Zika virus noncoding sfRNAs sequester multiple host-derived RNA-binding proteins and modulate mRNA decay and splicing during infection

Abstract

Insect-borne flaviviruses produce a 300-500-base long noncoding RNA, termed subgenomic flavivirus RNA (sfRNA), by stalling the cellular 5'-3'-exoribonuclease 1 (XRN1) via structures located in their 3' UTRs. In this study, we demonstrate that sfRNA production by Zika virus represses XRN1 analogous to what we have previously shown for other flaviviruses. Using protein-RNA reconstitution and a stringent RNA pulldown assay with human choriocarcinoma (JAR) cells, we demonstrate that the sfRNAs from both dengue type 2 and Zika viruses interact with a common set of 21 RNA-binding proteins that contribute to the regulation of post-transcriptional processes in the cell, including splicing, RNA stability, and translation. We found that four of these sfRNA-interacting host proteins, DEAD-box helicase 6 (DDX6) and enhancer of mRNA decapping 3 (EDC3) (two RNA decay factors), phosphorylated adaptor for RNA export (a regulator of the biogenesis of the splicing machinery), and apolipoprotein B mRNA-editing enzyme catalytic subunit 3C (APOBEC3C, a nucleic acid-editing deaminase), inherently restrict Zika virus infection. Furthermore, we demonstrate that the regulations of cellular mRNA decay and RNA splicing are compromised by Zika virus infection as well as by sfRNA alone. Collectively, these results reveal the large extent to which Zika virus-derived sfRNAs interact with cellular RNA-binding proteins and highlight the potential for widespread dysregulation of post-transcriptional control that likely limits the effective response of these cells to viral infection.

Keywords: 5′–3′-exoribonuclease (XRN1); RNA editing; RNA splicing; RNA turnover; RNA virus; Zika virus; flavivirus; post-transcriptional regulation; subgenomic flavivirus RNA (sfRNA).

Figure 1.

Accumulation of two sfRNA decay intermediates in ZIKV infection coincides with an increase in both the stability and abundance of normally short-lived host cell mRNAs. A, representative Northern blotting of total RNA from ZIKV-infected JAR cells isolated at the indicated time post-infection. RNAs were separated on a 5% denaturing acrylamide gel, probed with a radiolabeled probe complementary to the first 77 nt of the ZIKV 3′-UTR, and visualized by phosphorimaging. The position of size markers on the gel is indicated on the right. B, newly synthesized RNAs from mock-infected or ZIKV-infected JAR cells were metabolically labeled with 4SU at the indicated times post-infection. RNAs were separated into unlabeled and 4SU-labeled populations and subjected to quantitative ddRT-PCR analysis using primers to the FOS and JUN cellular mRNAs. The top graphs depict the abundance changes of the indicated mRNA relative to mock-infected cells, and the bottom graphs depict the mRNA stability relative to infected cells as determined by half-life calculations. Results are shown as the mean ± S.D. determined from three independent infections. Significance was determined using a Student's t test with * = p ≤ 0.05.

Figure 2.

ZIKV 3′-UTR stalls and represses the activity of the host cell 5′–3′-exoribonuclease XRN1. A, radiolabeled, 5′-monophosphorylated control (CTRL) RNA or RNAs containing the intact ZIKV 3′-UTR or with mutations that debilitate the first (MUT1), second (MUT2), or both (DM) structures that lead to the formation of sfRNAs in cells were incubated with either recombinant XRN1, C6/36 mosquito cytoplasmic extract, or HeLa cytoplasmic extract for the indicated amount of time. Reaction products were analyzed on a 5% denaturing acrylamide gel and quantified by phosphorimaging. The position of size markers is indicated on the left. B, 61 base-radiolabeled reporter RNA derived from pGEM4 was incubated with XRN1 (derived from HeLa extract) in the presence of a 20× excess of a cold competitor RNA derived from either pGEM4 (control RNA lanes), the DENV-2 3′ UTR (DENV 3′ UTR lanes), the ZIKV 3′ UTR (ZIKV 3′ UTR lanes), or the ZIKV 3′ UTR containing mutations that prohibit sfRNA formation (ZIKV DM lanes) for the times indicated. Radiolabeled reaction products derived from XRN1 acting on the reporter RNA were resolved on a 5% acrylamide gel containing urea and visualized by phosphorimaging. Gels from three independent experiments were quantified, and results are shown graphically in the lower panel. The asterisk represents a p value of <0.001 at both time points for viral 3′ UTR/fragments compared with the control as determined using Tukey's multiple comparisons test as a post hoc test. The gel in the top portion is representative data from these same experiments.

Figure 3.

ZIKV and DENV-2 sfRNAs interact with a common set of 28 proteins that impact a variety of aspects of cellular post-transcriptional gene regulation. A, biotinylated RNAs containing the DENV-2 3′ UTR, ZIKV 3′ UTR, or a size-matched control transcript were incubated with HeLa cytoplasmic extracts. Proteins that co-purified with the RNAs on streptavidin beads were identified by MS. A, Venn diagram depicting the number and commonality of human host-cell RNA-binding proteins identified via MS for each RNA. B shows a grouping of proteins that bound to both the ZIKV 3′-UTR and DENV 3′-UTR into the post-transcriptional process associated with each protein. C, ZIKV sfRNA directly binds to a variety of cellular RNA-binding proteins in infected cells. Left side, Western blotting documenting the specificity of the antibodies used in the panel. Right side, 293T cells were infected with ZIKV for 48 h; formaldehyde was added to the cultures to stabilize RNA–protein complexes, and total cell extracts were prepared. Antibodies to RNA decay factors DDX6 and EDC3, RNA-splicing factors PHAX and SF3B1, and the RNA-editing factor APOBEC3C or normal rabbit IgG were added to immunoprecipitated RNA–protein complexes. Co-purifying RNAs were analyzed by digital droplet RT-PCR using primers to the Zika virus sfRNA/3′-UTR. The abundance of ZIKV RNA co-precipitated using the various antibodies was determined relative to the amount pulled down in the normal rabbit IgG control. Results are shown as the average ± S.D. from three independent infections. D, biotinylated RNAs containing the 3′ UTRs of DENV-2 or ZIKV, as well as a size-matched control RNA, were incubated with cytoplasmic extracts. Associated proteins were eluted, separated on a 10% SDS-acrylamide gel, and probed with the antibodies indicated on the left by Western blotting.

Figure 4.

Host RNA decay factors DDX6 and EDC3 function to inhibit ZIKV replication and limit infectious virus production. A, representative Western blotting depicting endogenous DDX6 and EDC3 protein levels in ZIKV-infected 293T cells treated with either a control siRNA, a siRNA targeting DDX6, or a siRNA targeting EDC3. B, effects of siRNA-mediated knockdown of RNA decay factors DDX6 and EDC3 on ZIKV sfRNA production in infected 293T cells compared with siRNA Control knockdown is as determined via ddPCR. The average fold-change ± S.D. from three independent infections is shown. C, focus forming assay: Vero cells infected with the viral supernatant from the siRNA knockdown samples described in B. The average number of foci per ml ± S.D. from three independent infections is depicted. D–F, effects of overexpressing 3×FLAG-tagged DDX6 and EDC3 from a transfected plasmid in 293T cells during ZIKV infection. The average fold-change ± S.D. from three independent infections is represented on the graph. D, representative Western blotting showing 3×FLAG-tagged overexpression of DDX6 and EDC3 in 293T cells. Total protein from cells that were transfected as indicated above the three lanes was electrophoresed on a 10% SDS-acrylamide gel, blotted, and probed with anti-FLAG antibody. E, effects of overexpressing 3×FLAG-tagged DDX6 and EDC3 on ZIKV sfRNA production in infected 293T cells compared with an empty control vector as determined via ddPCR. The average fold-change ± S.D. was from three independent infections. F, focus-forming assay: Vero cells infected with the viral supernatant from the overexpression samples described in D. The average number of foci per ml ± S.D. from three independent infections is depicted. Significance in B, C, E, and F was determined by t test with * = p ≤ 0.05.

Figure 5.

Splicing factor PHAX and the nucleic acid deaminase APOBEC3C are restriction factors for ZIKV replication. A, siRNAs targeting PHAX or APOBEC3C or a nonspecific control siRNA were transfected into HEK293 cells. The levels of the indicated proteins were determined by Western blotting. The blots shown are representative of three independent experiments. B, relative levels of ZIKV RNA in cells transfected with either control, PHAX, or APOBEC3C targeting siRNAs were determined at 48 hpi. The data are presented as fold-change relative to the ZIKV levels present in cells transfected with the control siRNA. C, focus-forming assay: Vero cells infected with the viral supernatant from the siRNA knockdown samples described in B. The average number of foci per ml ± S.D. from three independent infections is depicted. D, FLAG-tagged versions of APOBEC3C and PHAX were constructed on expression vectors and transfected into HEK293 cells. The levels of the indicated proteins were determined by Western blotting. E, relative levels of ZIKV RNA in cells transfected with either empty vector, FLAG-PHAX, or FLAG-APOBEC3C expression plasmids were determined at 48 hpi. The data are presented as fold-change relative to the ZIKV levels present in cells transfected with the empty vector. F, focus-forming assay: Vero cells infected with the viral supernatant from the overexpression samples described in D. The average number of foci per ml ± S.D. is depicted. All data are the results of three independent biological experiments. * = p < 0.05 as determined by a Student's t test.

Figure 6.

Splicing of SF3B1 target gene is dysregulated during ZIKV infection. A, diagrammatic representation of the SRSF7 gene exon–intron organization between exons 1 and 8. The relative position of PCR primers (forward (F) and reverse (R)) used to amplify the region of interest is also shown. B, total RNA from uninfected 293T cells (lane C) or 293T cells ZIKV infected at 48 hpi (lane ZIKV) was obtained and subjected to RT-PCR analysis using primers to amplify the region between exons 2 and 8. Products were electrophoresed on a 1% agarose gel and visualized by ethidium bromide staining. C, PCR products from B were gel-purified, cloned into a pGEM-T easy vector, and sequenced. The position of the resulting splice sites and exons for the indicated intermediates is indicated by the diagrams.

Figure 7.

Transfection of sfRNA alone causes changes in cellular mRNA stability and splicing. A, HEK293T cells were transfected with RNAs containing the 3′ UTR of DENV-2, ZIKV, or a sized-matched unrelated control RNA. Total RNA was isolated 6 h post-transfection, and the abundance of the JUN and FOS mRNAs relative to the housekeeping GAPDH mRNA was assessed by quantitative RT-PCR. B, HEK293T cells were transfected with DENV-2 sfRNA or with a sized-matched unrelated control RNA (C lane). Total RNA was isolated 6 h post-transfection, and the splicing pattern of the SRSF7 mRNAs was analyzed as described in Fig. 6. M refers to a lane with size markers. The position of the resulting splice sites and exons for the indicated #2 splicing isoform is indicated by the diagram.

Figure 8.

ZIKV and DENV sfRNAs bind a plethora of diverse RNA-binding proteins that can impact many post-transcriptional processes in the cell.