Identification of host factors binding to dengue and Zika virus subgenomic RNA by efficient yeast three-hybrid screens of the human ORFeome

Abstract

Flaviviruses such as the dengue (DENV) and the Zika virus (ZIKV) are important human pathogens causing around 100 million symptomatic infections each year. During infection, small subgenomic flavivirus RNAs (sfRNAs) are formed inside the infected host cell as a result of incomplete degradation of the viral RNA genome by cellular exoribonuclease XRN1. Although the full extent of sfRNA functions is to be revealed, these non-coding RNAs are key virulence factors and their detrimental effects on multiple cellular processes seem to consistently involve molecular interactions with RNA-binding proteins (RBPs). Discovery of such sfRNA-binding host-factors has followed established biochemical pull-down approaches skewed towards highly abundant proteins hampering proteome-wide coverage. Yeast three-hybrid (Y3H) systems represent an attractive alternative approach. To facilitate proteome-wide screens for RBP, we revisited and improved existing RNA-Y3H methodology by (1) implementing full-length ORF libraries in combination with (2) efficient yeast mating to increase screening depth and sensitivity, and (3) stringent negative controls to eliminate over-representation of non-specific RNA-binders. These improvements were validated employing the well-characterized interaction between DDX6 (DEAD-box helicase 6) and sfRNA of DENV as paradigm. Our advanced Y3H system was used to screen for human proteins binding to DENV and ZIKV sfRNA, resulting in a list of 69 putative sfRNA-binders, including several previously reported as well as numerous novel RBP host factors. Our methodology requiring no sophisticated infrastructure or analytic pipeline may be employed for the discovery of meaningful RNA-protein interactions at large scale in other fields.

Keywords: ORF library; RNA-binding proteins; Subgenomic flavivirus RNA; Zika virus; dengue virus; host factors; yeast three-hybrid.

Figure 1.

Detecting RNA-binding proteins in a mating yeast three-hybrid ORF screen. (A) Primary hit calling. After transformation of yeast reporter strain YBZ2 (genotype: MATa, ura3, trp1, leu2, G418res, LexAop::HIS3) with a bait plasmid (prototrophic marker: LEU2) expressing the RNA of interest (ROI) and library strain Y187 (genotype: MATα, URA3, his3, trp1, leu2) with prey plasmids (prototrophic marker: TRP1) expressing the human ORF library, both strains are mated and resulting diploids plated on selective medium (SD/-LWUH supplemented with G418). The thus chosen growth conditions select for functional complementation of multiple markers, essentially resulting from the productive interaction between bait RNA and prey protein (HIS prototrophy). This interaction needs to be a consequence of fusion of cells from either yeast strain (G418res and URA3, respectively) by mating, by this means combining both bait (LEU2) and prey (TRP1) plasmids in one diploid cell. Each candidate RBP is identified by colony PCR using prey expression vector-specific primers (amplification of target ORF cDNA), Sanger sequencing and hit calling by BLAST analysis. (B) Elimination of false positives. In parallel, a negative control screen is performed the identical way, yet using an empty bait construct to eliminate non-specific RNA binders to be deselected as false-positives from the primary interaction screen. (C) Hit verification. Remaining hits may further be characterized quantitatively by Y3H, assessing their HIS3 and lacZ reporter gene expression levels. Hits can thus be verified by showing a marked increase in signal over several stringent negative controls, such as a matched antisense bait construct

Figure 2.

DDX6 has a proviral effect on DENV2 infection (A) and its binding to DENV2 sfRNA can be specifically detected by Y3H (B-D). (A) Genetic ablation of DDX6 expression in the human HAP1 cell line decreases DENV2 infectivity. Infectious virus yield in cell supernatant 2 days post infection (at an MOI of 1) is shown relative to HAP1-parental. n = 4 from two independent experiments. Unpaired t-test calculated in Graphpad Prism. ****p < 0.0001. (B + C) Elevated reporter gene expression in yeast resulting from functional Y3H interaction. Diploids expressing DDX6 as ‘prey’ and DENV2 sense sfRNA as ‘bait’ have markedly increased HIS3 and LacZ expression levels over diploids expressing antisense sfRNA employed as matched negative control ‘bait’. DDX6 was expressed from two different expression vectors to exclude vector-dependent effects. (B) Fold increase in LacZ expression over the antisense ‘bait’ control was determined in a coupled bioluminescent LacZ assay in two biological replicates. (C) HIS3 expression was titrated indirectly by measuring growth resistance of diploid Y3H cells to the competitive HIS3 inhibitor 3-AT. Growth was measured by absorbance (OD at 600 nm) in liquid cultures and normalized for SD/-LW (set as 100%) and SD/-LWH supplemented with 10 mM 3-AT (set as 0%). A 3-AT IC50 value was calculated with growth in SD/-LWH set as 100% (not shown). (D) Conserved dumbbell structures in the DENV2 sfRNA are required for DDX6-interaction in the Y3H. Different sfRNA variants of both YFV17D (construct 3) and DENV2 (construct 4) were assessed for DDX6 binding in Y3H. Diploids were grown overnight in SD/-LW medium and streaked for phenotypic analysis on SD/-LWH medium. In construct 6 (YFV ∆DB), the central dumbbell (DB) structure (orange) proposed to bind to DDX6 [27] has been deleted. In construct 8, the tandem repeat DB-I and DB-II structures (blue) from DENV2 sfRNA have been swapped for the homologous RNA elements in the YFV sfRNA. Constructs 5 (DENV2as) and 7 (YFVas ∆DB) represent antisense controls for respectively construct 4 (DENV2 sfRNA) and construct 6

Figure 3.

DDX6-sfRNA binding can be picked up in a Y3H pilot screen with high sensitivity. (A) Y187 clones expressing DDX6 as ‘prey’ were mixed with clones expressing a non-interacting protein (NIP) in five different ratios prior to mating with YBZ2 cells expressing the DENV2 sfRNA as bait. Diploids were plated on non-selective (-LW) and increasingly stringent selective media (-LWH, -LWUH + G418, and -LWUH + G418 + 1 mM 3-AT), the number of Y3H colonies were counted after 7 days, and the percentage of true (amplicon ~2kb) and false positives (~3 kb) determined by colony PCR. (B) Top: number of Y3H colony forming units (CFU) formed on different media. CFUs were corrected for mating efficacy using CFUs on -LW medium. Below: distribution of ‘prey’ ORFs identified using colony PCR on the plates where DDX6 was outnumbered 1:100 with NIP

Figure 4.

Identification of DENV2 and ZIKV sfRNA-binding proteins in a Y3H ORF screen. (A) 69 unique DENV2 and ZIKV sfRNA-binding proteins were identified. Left: Venn diagram representing the number of unique hits detected in each screen. Right: Pie charts showing the distribution of prey proteins identified in positive colonies in both ORFeome screens. Proteins repeatedly identified in a negative control screen using only the MS2 RNA without RNA insert as ‘bait’ were classified as false positives. (B) Enrichment for P-body components. Overview of the cellular component gene ontologies for proteins that were picked up in the DENV2 and ZIKV sfRNA Y3H screens. The resulting hit list was significantly enriched for proteins that are part of P-bodies (GO:0000932). Hits run as an ordered query, according to their screen hit frequency, in the g:GOst tool in g:profiler [42]. **p ≤ 0.01 (C) Correlation between hit calling frequencies and reporter gene expression levels. A panel of yeast diploids selected in the DENV2 screen were assessed for LacZ expression at two different yeast cell densities, all hits showed a marked increase over the NIP control. A positive correlation between screen hit frequency (ordered from left to right) and respective LacZ signal intensities could be observed with the exception of DPYSL3 (Excluding DPYSL3, Spearman r: 0.8986, two-tailed p = 0.0278). (D) Replica plating confirms HIS3 reporter gene expression. A panel of yeast diploids picked up in the DENV2 screen were replica plated on selective medium to confirm HIS3 expression. Results are shown for diploids expressing protein EDC3, the most frequent hit, as prey. (E) Comparison with other genome-wide approaches to identify DENV and ZIKV host factors. Venn diagram showing the overlap of hits identified by Y3H with DENV2 and ZIKV 3ʹUTR/sfRNA-binding proteins previously identified in orthogonal screens, i.e. by (1) a mammalian three-hybrid assay [16] and (2) RNA pulldown screens [22,30] and (3) host dependency or restriction factors of DENV and ZIKV replication identified by two independent RNAi screens [43,44] with no bias for RBP. (F) RNA-binding domains involved. Venn diagram showing hits with known RNA-binding activity based on (1) the presence of a classical RNA-binding domains (n = 17) and/or (2) detection in a comprehensive RNA-interactome screen (n = 13) [3] and/or (3) RNA-binding gene ontology (n = 19)