Cell Lines, Virus Propagation and Plaque Titration
BHK-21, Vero and 293T cell lines were cultured in HyQ MEM/EBSS media with 10% fetal bovine serum (FBS). Aedes aegypti Aag2 cells were maintained in Schneider’s Drosophila medium supplemented with 10% FBS. HeLa S3 spinner cells were maintained in JMEM with 10% Horse Serum. C6/36 Aedes albopictus suspension cells were cultured in SF-900II (Gibco) serum free media.
Full length SinV genomic RNAs were produced by in vitro
transcription of either wild type SinV AR339 or the temperature sensitive ts6SinV AR339 clone (Barton et al., 1988
; Bick et al., 2003
), as previously described (Garneau et al., 2008
). The ΔURE SinV variant which contained a 30 base deletion of the URE was constructed using the primers 5’-ATTTTGTTTTTAACATTTCA(37)
GGGAATTC and 5’-TTATGCAGACGCTGCGTGGCATTATGC to jump from the CSE to the region upstream of the URE in the pToto1101/ts6 SinV AR339 vector using PCR (Garneau et al, 2008
). Viral titers were determined by plaque titration on Vero cells.
Construction of a Mosquito-Specific shRNA Vector
The Hygromycin Phosphotransferase (hph) gene was isolated from pHyg (Gritz and Davies, 1983
) via PCR using the primers 5’-CATACATGTTCATGAAAAAGCCTGAACTCACCGCG and 5’-CATCTCGAGCTATTCCTTTGCCCTCGGACGAGTG. PCR products were cut with PciI
and inserted into pBiEx-1 (Promega) to create pBiEx-hph. An Aedes aegypti
U6 promoter-driven shRNA expression cassette was generated via PCR from pAedes1 (Konet et al., 2007
) using the primers 5’-CATGGGCCCGAATGAATCGCCCATCGAGTTGATACGTC and 5’-CATGGCGCCAAAAAAAAAAGCTTCAGCTGGGTACCGGATCCATTTCACTACT CTTGCCTCTGCTCTTATATAG. The PCR product was cut with ApaI
and ligated into pBiEx-hph to create a selectable mosquito shRNA vector pAeSH that allows for the insertion of any shRNA into the multiple cloning site present downstream of the U6 promoter. Targeted shRNAs to Aedes aegypti
aeHuR were designed and the following oligos were inserted into the BamHI
sites of pAeSH: 5’-GATCCCAAAGTGCTAGCAGCCGTATTCAAGAGATACGGCTGCTAGCACTTG TTA and 5’ AGCTTAACAAAGTGCTAGCAGCCGTATCTCTTGAATACGGCTGCTAGCACTT TGG to create pAeSH-aeHuR1.
Preparation of RNA Substrates
Oligos containing sequences derived from the 3’ UTRs of VEE, EEE, WEE, and SFV (Fig. S1B
) were inserted into the EcoRI
sites of pGEM4-A60 (Garneau et al., 2008
). Transcription templates were generated via digestion with HindIII
, for the non-adenylated and adenylated species respectively. Internally radiolabeled, capped RNA substrates were transcribed by SP6 polymerase and purified as described (Wilusz and Shenk, 1988
Viral RNA Decay Analysis
293T cells were either transfected with a Mission shRNA vector (Sigma Aldrich) specific to HuR (TRCN0000017277) or mock transfected (lacking specific shRNA but still containing transfection reagent) using FuGene 6. At 12 hours post transfection cells were infected with ts6SinV at an MOI of 5. Following a 30 min adsorption period, fresh media was added and the cells were placed at 28°C for 10 hours. Pre-warmed media was then added and the cells were transferred to 40°C to shut off viral transcription. At the times indicated post shut off, total RNA was harvested using Trizol and quantified by qRT-PCR (Garneau et al., 2008
) using the primers listed in Fig. S1B
. Note that since the analysis was done at 10 hpi, shRNA knockdown of HuR was allowed to proceed for 22hrs post transfection.
Determination of SinV RNA decay in Aag2 cells was performed as described above using stable aeHuR-knockdown cell lines selected following transfection of the pAeSH-aeHuR1 or empty pAeSH vector using FuGene6. Stable cell pools were selected using 300U of Hygromycin B.
From the half life regression curves for each independent in vivo RNA decay experiment, the average genomic and subgenomic half lives observed in the 293T and HuR-deficient cell lines were determined. These average half lives were subjected to statistical analysis via a two-tailed Student’s T-test.
Abundance of aeHuR and HuR mRNAs was examined using qRT-PCR and the primers listed in Fig. S1B
. The relative abundances between control transfected cells and shRNA treated cells were compared at time point zero.
Cell-free RNA Deadenylation Assays
Cytoplasmic extracts were derived from C6/36 cells and HeLa S3 cells as previously described (Sokoloski et al., 2008a
). 100,000 CPM (4-50 fmoles) of internally radiolabeled, polyadenylated RNA was incubated in the presence of mosquito cytoplasmic extract as previously described (Opyrchal et al., 2005
), aliquots were removed at the desired time points and recovered RNAs were analyzed on 5% acrylamide gels containing 7M urea. RNAs were analyzed by phosphorimaging.
Ultraviolet Cross-linking and Immunprecipitation Assays
100,000 CPM (4-50 fmoles) of RNA substrate were incubated in cell-free RNA decay assays for 1 min. using either HeLa or C6/36 extracts in the presence of 0.5 mM EDTA to inhibit RNA decay (Sokoloski et al., 2008b
). Following cross-linking of RNA-protein complexes by 254nm UV light, the mixture was treated with RNAse A and RNAseONE, proteins cross-linked to short radiolabeled RNA oligomers were resolved using 10% SDS-PAGE and visualized by phosphorimaging.
For RNA cross-linking / immunoprecipitation assays, samples were incubated, irradiated and treated with RNAse as described above. Samples were diluted with NET2 buffer (40mM Tris-HCl, pH 7.5 / 200mM NaCl / 0.05% (v/v) NP-40) and clarified by centrifugation. Samples received either control sera or target-specific antibody and were incubated for one hr at 4°C. Protein G sepharose or Pansorbin were then added, antibody-antigen complexes were washed ~5 times with NET2 buffer and immunoprecipitated proteins were analyzed by 10% SDS-PAGE.
Cell-Based Co-Immunoprecipitation Assays
293T or Aag2 cells were infected with wild type SinV at an MOI of 5. Cells were released using trypsin (293T) or mechanical scraping (Aag2), washed with PBS, resuspended in a 1% formaldehyde solution in PBS and incubated for 10 min. at room temperature. The reaction was quenched using 0.25M glycine. Cell pellets were washed with PBS and resuspended in RIPA Buffer (50 mM Tris-Cl, pH 7.5 / 1% (v/v) NP-40 / 0.5% (w/v) Sodium Deoxycholate / 0.05% (w/v) SDS / 1 mM EDTA / 150 mM NaCl). Cells were disrupted by sonication on ice and insoluble materials removed via centrifugation. Aliquots received either anti-HuR, anti-aeHuR or control (pre-immune) antibodies and incubated at 4°C for one hr. Antibody bound complexes were recovered using protein G or protein A sepharose after 6X washes with RIPA buffer containing 1M urea. Formaldehyde cross-links were reversed by heating at 70°C for 45 min. Isolated RNAs were analyzed by RT-PCR to assess total SinV RNA.
Affinity Purification and Mass Spectrometry
5’ Biotinylated RNA oligos consisting of either the SinV URE/CSE fragment (5’-Bio- UAAUCAUUUAUUAUUUUCUUUUAUUUUAUUCACAUAAUUUUGUUUUUAA) or a nonspecific sequence (5’-Bio-GAAUCGAGCUCGGUACCCGGGGAUCCUCUAGAGUCGACCUGCAG) were incubated with 2.5 mgs of cytoplasmic extract. Protein-RNA complexes were bound to Streptavidin resin, pelleted by centrifugation and washed five times with 20mM HEPES, pH 7.9 / 225mM KCl / 2.5mM MgCl2 / 0.1% (v/v) NP-40 / 1mM DTT. Proteins were eluted by boiling in HSCB buffer, concentrated using methanol and resolved via 10% SDS-PAGE. Gels were stained and excised bands were analyzed by MALDI-TOF/TOF.
Expression of Recombinant Proteins
The Aedes aegypti
open reading frame corresponding to AaeL_AAEL008164 was PCR-amplified using the primers 5’-CATGGATCCATGACCAACAAAGTGCTAGCAGCC and 5’-CATGAATTCTTAATGATCGGCCATTTCGGCG. The gel-purified fragment was cut with BamHI
and inserted into pGEX-2TZQ (Qian and Wilusz, 1994
) to make pGEX-aeHuR. Recombinant protein was prepared from BL21 DE3 E. coli
and the GST tag was removed by thrombin cleavage. Recombinant human HuR was a gift from N. Curthoys (Colorado State University).
Cell Fractionation and Western Blotting
Separation of cells into nuclear and cytoplasmic fractions was performed as described in Hel et al (1998). Following separation from nuclei, the cytoplasmic fraction was centrifuged at 16,000 × g for 10 min at 4°C prior to analysis by western blotting. Antibodies to PABPN1 (K-18) and HuR (3A2) were purchased from Santa Cruz Biotechnology; GAPDH antibodies were obtained from Millipore. A polyclonal aeHuR antisera was raised by Bioo Scientific in rabbits using the above recombinant aeHuR. Serum specificity was determined via western blot analysis of C6/36 cytoplasmic extract separated on 10% SDS-PAGE.
Immunofluorescence and FISH analysis
Aag2 and 293T cells were grown on glass coverslips, fixed in 4% paraformaldehyde, permeabilized in methanol and rehydrated in 70% ethanol. Coverslips were blocked in 6% bovine serum albumin fraction V (BSA) in PBS for at least one hour and washed in PBS. Primary antibody (diluted in 0.6% (w/v) BSA in PBS) was added for one hr, washed in PBS and secondary antibody (diluted as above) was applied for one hr. After washing, the coverslips were mounted using ProLong Gold antifade reagent with DAPI. Antibodies used were HuR (3A2) (Santa Cruz Biotechnology (SCBT)), Nucleophosmin NA24 (Thermo Scientific), anti-aeHuR polyclonal serum (this study), Cy2 donkey anti-mouse Ig (Jackson ImmunoResearch), Cy5 goat anti-rabbit Ig (GE Healthcare) and Texas red anti-mouse Ig (SCBT).
For FISH analyses, cells were grown, fixed, permeabilized and rehydrated as above. Coverslips were washed in PBS followed by a treatment of 40% formamide/2X SSC. The oligonucleotide (5’Alexa488 labeled 5’-TGACATTTCAAGGAGCCGCAGCATTT) was diluted in 40% formamide/2X SSC/0.2% (w/v) BSA and added to the coverslip for two hrs and washed in 1X SSC.
RNA Electrophoretic Mobility Shift Assays
Internally radiolabeled unadenylated alphavirus URE/CSE fragments (~1.25 fmols) were incubated in the presence of recombinant aeHuR or HuR at the indicated concentrations in Gel Shift Buffer (15mM HEPES, pH 7.9, 100mM KCl, 2.25mM MgCl2, 5% (v/v) Glycerol). The complexes were allowed to form for five minutes at room temperature prior to the addition of 2.6 ug/ul Heparin Sulfate. The addition of heparin prior to the incubation with recombinant protein gave similar results. Following a 5 min. incubation on ice, protein-RNA complexes were resolved on a 5% native acrylamide gel and analyzed by phosphorimaging. Values obtained for bound versus free RNA were plotted and dissociation constants were calculated from the slope of the linear regression line fitted to the data.