cells transfected with a DENV replicon RNA expressing Renilla luciferase. Renilla luciferase activity was monitored at the indicated time point. The data shown are the means ± SEM of three independent experiments performed in triplicate. Significance was calculated using two-way ANOVA with Dunnett’s multiple-comparison test. n.s, not significant; ****, P < 0.0001; AU, arbitrary units; Gluc, Gaussia luciferase; Rluc, Renilla luciferase.
FIG 6
FIG 6
Northern blot analysis of the impact of RACK1, Vigilin, and SERBP1 knockout on DENV genomic RNA (gRNA) stability. (A) Indicated cells were infected at an MOI of 1 with DENV2-16681. Total RNA was extracted 48 h.p.i. at the indicated time after treatment with MK0608 replication inhibitor. The data shown are the means ± SEM of three independent experiments performed in triplicate. (B) vRNA stability is expressed as a percentage relative to the signal monitored at time point 0 h after MK0608 treatment. Ethidium bromide serves as a loading control, showing 28S and 18S rRNA. Statistics were performed using two-way ANOVA. hpt, h posttransfection; ns, nonsignificant.
FIG 7
FIG 7
Characterization of Vigilin and SERBP1 mutants (Mut) deficient for RACK1 binding. (A) Schematic representations of Vigilin mutant (upper diagram) and SERBP1 mutant (lower diagram) constructs. (B) Evaluation of FLAG-Vigilin mutant (left) or Myc-SERBP1 mutant (right) interaction with RACK1. Cell extracts from 293T expressing the WT or mutated forms of Vigilin and SERBP1 were subjected to affinity purification using anti-FLAG- or -Myc-coated beads, respectively. Input and eluates were resolved by SDS-PAGE, and interacting proteins were revealed by Western blotting using corresponding antibodies. Shown is a representative Western blot of three independent experiments. (C) Analysis of Vigilin (WT and Mut) and SERBP1 (WT and Mut) interactions with the DENV RNA by RNA immunoprecipitation assay (RIP). The cells were infected at MOI 1 by DENV2-16681 and harvested 48 hpi. Tagged proteins were immunoprecipitated after UV cross-link at 254 nm using anti-FLAG- or -Myc-coated beads. The enrichment of DENV RNA or Actin RNA over the negative-control condition were determined by quantitative reverse transcription-PCR using specific primers and quantified using the ΔΔCt method. The data shown are the means ± SEM of three independent experiments performed in triplicate. Significance was calculated using a two-way ANOVA with Dunnett’s multiple-comparison test. Cter, C terminus; Nter, N terminus; n.s, not significant; ****, P < 0.0001.
FIG 8
FIG 8
Vigilin and SERBP1 interaction with RACK1 is important in DENV infection. (A) Stable expression of Vigilin WT or Mut and SERBP1 WT or Mut in Vigilin KO or SERBP1 KO HAP1 cells, respectively. Western blot analysis of Vigilin or SERBP1 expression is shown. The data shown are representative of three independent experiments. (B) The indicated cells were infected at MOI 1 with DENV2-16681. Levels of infection were determined by flow cytometry at 48 hpi using the 2H2 MAb. The data shown are the means ± SEM of three technically independent experiments performed in duplicate. Significance was calculated using one-way ANOVA with Dunnett’s multiple-comparison test. n.s, not significant; ****, P < 0.0001.
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