Double-stranded RNA (dsRNA) is a viral-associated molecular sample that initiates innate immune packages in mammalian cells (1, 2). In response to dsRNA, cells induce the expression of antiviral proteins resembling cytokines, which prime an antiviral state in contaminated and noninfected cells. Concurrently, cells restrict translation to scale back viral protein manufacturing. For many years, activation of the cytoplasmic endoribonuclease, RNase L, in response to viral dsRNA has been thought to restrict viral protein synthesis by cleaving host ribosomal RNA (rRNA), leading to arrest of translation (3–7). Nevertheless, two current research point out that RNase L–mediated cleavage of rRNA doesn’t arrest translation. First, flaviviruses, together with Zika virus and dengue virus (DENV), can replicate regardless of activating RNase L–mediated rRNA decay (8). Second, speedy and widespread decay of host mRNAs by RNase L accounts for RNase L–mediated discount in translation (9, 10). In gentle of those findings, it’s unclear how RNase L limits viral protein synthesis, how some viruses escape the consequences of RNase L, and the way RNase L activation impacts host antiviral protein manufacturing.
DENV mRNAs escape RNase L–mediated mRNA decay and produce protein
DENV is a flavivirus [+single-stranded RNA (+ssRNA) virus] that replicates within the cytoplasm. To research the mechanism by which DENV evades the consequences of RNase L (8), we contaminated parental [wild-type (WT)] and RNase L–knockout (KO) (RL-KO) A549 cells with DENV serotype 2 and carried out single-molecule fluorescence in situ hybridization (smFISH) for DENV mRNA and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA, which is degraded when RNase L is lively (9) and in addition stained for DENV NS3 protein manufacturing by immunofluorescence.
DENV-infected cells continuously activated RNase L, as noticed by a robust discount of GAPDH mRNA in most DENV-infected WT however not RL-KO cells (Fig. 1, A and B, and fig. S1). DENV-infected cells that activated RNase L confirmed a small, however not statistically vital, discount in DENV mRNA ranges as in comparison with WT cells that didn’t activate RNase L or RL-KO cells (Fig. 1C). Furthermore, DENV protein manufacturing was strongly correlated with DENV mRNA ranges (Fig. 1D) and solely confirmed small decreases in cells with activated RNase L (Fig. 1E). Thus, DENV RNA is basically unaffected by RNase L–mediated mRNA decay. Consequently, this allows DENV protein manufacturing, in line with cytoplasmic mRNAs having the capability to be translated throughout the RNase L response (9, 10).
The host immune response inhibits mRNA export by way of RNase L activation
Examination of host cytokine mRNAs induced by DENV an infection in WT and RL-KO A549 cells revealed two necessary factors. First, we noticed that antiviral cytokine mRNAs largely escape RNase L–mediated mRNA decay. That is based mostly on the statement that in some WT cells that activated RNase L–mediated mRNA decay (degraded GADPH mRNA), IFN-λ1 (interferon-λ1) and IFN-β mRNAs had been ample within the cytoplasm, usually at ranges akin to these noticed in RL-KO cells (Fig. 2, A and B, and figs. S2, A and B, and S3, A and B). Furthermore, whereas median GAPDH mRNA ranges had been diminished greater than 10-fold by RNase L (Fig. 1B), median IFN mRNA ranges had been solely diminished 2-fold by RNase L (Fig. 2C and fig. S3C). Thus, cytokine mRNAs that attain the cytoplasm largely escape RNase L–mediated mRNA decay throughout DENV an infection, in line with our observations throughout the dsRNA response (9).
Unexpectedly, we additionally noticed accumulation of IFN-β and IFN-λ1 mRNAs throughout the nucleus of some DENV-infected WT cells (Fig. 2, A to D, and figs. S2, A and B, and S3, A to D). In precept, this mRNA export block could possibly be mediated by DENV or could possibly be a consequence of the mobile response to dsRNA. To differentiate these potentialities, we transfected cells with polyinosinic:polycytidylic acid [poly(I:C)], a viral dsRNA mimic, and examined whether or not mRNA export was affected. We noticed that poly(I:C) transfection was enough to activate RNase L, to induce IFN mRNAs, and to dam mRNA export in a fraction of WT cells (Fig. 3, A to C, and fig. S4, A to C). This demonstrates a beforehand unidentified facet of the host innate immune response that blocks nuclear mRNA export.
A number of observations reveal that this block to mRNA export requires activation of RNase L–mediated mRNA degradation. First, nuclear retention of IFN mRNAs was solely noticed in DENV-infected WT cells that activated RNase L–mediated decay of GAPDH mRNA (Fig. 2, A to D). In distinction, neither WT cells that didn’t activate RNase L (as assessed by the dearth of GAPDH mRNA degradation) nor RL-KO cells that induced cytokine mRNAs displayed the mRNA export block (Fig. 2, A to D). Second, the mRNA export block in response to poly(I:C) was additionally RNase L–dependent, as we didn’t observe IFN mRNA accumulation within the nucleus of RL-KO cells (Fig. 3, A to C, and fig. S4, A to C). Third, the block to mRNA export requires RNase L catalytic exercise since rescuing the RL-KO cells with RNase L, however not RNase L-R667A (a catalytic mutant), restored nuclear retention of IFN-β mRNA in response to poly(I:C) (Fig. 3D). Nuclear accumulation of IFN-β mRNAs was additionally noticed by quantitative reverse transcription polymerase chain response (RT-qPCR) of nuclear and cytoplasmic biochemical fractions (Fig. 3, E and F). Thus, activation of RNase L is enough to induce a block to nuclear mRNA export.
Immunofluorescence assay confirmed that nuclear-retained IFN-β mRNA is inner to the nuclear pore complicated (NPC) and never retained on the website of transcription (Fig. 3G), which could be noticed with many RNA processing defects (11). The block to nuclear export is impartial of PKR (protein kinase RNA-activated), since nuclear accumulation of mRNAs after poly(I:C) therapy can also be noticed in PKR-KO cells (fig. S4D). The nuclear block seems impartial of RNase L–mediated apoptosis because the Z-VAD-FMK caspase inhibitor didn’t inhibit it (fig. S4E).
Our observations point out that RNase L activation inhibits the chromosomal area upkeep 1 (CRM-1) mRNA export pathway, which is a specialised mRNA export pathway utilized by mRNAs with out introns and/or that comprise AU-rich components (AREs) (12). The IFN-β mRNA, which lacks introns and accommodates AREs, is believed to exit the nucleus by the CRM-1 mRNA export pathway (13). Remedy of RL-KO cells with the CRM-1 inhibitor, leptomycin B, phenocopied the RNase L–dependent nuclear retention of IFN-β mRNA noticed in WT cells (fig. S4F). Thus, nuclear accumulation of IFN-β mRNA is a results of RNase L–dependent inhibition of CRM-1–mediated mRNA export.
RNase L activation additionally inhibits the NXF1-TREX (nuclear RNA export issue 1-transcription export complicated) bulk mRNA export pathway that acts on spliced mRNAs since IFN-λ1 mRNA accommodates introns and is insensitive to leptomycin B (fig. S4G). Furthermore, the GAPDH mRNA, which can also be spliced and presumably exported by the NFX-TREX pathway, additionally exhibits nuclear retention in cells with activated RNase L (Fig. 3D). These observations argue that RNase L activation inhibits each the CRM-1 and the NFX1-TREX mRNA export pathways.
Activation of RNase L inhibits influenza virus mRNA export and protein synthesis
We hypothesized that RNase L–mediated inhibition of mRNA export could also be necessary for limiting viral protein synthesis for viruses that should export their mRNAs from the nucleus. For instance, influenza (–ssRNA virus) replicates within the nucleus and should export its mRNAs to the cytosol and is inhibited 100- to 1000-fold by RNase L (14). To check whether or not RNase L activation blocked influenza mRNA export, we contaminated WT and RL-KO A549 cells with influenza A/Udorn/72 virus (IAV). For the reason that IAV NS1 protein (non-structural protein 1) strongly attenuates RNase L activation (fig. S5A) (14), we transfected cells with poly(I:C) 1 hour after an infection to advertise RNase L activation. We carried out smFISH 7 hours after an infection, when IAV output is in log section development and when RNase L reduces viral output by higher than 100-fold (14).
An necessary end result was that almost all WT cells that activated RNase L (as assessed by degradation of the GAPDH mRNA) restricted the export of IAV NA (neuraminidase) and NS1 mRNAs (Fig. 4, A and B, and fig. S5, B to E). Nuclear retention of those mRNAs didn’t happen in WT cells that didn’t activate RNase L–mediated mRNA decay or in RL-KO cells. This demonstrates that RNase L activation results in the inhibition of IAV mRNA export.
We noticed that in cells that activated RNase L–mediated mRNA decay however didn’t set off the mRNA export block, influenza mRNAs had been ample within the cytoplasm (Fig. 4, A to C, and figs. S5, B to F, and S6A), at ranges akin to these noticed in WT cells that didn’t activate RNase L and RL-KO cells (Fig. 4D and figs. S5, B to F). This argues that IAV mRNAs escape RNase L–mediated mRNA decay and will probably be translated for protein manufacturing, just like DENV mRNAs (Fig. 1). This urged that the RNase L–mediated mRNA export block was the mechanism by which RNase L limits influenza protein synthesis.
To check this speculation, we examined the connection between influenza NS1 protein manufacturing and the NS1 mRNA export block in particular person cells (Fig. 4C and fig. S6, A to C). We noticed that cells displaying RNase L activation (GADPH mRNA degradation), however profitable NS1 mRNA export, produced NS1 protein at ranges comparable, albeit barely much less, to WT cells that didn’t activate RNase L and RL-KO cells (Fig. 4, C to E). In distinction, WT cells during which NS1 mRNA export was inhibited by RNase L activation displayed considerably much less NS1 protein manufacturing (Fig. 4, C to E). Final, cytoplasmic ranges of NS1 mRNA strongly correlated with NS1 protein ranges with or with out RNase L activation (fig. S6B). These observations reveal that RNase L–mediated inhibition of mRNA export is a main mechanism by which RNase L reduces influenza protein manufacturing.
RNase L–mediated inhibition of mRNA export limits antiviral protein manufacturing
RNase L–mediated inhibition of mRNA export would even be anticipated to restrict translation of transcriptionally induced antiviral mRNAs, which could possibly be detrimental to the antiviral response. Nevertheless, since this response was heterogeneous with respect to particular person cells (Figs. 2 and 3), we suspected that the RNase L–mediated mRNA export block would cut back, however not abolish, antiviral cytokine manufacturing. Such a operate could be necessary for guaranteeing cytokine manufacturing whereas probably stopping the overproduction of cytokines, which might trigger cytokine storm phenomena.
Three main observations assist these hypotheses. First, RNase L expression diminished, however didn’t abolish, secretion of IFN-β and IFN-λ1 proteins in a way depending on its catalytic exercise in response to poly(I:C) (Fig. 5A and fig. S7), in line with a earlier examine (15).
Second, nuclear retention of IFN-β and IFN-λ1 mRNAs was differential with respect to time (Fig. 5, B and C, and figs. S8, A to D, and S9, A to D), and this correlated with RNase L–dependent repression of protein manufacturing (Fig. 5A). Particularly, ~75% of WT cells displayed nuclear retention of IFN-β mRNA earlier than 12 hours post-poly(I:C) lipofection (Fig. 5C) when RNase L inhibited IFN-β ranges by 10-fold (Fig. 5A). At 12 and 16 hours after poly(I:C), cytoplasmic IFN-β mRNA ranges elevated in WT cells (Fig. 5C), which preceded/coincided with a rise in IFN-β (Fig. 5A). In distinction, fewer cells (~35%) displayed nuclear retention of IFN-λ1 mRNA at early instances after poly(I:C) (Fig. 5C), and this correlated with comparable IFN-λ1 ranges from WT and RL-KO cells at 8 and 12 hours after poly(I:C) (Fig. 5A). Nevertheless, localization of the IFN-λ1 mRNA progressively elevated to nucleus over time in WT cells (Fig. 5C), correlating with a cessation of IFN-λ1 secretion at late instances after poly(I:C) (Fig. 5A). These knowledge present that RNase L–mediated inhibition of IFN mRNA nuclear export correlates with discount in IFN protein manufacturing.
Third, a number of observations point out that RNase L inhibits IFN-β protein manufacturing at 12 hours after poly(I:C), particularly by way of the export block versus mRNA decay. The twofold discount of IFN-β mRNA by RNase L earlier than 12 hours post-poly(I:C) lipofection is inadequate to account for the 10-fold discount of IFN-β protein noticed at 12 hours after poly(I:C) (Fig. 5, A to D, and fig. S9C). Furthermore, regardless of an equal ~2-fold discount in IFN-λ1 and IFN-β mRNAs by RNase L throughout this time, IFN-λ1 protein ranges had been largely unaffected by RNase L (Fig. 5A and fig. S9, C and D), correlating with IFN-λ1 mRNA being predominantly cytoplasmic, whereas IFN-β mRNA was primarily nuclear at early instances after poly(I:C) (Fig. 5C). These knowledge argue that nuclear retention, versus RNA decay, of the IFN-β mRNA accounts for diminished IFN-β protein manufacturing.
We additionally thought-about the likelihood that RNase L limits IFN-β protein manufacturing by activating eukaryotic initiation issue–2α (eIF2α) kinases (9), significantly PKR (16), which might promote phosphorylation of eIF2α (p-eIF2α) and repress translation. Nevertheless, the cumulative enhance of IFN-β manufacturing in RNase L/PKR double KO cells compared to PKR-KO or RL-KO cells argues that RNase L inhibits IFN-β manufacturing independently of PKR (Fig. 5E). We word that neither PKR nor RNase L markedly affected IFN-β mRNA ranges (Fig. 5D). Furthermore, built-in stress response inhibitor (ISRIB) therapy, which counteracts the consequences of p-eIF2α (17), failed to extend IFN-β in cells that categorical RNase L (Fig. 5F). Thus, RNase L reduces translation of IFN-β mRNA independently of p-eIF2α.
Final, RNase L–mediated apoptosis (18, 19) shouldn’t be chargeable for limiting IFN-β manufacturing at 12 hours after poly(I:C), since we didn’t observe a notable variety of apoptotic cells or caspase-mediated poly[adenosine 5′-diphosphate (ADP)–ribose] polymerase (PARP) cleavage earlier than 12 hours after poly(I:C) (Fig. 5, G and H), in line with live-cell imaging research (20). Furthermore, we didn’t observe a considerable distinction within the charge of cells initiating apoptosis or within the charge of caspase-mediated PARP cleavage between WT and RL-KO cells (Fig. 5, G and H).
Collectively, these observations argue that world reductions in translation by way of mRNA decay, translation arrest, or apoptosis don’t account for RNase L–mediated inhibition of IFN-β. As a substitute, our knowledge reveal that RNase L reduces kind I and kind III IFN manufacturing by blocking the export of their mRNAs to the cytoplasm.
We current a number of observations documenting new points of the innate immune response. First, we present that cytoplasmic DENV and IAV mRNAs escape RNase L–mediated decay and, consequently, produce protein throughout the RNase L response (Figs. 1 and 4). These knowledge point out that RNase L activation doesn’t arrest translation by way of rRNA cleavage, in line with current research (9, 10), and elucidate how some viruses, resembling ZIKA virus, can synthesize proteins regardless of strong RNase L–mediated rRNA decay (8). Just like host antiviral mRNAs (9, 10), the excessive transcriptional charges of viral mRNAs and their construction, in addition to their localization to replication factories and affiliation with viral proteins (8), seemingly contribute to their capacity to flee the consequences of RNase L–mediated mRNA decay.
We additionally recognized a brand new mechanism by which RNase L reduces host and viral gene expression, whereby RNase L activation inhibits mRNA export of host and viral mRNAs, which reduces their translation by stopping their affiliation with ribosomes within the cytoplasm. That is based mostly on the observations that host and viral mRNAs accumulate within the nucleus of cells which have activated RNase L, and this correlates to diminished protein expression from these transcripts (Figs. 2 to 5).
The mechanism by which activation of RNase L inhibits mRNA export stays to be found. A number of observations are in line with RNA export being inhibited following widespread cytoplasmic RNA decay as a consequence of an inflow of cytoplasmic RNA binding proteins (RBPs), which then restrict the interplay of RNA export components with nuclear mRNAs. For instance, along with RNase L–mediated mRNA decay, widespread degradation of host cytoplasmic mRNAs by the SOX and VHS nucleases encoded by Kaposi’s sarcoma–related herpesvirus and herpes simplex virus 1, respectively, results in the import of cytosolic RBPs into the nucleus at the side of mRNA export inhibition (9, 20–24). Additional supporting this mannequin, RNase L–mediated inhibition of mRNA export relies on its catalytic exercise and happens after mRNA decay (Fig. 3, A to D). Elucidating the mechanism by which RNase L–mediated mRNA decay inhibits mRNA export will likely be a key focus of future research.
One operate of RNase L–mediated inhibition of mRNA export is to restrict viral protein manufacturing by limiting gene expression from viruses that replicate within the nucleus. Our knowledge counsel that it is a main mechanism by which RNase L limits influenza protein manufacturing since nuclear accumulation of influenza mRNAs is required for a discount of their translation in cells with activated RNase L (Fig. 4). Since most DNA viruses should export their mRNAs from the nucleus and might activate RNase L (25–28), this could possibly be a broad antiviral mechanism.
A second operate of RNase L–mediated inhibition of mRNA export is to manage cytokine manufacturing. We noticed that the discount of IFN-β protein as a consequence of RNase L correlates with the mRNA export block and can’t be defined by RNase L–mediated world discount in translation, mRNA degradation, or enhanced apoptosis (Fig. 5). Thus, RNase L activation limits cytokine manufacturing by trapping cytokine mRNAs within the nucleus in a fraction of the cells (Fig. 5). We propose that the heterogeneous and temporal nature between particular person cells permits for enough manufacturing of cytokines to restrict viral replication (Figs. 3, A and B, and 5, A to C, and figs. S7 to S9) (29). Furthermore, our knowledge point out that kind III IFNs are much less repressed by RNase L than kind I IFNs at early instances after dsRNA (Fig. 5, A to C, and figs. S7 to S9). This could promote localized epithelial cell–particular antiviral signaling earlier than systemic kind I IFN signaling (30). Mixed, these capabilities might stop systematic overproduction of cytokines, which might result in cytokine storm, sepsis, and autoimmune issues in response to viral an infection (31–33).
MATERIALS AND METHODS
The A549 cell line was offered by C. Sullivan (The College of Texas at Austin). The A549 RL-KO cell line and RNase L and RNase L-R667A RL-KO cell strains are described in (9). The era of PKR-KO and RL/PKR-KO and contours are described in (20). Cells had been maintained at 5% CO2 and 37°C in Dulbecco’s modified Eagle’s medium supplemented with fetal bovine serum (10% v/v) and penicillin-streptomycin (1% v/v). Cells examined damaging for mycoplasma contamination by the BioFrontiers Cell Tradition Core Facility. Cells had been transfected with poly(I:C) HMW (excessive molecular weight) (InvivoGen: tlrl-pic) utilizing 3 μl of Lipofectamine 2000 (Thermo Fisher Scientific) per 1 μg of poly(I:C). Cells had been handled with caspase inhibitor Z-VAD-FMK (Promega: G7231) at 20 μM focus.
A549 cells had been contaminated with DENV serotype 2 16681 pressure at a multiplicity of an infection (MOI) of 0.1. Cells had been mounted 48 hours after an infection. Cells had been contaminated with IAV pressure at an MOI of 0.5. Cells had been mounted 7 hours after an infection.
Immunoblot evaluation was carried out as described in (9). Rabbit anti-GAPDH (Cell Signaling Expertise: 2118L) was used at 1:2000. Anti-rabbit immunoglobulin G (IgG), horseradish peroxidase (HRP)–linked antibody (Cell Signaling Expertise: 7074S) was used at 1:3000. Anti-mouse IgG, HRP-linked antibody (Cell Signaling Expertise: 7076S) was used at 1:10,000. Rabbit anti-PARP (Cell Signaling Expertise: 9452S) was used at 1:1500. Histone H3 antibody (Thermo Fisher Scientific; NB500-171) was used at 1:1000.
Immunofluorescence and smFISH
smFISH was carried out following the producer’s protocol (https://biosearchassets.blob.core.windows.net/assets/bti_custom_stellaris_immunofluorescence_seq_protocol.pdf). GAPDH smFISH probes labeled with Quasar 570 dye (SMF-2026-1) or Quasar 670 dye (SMF-2019-1) had been bought from Stellaris. Customized IFNB1, IFNL1, IAV, and DENV smFISH probes had been designed utilizing Stellaris smFISH probe designer (Biosearch Applied sciences) obtainable on-line at http://biosearchtech.com/stellaris-designer. Reverse complement DNA oligos had been bought from IDT (knowledge file S1). The probes had been labeled with ATTO-633 utilizing ddUTP-Atto633 (Axxora: JBS-NU-1619-633), ATTO-550 utilizing 5-Propargylamino-ddUTP (Axxora; JBS-NU-1619-550), or ATTO-488 utilizing 5-Propargylamino-ddUTP (Axxora; JBS-NU-1619-488) with terminal deoxynucleotidyl transferase (Thermo Fisher Scientific: EP0161) as described in (34).
For immunofluorescence detection of IAV NS1, cells had been incubated with the anti–influenza A virus NS1 rabbit antibody (GeneTex; GTX125990) at 1:1000 for two hours, washed thrice, after which incubated with the goat anti-rabbit IgG H&L (heavy and light-weight) fluorescein isothiocyanate (FITC) (Abcam; ab6717) at 1:2000 for 1 hour. After three washes, cells had been mounted, after which smFISH protocol was carried out. For detection of DENV NS3, cells had been incubated with anti-DENV NS3 protein antibody (GeneTex: GTX124252) at 1:1000 for two hours after which incubated with goat anti-rabbit IgG H&L (Alexa Fluor 647) (Abcam: ab150079) for 1 hour. Mouse anti-NPC protein antibody (Abcam; ab24609) was used at 1:500 and detected with goat anti-mouse IgG H&L (FITC) (Abcam; ab97022) used at 1:2000.
Microscopy and picture evaluation
Coverslips had been mounted on slides with VECTASHIELD Antifade Mounting Medium with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories; H-1200). Pictures had been obtained utilizing a wide-field DeltaVision Elite microscope with a 100× goal utilizing a PCO Edge sCMOS digital camera. Between 10 and 15 Z planes at 0.2 μm per part had been taken for every picture. Deconvoluted photographs had been processed utilizing ImageJ with FIJI plugin. Z planes had been stacked, and minimal and most show values had been set in ImageJ for every channel to correctly view fluorescence. Fluorescence depth was measured in ImageJ. Single cells had been outlined by figuring out the cell boundaries by way of background fluorescence and imply depth, and built-in depth was measured within the related channels. Imaris Picture Evaluation Software program (Bitplane) (College of Colorado Boulder, BioFrontiers Superior Mild Microscopy Core) was used to quantify smFISH foci in nucleus and cytoplasm. Single cells had been remoted for evaluation by defining their borders by way of background fluorescence. Whole foci above background threshold depth had been counted. Afterward, the nucleus marked with DAPI was masked, and foci had been counted within the cell on the identical depth threshold cutoff, yielding the cytoplasmic foci depend, from which the nuclear foci quantity could possibly be decided.
RT-qPCR for IFN-B1 was carried out as described in (9). WT and RL-KO A549 cells (12-well format; 60% confluent) had been transfected with or with out poly(I:C). Six hours after transfection, crude nuclear and cytosolic fractions had been obtained as described in (35). RNA was extracted from every fraction, handled with deoxyribonuclease (DNase) I (NEB) for 15 min, repurified by way of ethanol (75%) sodium acetate (0.3 M) precipitation, and resuspended in 15 μl of water. Equal volumes of RNA from every fraction had been then reverse-transcribed utilizing SuperScript III reverse transcriptase (Thermo Fisher Scientific) and polydT(20) primer (Built-in DNA Applied sciences). cDNA was diluted to 100 μl. cDNA (2 μl) was added to qPCR response containing iQ SYBR inexperienced grasp combine (Bio-Rad) and 10 pmol of gene-specific primers. Reactions had been run in duplicate or triplicate (technical replicates) on a CFX96 qPCR machine (Bio-Rad) utilizing the usual two-step cycle.
Quantification of secreted cytokines
WT and RL-KO A549 cells (six-well format, 1 ml or medium, and 70% confluency) had been transfected with poly(I:C). At 6 and 12 hours after poly(I:C), 50 μl of medium was faraway from the effectively and instantly assayed by way of enzyme-linked immunosorbent assay (ELISA) following the producer’s directions. IFN beta human ELISA Package (Thermo Fisher Scientific; 414101) was used to quantify IFNB1. Human IL-29/IFN-lambda 1 ELISA Package (Novus Biologicals; NBP1-84819) was used to quantify IFNL1. Time zero was taken by eradicating 50 μl of medium earlier than poly(I:C) transfection.