An RNA-centric dissection of host complexes controlling flavivirus infection

Abstract

Flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), cause severe human disease. Co-opting cellular factors for viral translation and viral genome replication at the endoplasmic reticulum is a shared replication strategy, despite different clinical outcomes. Although the protein products of these viruses have been studied in depth, how the RNA genomes operate inside human cells is poorly understood. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we took an RNA-centric viewpoint of flaviviral infection and identified several hundred proteins associated with both DENV and ZIKV genomic RNA in human cells. Genome-scale knockout screens assigned putative functional relevance to the RNA-protein interactions observed by ChIRP-MS. The endoplasmic-reticulum-localized RNA-binding proteins vigilin and ribosome-binding protein 1 directly bound viral RNA and each acted at distinct stages in the life cycle of flaviviruses. Thus, this versatile strategy can elucidate features of human biology that control the pathogenesis of clinically relevant viruses.

Fig. 1. ChIRP-MS reveals the protein interactome of DENV and ZIKV RNA genomes.

a, Outline of the ChIRP-MS method. Uninfected, DENV, or ZIKV infected Huh7.5.1 cells were formaldehyde crosslinked and solubilized by sonication. Target viral RNA was pulled-down by biotinylated anti-sense oligonucleotides, associated proteins were eluted, and subjected to LC-MS/MS. b, Cartoon of the topology of the flaviviral polyprotein inserted in the ER membrane. c, and d, Map of DENV (c) and ZIKV (d) genome organizations with corresponding MS spectral counts determined by ChIRP-MS. e, Scatter plot depicting enrichment ratio of host proteins identified by ChIRP-MS with DENV and ZIKV RNA over uninfected background. ChIRP-MS was performed in triplicates for each virus and x- and y-axis represent the mean of Peptide Spectrum-Matches (PSM) scores enrichment over background for DENV and ZIKV, respectively. A total number of 464 enriched proteins were identified for DENV and ZIKV and several of the most enriched hits are indicated in the top panel. The bottom panel demonstrated that 3¼64 enriched proteins are ER-annotated proteins and the remaining 43¾64 proteins are implicated in other non-ER sub-cellular localizations. A full list of the enriched proteins is presented in Supplementary Table 2. f, GO cellular component analysis of high confidence host factors enriched by ChIRP-MS. g, GO protein domain analysis of high confidence host factors enriched by ChIRP-MS. For panel f and g, FDR calculation was performed using the Benjamini Hochberg method on the combined ChIRP-MS enriched hits (n = 1 merged dataset).

Fig. 2. Intersection of ChIRP-MS with genome-wide CRISPR screens nominates functionally relevant pro-viral host proteins.

a, Genome-scale CRISPR knockout screens for all four DENV serotypes (DENV-1^276RKI, DENV-2⁴²⁹⁵⁵⁷, DENV-3^{Philippines/H871856}, and DENV-4^BC287/97) in Huh7.5.1 cells. The genetic screens were independently performed for each serotype, analyzed with MAGeCK, and combined to obtain significance scores (y-axis). The 50 most enriched genes were colored and grouped by function. b, Scatter plot depicting enrichment scores of high-confidence ChIRP-MS DENV hits (x-axis) and the 200 top scoring hits from DENV CRISPR genetic screens (y-axis). Common hits shared by both DENV genetic screens and DENV ChIRP-MS were colored in red (vigilin), blue (RRBP1), and purple (others). c, Western blot (WB) analysis of wild-type (WT) or clonal RRBP1 knock-out (KO) (upper panel) and vigilin-KO (lower panel) cells in Huh7.5.1 cells. Representative WB of n = 2 biologically independent replicates showing similar results. d, qRT-PCR analysis of DENV infected WT and RRBP1-KO Huh7.5.1 cells (48 hours post-infection (hpi), MOI of 0.1) or ZIKV^PRVABC59, POWV^LB (48 hpi, MOI 0.1) and CHIKV^18½5 (24 hpi, MOI of 0.01) infected WT and RRBP1-KO Huh7.5.1 cells. e, qRT-PCR analysis as in (d) here with vigilin-KO. Note: The WT datasets for POWV in panel d and e derived from the same experiments. f, WB analysis of DENV-2⁴²⁹⁵⁵⁷ infected (MOI of 0.1, 72 hpi) WT, RRBP1-KO, and vigilin-KO Huh7.5.1 cell lysates, probed with DENV prM and NS3 antibodies. Representative WB of n = 4 biologically independent replicates showing similar results. g, Titers of infectious particles production from WT, RRBP1-KO, and vigilin-KO Huh7.5.1 cells infected with DENV-2⁴²⁹⁵⁵⁷ at MOI of 0.1 for 72 h. For panel d, e, and g, the datasets represent the mean with standard error of the mean (SEM) of n = 3 independent biological replicates, except for POWV, n = 4 independent biological replicates. All P-values were determined by two-tailed, unpaired t-test using GraphPad Prism (GraphPad Software), where * = P <0.05 and n.s. = non-significant.

Fig. 3. RRBP1 and vigilin interact at the endoplasmic reticulum.

a, Single cell quantification and correlation between, RRBP1, vigilin, ER-GFP (an ER marker), and 4′,6-diamidino-2-phenylindole (DAPI) immunofluorescence signals. Total number of cells that were randomly chosen for each analysis, with mean and SEM are indicated. b, Western blot analysis of ER and cytosolic cell fractions probed with GAPDH or Tubulin (cytosolic markers), RPN1 (ER marker), RRBP1, and vigilin antibodies. Representative WB of n = 3 biologically independent replicates showing similar results. c, Western blot analysis of three independent co-IP experiments from non-infected Huh7.5.1 cells with RRBP1 as the bait showing similar results. Samples were treated with or without RNase A. d, and e, Representative IF of RRBP1 (d), vigilin (e) co-stained with RNA fluorescent in situ hybridization targeting (RNA-FISH) of DENV or ZIKV positive stranded RNA genomes. Representative images of n = 2 biologically independent replicates showing similar results. Scale bars, 10 μm.

Fig. 4. DENV and ZIKV co-opt the RNA binding properties of RRBP1 and vigilin in human cells.

a, RRBP1 (left) and vigilin (right) irCLIP reverse transcriptase (RT) stop mapping statistics annotated to the human, DENV, or ZIKV genomes and the ribosomal RNAs (rRNA) from Huh7.5.1 cells infected with MOI of 0.1 for 48 h. b, Histogram of RT stops mapping to the rRNAs from the RRBP1 (top) and vigilin (bottom) irCLIP in uninfected Huh7.5.1 cells. The three cytosolic rRNAs are highlighted. Red dashed line denotes vigilin’s strongest binding site, which is adjacent to RRBP1’s. c, Annotation of peaks called from RRBP1 (top) and vigilin (bottom) irCLIP RT stops mapping to functional elements of human mRNAs including 5’UTR, exons, 3’UTR, and introns. Enrichment values are calculated based on the size of each function domain relative to the human genome. d, RRBP1 (top) and vigilin (bottom) irCLIP RT stops mapped at base resolution to the DENV genome. RT stop intensity was normalized to the total number of unique reads mapping to the viral genome. The 5’UTR and 3’UTR regions are highlighted in red and blue, respectively.

Fig. 5. RRBP1 and vigilin modulate DENV translation and replication.

a, and b, Time-course DENV-Luc infection assays. WT, RRBP1-KO, and RRBP1-KO + RRBP1 cDNA rescue (a) or WT, vigilin-KO, and vigilin-KO + vigilin cDNA rescue (b) HEK293FT cells were infected with DENV-Luc (MOI of 0.01) and harvested at indicated time points. Virus infectivity was then determined by measuring Renilla luciferase expression from infected cells. c, and d, Time-course CVB3-Luc infection assays. WT, RRBP1-KO, and RRBP1-KO + RRBP1 cDNA rescue (c) or WT, vigilin-KO, and vigilin-KO + vigilin cDNA rescue (d) HEK293FT cells were infected with CVB3-Luc (MOI of 1) and harvested at indicated time points. e, and f, Luciferase expression of luciferase-encoding DENV replicon RNA in WT and RRBP1-KO (e) or WT and vigilin-KO (f) HEK293FT cells over indicated time points post-electroporation of replicon RNA. The data in each panel (a-f) represent the mean with SEM of of n = 3 independent biological replicates. g, Luciferase expression 8 hours (left) post-DENV-Luc (MOI of 0.025) infection in the presence of the replication inhibitor MK0608 (50 μM final concentration) or 36 hours (right) post DENV-Luc infection in the presence of DMSO of WT, RRBP1-KO, and vigilin-KO HEK293FT cells. The data in each panel represents the mean with SEM of 10 biologically independent infections. Fold change between datasets is indicated. All P-values stated in this figure were determined by two-tailed, unpaired t-test using GraphPad Prism, where * = P < 0.05 and n.s. = non-significant.

Fig. 6. RRBP1 and vigilin promote DENV infection and viral RNA stability.

a, Western blot analysis of WT Huh7.5.1, vigilin-KO, RRBP1-KO, and RRBP1-vigilin double-KO cells. Representative WB of n = 2 biologically independent replicates showing similar results. b, Luciferase expression at 8 hpi and 24 hpi upon DENV-Luc infection (MOI 0.01) of WT Huh7.5.1, vigilin-KO, RRBP1-KO, and RRBP1-vigilin double-KO cells. The data in each panel represent the mean and SEM of 9 biologically independent infections. The P-values were determined by two-tailed, unpaired t-test using GraphPad Prism, where * = P < 0.05 and n.s. = non-significant. c, Northern blot analysis of dengue genomic RNA extracted from WT Huh7.5.1 and RRBP1-vigilin double-KO cells that were first infected with DENV-2¹⁶⁶⁸¹ (MOI of 0.1) for 48 hours, followed by MK0608 replication inhibitor treatment for indicated time frames (top panel). Quantification of DENV genomic RNA (i.e. northern blot signal) from 3 independent experiments (error bars are SEM) as a percentage relative to time point 0 hour after MK0608 treatment (bottom).