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Pathogen-Associated Molecular Pattern Recognition of Hepatitis C Virus Transmitted/Founder Variants by RIG-I Is Dependent on U-Core Length

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Pathogen-Associated Molecular Pattern Recognition of Hepatitis C

Virus Transmitted/Founder Variants by RIG-I Is Dependent on

U-Core Length

Alison Kell,aMark Stoddard,bHui Li,bJoe Marcotrigiano,cGeorge M. Shaw,bMichael Gale, Jr.a

Center for Innate Immunity and Immune Disease, Department of Immunology, School of Medicine, University of Washington, Seattle, Washington, USAa ; Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USAb

; Center for Advanced Biotechnology and Medicine, Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, New Jersey, USAc

ABSTRACT

Despite the introduction of direct-acting antiviral (DAA) drugs against hepatitis C virus (HCV), infection remains a major pub-lic health concern because DAA therapeutics do not prevent reinfection and patients can still progress to chronic liver disease. Chronic HCV infection is supported by a variety of viral immune evasion strategies, but, remarkably, 20% to 30% of acute infec-tions spontaneously clear prior to development of adaptive immune responses, thus implicating innate immunity in resolving acute HCV infection. However, the virus-host interactions regulating acute infection are unknown. Transmission of HCV in-volves one or a few transmitted/founder (T/F) variants. In infected hepatocytes, the retinoic acid-inducible gene I (RIG-I) pro-tein recognizes 5=triphosphate (5=ppp) of the HCV RNA and a pathogen-associated molecular pattern (PAMP) motif located within the 3=untranslated region consisting of poly-U/UC. PAMP binding activates RIG-I to induce innate immune signaling and type 1 interferon antiviral defenses. HCV poly-U/UC sequences can differ in length and complexity, suggesting that PAMP diversity in T/F genomes could regulate innate immune control of acute HCV infection. Using 14 unique poly-U/UC sequences from HCV T/F genomes recovered from acute-infection patients, we tested whether RIG-I recognition and innate immune acti-vation correlate with PAMP sequence characteristics. We show that T/F variants are recognized by RIG-I in a manner dependent on length of the U-core motif of the poly-U/UC PAMP and are recognized by RIG-I to induce innate immune responses that re-strict acute infection. PAMP recognition of T/F HCV variants by RIG-I may therefore impart innate immune signaling and HCV restriction to impact acute-phase-to-chronic-phase transition.

IMPORTANCE

Recognition of nonself molecular patterns such as those seen with viral nucleic acids is an essential step in triggering the im-mune response to virus infection. Innate immunity is induced by hepatitis C virus infection through the recognition of viral RNA by the cellular RIG-I protein, where RIG-I recognizes a poly-uridine/cytosine motif in the viral genome. Variation within this motif may provide an immune evasion strategy for transmitted/founder viruses during acute infection. Using 14 unique poly-U/UC sequences from HCV T/F genomes recovered from acutely infected HCV patients, we demonstrate that RIG-I bind-ing and activation of innate immunity depend primarily on the length of the uridine core within this motif. T/F variants found in acute infection contained longer U cores within the motif and could activate RIG-I and induce innate immune signaling suffi-cient to restrict viral infection. Thus, recognition of T/F variants by RIG-I could significantly impact the transition from acute to chronic infection.

R

ecognition of nonself molecular patterns is the first step in the immune response to infection and serves to trigger innate immune defenses. During acute virus infection, cellular pattern recognition receptors (PRRs) are responsible for identifying in-coming transmission/founder (T/F) genomes and/or their prod-ucts as pathogen-associated molecular patterns (PAMPs). The PAMP/PRR interaction then induces signaling of cell-intrinsic in-nate immunity, resulting in an antiviral state within the infected cell and the production of antiviral and proinflammatory cyto-kines that activate neighboring cells for antiviral immune defense. Retinoic acid-inducible gene I (RIG-I) is a cytosolic PRR that is expressed at a low level in most cells of the body and increases in abundance in response to interferon (IFN). RIG-I recognizes viral RNA (vRNA) PAMPs containing exposed 5=phosphates, includ-ing triphosphate (5=ppp), with double-stranded RNA (dsRNA) motifs and/or poly-uridine/cytosine (pU/UC) motifs (1–4; re-viewed in references5and6). Upon engaging a PAMP, RIG-I

undergoes an ATP-dependent conformational change (7,8), re-leasing the two N-terminal caspase activation and recruitment domains (CARDs) for interaction with the mitochondrial antivi-ral signaling (MAVS) mitochondrion-associated membrane pro-tein, to initiate downstream innate immune signaling (9–11). Sig-naling through MAVS results in phosphorylation of transcription

Received4 August 2015 Accepted19 August 2015

Accepted manuscript posted online26 August 2015

CitationKell A, Stoddard M, Li H, Marcotrigiano J, Shaw GM, Gale M, Jr. 2015.

Pathogen-associated molecular pattern recognition of hepatitis C virus transmitted/founder variants by RIG-I is dependent on U-core length. J Virol 89:11056 –11068.doi:10.1128/JVI.01964-15.

Editor:R. M. Sandri-Goldin

Address correspondence to Michael Gale, Jr., mgale@uw.edu.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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factors interferon regulatory factor 3 (IRF3) and IRF7 and activa-tion of NF-␬B, driving the expression of interferon beta (IFN-␤) and other IFN-stimulated genes (ISGs) with antiviral and immu-nomodulatory functions (6,12). RIG-I is essential to control nu-merous viral infections, including infections by members of the orthomyxovirus, paramyxovirus, reovirus, and flavivirus families (4,13–19; reviewed in reference5). The importance of this path-way in determining the outcome of viral infection is demonstrated by the variety of viral antagonists shown to interfere with RIG-I-and MAVS-dependent signaling (20–31). Further, careful dis-crimination between self and nonself molecular patterns is essen-tial to prevent triggering of an innate immune response to the host. Thus, PAMPs that trigger RIG-I activation are shown to contain two or more recognition motifs, including exposed 5=ppp with specific RNA structure or sequence motifs, to fully define a nonself RNA molecule (5).

Hepatitis C virus (HCV) is a hepatotropic, positive-sense, sin-gle-stranded RNA virus that establishes a chronic infection in most people after acute exposure. Chronic HCV infection is tightly linked to the development of hepatic disease and liver can-cer and is a chief cause of illness requiring liver transplantation in the United States and Japan (32,33). Liver inflammation, fibrosis, and cirrhosis are the consequences of persistent HCV replication and dispose patients to the development of hepatocellular carci-noma. Early recognition of HCV and responses by the innate im-mune system are hypothesized to explain the occasional sponta-neous resolution of disease in acute-infection patients (34–36). Thus, evasion of this innate response by the virus may lead to a transition from acute to chronic infection (35,36).

Previous studies showed that RIG-I recognition of HCV is de-pendent on a poly-uridine/cytosine-rich motif found in the 3=

untranslated region (3=UTR) (1,2). Those studies used artificial RNA sequences to show that HCV PAMP recognition is both length dependent and sequence dependent and also requires a 5=ppp. This pU/UC motif is approximately 100 nucleotides (nt) in length and is essential for viral replication (37–39). While the presence and location of the pU/UC motif in the 3=UTR of HCV are highly conserved, significant variability in the pU/UC se-quence and in the length of poly-uridine stretches is observed (2). Reducing the poly-uridine sequence, or “U core,” to below 17 nucleotides, or altering the ribocytosine composition, ablates RIG-I recognition of the pU/UC motif as a PAMP, and such RNAs do not trigger innate immune signaling when introduced into human cells (1,2). Thus, the U core and a 5=ppp are considered to be the essential determinants of RIG-I recognition of HCV infec-tion. Importantly, priming of human hepatocytes through trans-fection of the native pU/UC RNA sequence alone induces RIG-I signaling and a potent innate immune response that restricts sub-sequent viral infection (1,2). To counteract the effects of RIG-I-dependent innate immune signaling, the HCV-encoded NS3/4A protease targets and cleaves MAVS to disrupt CARD-CARD in-teractions with RIG-I, thus abrogating the RIG-I pathway and the cell-intrinsic innate immune response to infection (36,40). Our studies have shown that NS3/4A cleavage of MAVS and RIG-I pathway inactivation are features of chronic HCV infection (36) wherein NS3/4A targeting of MAVS is necessary to support per-sistent viral RNA replication (35). In terms of innate antiviral immunity, however, MAVS cleavage itself is not sufficient for cau-sation of chronic infection, as hepatitis A virus also directs MAVS cleavage but, in contrast to HCV, mediates an acute, self-limiting

infection (41). Thus, along with NS3/4A control of the RIG-I pathway, additional parameters of innate immune regulation op-erate to support the transition from acute to chronic HCV infec-tion.

HCV is most often transmitted via parenteral exposure to blood or blood products, although mucosal exposures, especially in the setting of preexisting human immunodeficiency virus type 1 (HIV-1) infection, are responsible for a significant number of new cases (42). Establishment of infection generally results from the acquisition of one or more transmitted/founder (T/F) viruses, which can be identified unambiguously by single-genome se-quencing of acute-infection plasma vRNA and phylogenetic infer-ence. In a study of normal human plasma donors who were in-fected with HCV as a result of either mucosal or intravenous exposure, the numbers of T/F viruses leading to productive clini-cal infection ranged from 1 to more than 30, with a median of 4 (43). Follow-up studies of other categories of plasma donors again showed a wide range in the numbers of T/F viruses resulting in productive infection from 2 to 12 or more (reference44and un-published data), whereas more-recent studies in HIV-1-positive men who have sex with men found a lower number (median of 1) of T/F genomes, likely as a consequence of mucosal acquisition of the virus. Widely differing numbers of HCV virions in an inocu-lum leading to widely different numbers of T/F viruses could re-sult in challenges to the innate immune system to constrain early virus replication. Differential innate immune inductions and the onset of viral evasion strategies could contribute to various rates of infection and the virus population bottleneck observed at trans-mission (43).

In order to study the molecular, biological, and immuno-pathogenic properties of HCV T/F viruses and their contributions to early virus-host interactions, single-genome sequence (SGS) analyses were conducted to include full-length T/F genomic anal-yses of HCV, including the 5=and 3=UTR (44). Six subjects from whom a total of 7 unique full-length T/F genomes were inferred on the basis of SGS analyses of the 5=UTR, all structural and regulatory genes, and the X region were previously reported (44). The sequences of these T/F genomes, exclusive of polyU/UC se-quences, were unambiguous because, in each case, the plasma vRNA/cDNA sequences conformed to a model of random varia-tion from which a coalescent T/F genome could be inferred. The corresponding pU/UC sequences exhibited length variations due to polymerase slippage exhibited by the Moloney murine leuke-mia virus (MuLV) reverse transcriptase at polyU sequences dur-ing the cDNA step of the analysis. Extensive analysis of polyU/UC sequences from the six acute-infection subjects included sequen-tial sampling and analysis of plasma vRNA throughout the acute-infection period. Results from these analyses indicated that MuLV reverse transcriptase and, to a lesser extent,Taqpolymerase were associated with artificially shortened polyU/UC sequences of T/F genomes (44). HCV RNA-dependent RNA polymerase slippage could also occur but was assumed to contribute less given the known structure-function relationships of this polymerase and its processivity. On the basis of this information, a total of 14 full-length HCV genomic clones, corresponding to 7 T/F genomes from 6 acutely infected subjects, were generated. Each of the 7 T/F genomes was constructed to contain the longest polyU/UC tract observed in the respective subjects and representing the native viral T/F genome. Additional genomes were constructed that con-tained as many as one to three polyU/UC tracts of shorter length,

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taking advantage of the slippage characteristics associated with the MuLV reverse transcriptase (see Fig. S4 in reference44). We hy-pothesized that variation within these pU/UC sequences would impart differential recognition by RIG-I and that those variants with a shorter U-core length would evade RIG-I interactions and signaling whereas sequences with longer pU/UC tracts represent-ing authentic T/F virus variants should be recognized by RIG-I and result in robust innate immune stimulation in human hepa-toma cells. Together, these sequences provide a set of RNA ligands with diverse properties that can be used to determine the defining features of RIG-I pU/UC PAMP ligands of acute HCV infection.

Here, we quantitatively measured the RIG-I binding, RNA-induced conformational change of RIG-I that programs its signal-ing properties and the RIG-I ATPase-inducsignal-ing activities of the 14 pU/UC sequences. We also assessed the innate immune signaling and antiviral response induced by the pU/UC sequences to ascer-tain how each sequence can impart RIG-I signaling to control the outcome of acute HCV infection. Our studies show that RIG-I is indeed essential for recognition of incoming HCV PAMP RNA but that recognition by RIG-I is subject to variation linked to the composition and length of the U core within each pU/UC motif. Our observations suggest that the incoming T/F virions are ro-bustly recognized by RIG-I during acute HCV infection and that length-dependent differences in the U core remain the strongest determining factor in RIG-I activation and signaling.

MATERIALS AND METHODS

Cells and viruses.Huh7 cells and Huh7.5 cells were cultured in Dulbec-co’s modified Eagle medium (DMEM) supplemented with 10% fetal bo-vine serum (FBS) and 100 mg/ml of penicillin and streptomycin. Huh7.5 cells encode a mutant RIG-I protein that cannot signal and are therefore highly permissive of HCV infection (45,46). The hepatitis C virus (HCV) used in these studies was a cell culture-adapted variant that was produced from the synthetic JFH-1 HCV 2a infectious clone as previously described (36,47).

Plasmids and proteins.Plasmid pJFH-1 (47,48) has been previously described. Plasmids encoding the full sequences of transmitted/founder viruses were previously described (see Fig. S4 in reference44). Sequences for HCV strains 105431TF1.UC1, 105431TF2.UC1, and 105431TF2.UC2 were provided in the pCRXL-TOPO vector, while the sequences for all other strains were provided in the pBR322 vector. Purified recombinant full-length RIG I protein was kindly provided by J. Marcotrigiano (Rut-gers University) and was produced as previously described (7).

RNA methods.Allin vitro-transcribed RNAs contained a 5= triphos-phate (5=-ppp) and three guanine nucleotides at the 5=end to enhance T7 polymerase transcription. 5=-ppp RNAs were synthesized from T7 pro-moter-linked complementary oligonucleotides (Integrated DNA Tech-nologies) for the HCV Con1 pU/UC RNA (5=-TAATACGACTCACTAT AGGCCATCCTGTTTTTTTCCCTTTTTTTTTTTCTTTTTTTTTTTTT TTTTTTTTTTTTTTTTTTTTTCTCCTTTTTTTTTCCTCTTTTTTTCC TTTTCTTTCCTTT-3=) and HCV Con1 X RNA (5=-TAATACGACTCA CTATAGGTGGCTCCATCTTAGCCCTAGTCACGGCTAGCTGTGAA AGGTCCGTGAGCCGCTTGACTGCAGAGAGTGCTGATACTGGC CTCTCTGCAGATCAAGT-3=). 5=-ppp RNAs for the HCV T/F pU/UC regions were synthesized from T7 promoter-linked PCR products derived from plasmids encoding full-length T/F virus sequences. The amplified PCR product was purified by agarose gel extraction using a QIAquick gel extraction kit (Qiagen) per the manufacturer’s protocol. All RNA prod-ucts were generated using T7 RNA polymerase and a T7 MEGAshortscript kit (Ambion) according to the manufacturer’s instructions. Followingin vitrotranscription, DNA templates were removed with DNase treatment and unincorporated nucleotides and protein were removed from the re-action mixture by phenol-chloroform extrre-action. RNA was then

precipi-tated using ethanol and ammonium acetate as described by the manufac-turer and resuspended in nuclease-free water. RNA concentrations were determined by absorbance using a Nanodrop spectrophotometer. RNA quality (Fig. 1) was assessed on denaturing 2% agarose formaldehyde gels.

Electromobility shift assay.Various amounts of purified recombi-nant full-length RIG-I protein (0 to 40 pmol) were mixed with 6 pmol RNA and 10␮l ATPase reaction buffer (20 mM Tris-HCl [pH 8.0]; 1.5 mM MgCl2; 1.5 mM dithiothreitol [DTT]). Reaction mixtures were incu-bated at 37°C for 15 min, and then 4⫻native sample buffer (25 mM Tris-HCl [pH 6.8]; 0.02% bromophenol blue; 60% glycerol) was added to samples. Products were separated on a 2% agarose gel (Tris-acetate-EDTA [TAE; pH 7.2]), and RNA was visualized using SYBR gold nucleic acid stain (Invitrogen). Gel shift images were analyzed using ImageJ soft-ware (National Institutes of Health), and RNA-protein binding curves were graphed using Prism 5 software (GraphPad). Percent RNA bound was calculated by dividing the signal intensity of the shifted band by the sum of the signal intensities from the shifted and unshifted bands. The background intensity value was subtracted before analysis. Values corre-sponding to 10%, 50%, and 90% effective concentrations (EC10, EC50, and EC90values, respectively) were obtained by applying nonlinear

re-gression analysis to the data with a Hillslope set at 1.0 and fixed upper and lower limits.

ATPase assay.Various amounts (0 to 1 pmol) of RNA were mixed with 5 pmol purified RIG-I protein in a total of 25␮l of ATPase reaction buffer (20 mM Tris-HCl [pH 8.0]; 1.5 mM MgCl2; 1.5 mM DTT).

Reac-tion mixtures were incubated at 37°C for 15 min, ATP (NEB) was added to the reaction mixture at a final concentration of 1 mM, and the reaction mixtures were incubated at 37°C for 15 min. The free-phosphate concen-tration was determined using Biomol Green reagent (Enzo Life Sciences) in a microplate format, and absorbance was measured at an optical den-sity at 630 nm (OD630). EC50and EC90values were obtained by applying

nonlinear regression analysis to the data with a Hillslope set at 1.0 and fixed upper and lower limits.

Limited trypsin proteolysis of RIG-I/RNA complexes. Various amounts (0 to 10 pmol) of RNA were mixed with 30 pmol of purified RIG-I protein, 2␮l of 5⫻reaction buffer (20 mM Tris-HCl [pH 8.0]; 1.5 mM MgCl2; 1.5 mM DTT; 70 mM KCl), 0.67␮l of adenylyl

imidodiphos-phate lithium salt hydrate (AMP-PNP) (10 mg/ml), and sufficient nu-clease-free water to achieve a 10-␮l total volume. Reaction mixtures were incubated for 5 min at room temperature. Sequencing-grade tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated modified trypsin (Promega) was added to the RIG-I/RNA mixtures at a protease/protein ratio of 1:20 (wt/wt), and the reaction mixtures were incubated at 37°C for 15 min. Proteolysis was stopped by adding 0.5␮l of TLCK (N␣-p

-tosyl-L-lysine chloromethyl ketone) (1 mg/ml) and by incubating reaction

mix-tures for 5 min at room temperature. SDS-PAGE Laemmli sample buffer (Bio-Rad) was then added to the samples, and reaction products were analyzed by SDS-PAGE followed by visualization with QC colloidal Coo-massie stain (Bio-Rad) according to the manufacturer’s instructions. Im-ages were analyzed using ImageJ software, the signal intensity of the band representing the repressor domain (RD) was quantified, and the results are represented as percentages of the signal intensity of Con1-treated pro-tein.

Immunoblotting and antibodies.Huh7 and Huh7.5 cells were plated on 12-well plates and incubated for 18 to 24 h at 37°C. Transfections were performed using 25 pmol RNA and a TransIT-mRNA transfection kit (Mirus) according to the manufacturer’s instructions. Protein extracts were prepared and analyzed by immunoblotting as previously described (40) using antibodies specific to phospho-IRF3 Ser396 (Cell Signaling Technology), IRF3 (from A. Rustagi at University of Washington, Seattle, WA), RIG-I (Enzo Life Sciences), ISG56 (from G. Sen at Cleveland Clinic Foundation, Cleveland, OH), Mx-1 (W. Lai, Antibody Production Core, University of Texas Southwestern Medical Center), and tubulin (Sigma). All secondary antibodies were obtained from Jackson ImmunoResearch,

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and immunoreactive bands were detected with Amersham ECL Plus Western blotting detection reagents (GE Healthcare).

Real-time PCR.Huh7 and Huh7.5 cells were plated on 24-well plates and incubated for 18 to 24 h at 37°C. Transfections were performed using 15 pmol RNA with a TransIT-mRNA transfection kit (Mirus) according to the manufacturer’s instructions. Huh7 and Huh7.5 cells were har-vested, and RNA was extracted using an RNeasy kit (Qiagen) according to the manufacturer’s instructions. Synthesis of cDNA was conducted using an iScript Select cDNA synthesis kit (Bio-Rad) with both oligo(dT) and random primers following the manufacturer’s instructions. One-step real-time quantitative PCR was performed with SYBR green master mix (Applied Biosystems) using an ABI Prism 7300 real-time PCR system. Gene-specific primers for human IFN-␤, tumor necrosis factor alpha (TNF-␣), ISG56, interleukin-6 (IL-6), Mx-1, RIG-I, and GAPDH

(glyc-eraldehyde-3-phosphate dehydrogenase) were purchased from Inte-grated DNA Technologies. Results were normalized to the expression of GAPDH mRNA. Results were analyzed and graphed using Prism 5 soft-ware (GraphPad).

HCV infection.Huh7 cells were plated on 48-well plates and incu-bated for 12 to 24 h at 37°C. Cells were transfected with 15 pmol RNA using a TransIT-mRNA transfection kit (Mirus) per the manufacturer’s instructions and incubated at 37°C for 18 h. The transfection medium was removed, and the cells were washed gently with complete DMEM. Trans-fected cells were then inTrans-fected with cell culture-adapted JFH-1 HCV (mul-tiplicity of infection [MOI]⫽1) in a 100-␮l total medium volume and incubated at 37°C for 3 h. The virus inoculum was then removed, 500␮l complete DMEM was added, and the cells were incubated at 37°C for 48 h. HCV-infected cellular RNA was harvested using an RNeasy kit (Qiagen). FIG 1RIG-I binds T/F pU/UC RNA. (A) T/F pU/UC RNA on denaturing agarose gel. (B) Electrophoretic mobility shift assays using 6 pmol RNA incubated with recombinant RIG-I protein. (C) Densitometric analysis was performed to measure the relative quantities of RNA from the shifted region and the unshifted region; data are plotted as percent RNA shifted. (D) Effective millimolar concentrations of RIG-I at which 10%, 50%, or 90% of RNA was shifted in the gel. An asterisk (*) denotes anin vivoT/F pU/UC sequence; “-V” denotes anin vitroU-core variant.

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The copy number of HCV RNA was measured by quantitative real-time PCR with a TaqMan Fast Virus 1-step kit and primers specific for the 5=

UTR (Pa03453408_s1; Life Technologies). The copy number was calcu-lated by comparison to a standard curve ofin vitro-transcribed full-length HCV RNA. HCV-infected cell supernatants were collected and subjected to titration on Huh7.5 cells as previously described (2).

Correlation analyses.Each pU/UC RNA was assigned an “activity rank.” This designation was applied based on the EC50values determined

by electrophoretic mobility shift assay (EMSA) and ATPase assay, the percentage of RD accumulation following limited proteolysis, immune stimulation as measured by Western blot analysis and reverse transcrip-tase PCR (RT-PCR), and induction of the innate immune response for infection restriction in Huh7 cells. The ranking system gives RNAs with high RIG-I stimulatory activity (low EC50values, strong innate immune

induction) low numerical values and those with decreasing stimulatory activity increasing numerical values, from 1 to 15. By assigning a simple numerical score to the raw data, we were able to perform correlation analyses to identify motifs within the pU/UC sequences that are impor-tant for RIG-I recognition. A two-tailed Spearman correlation analysis was performed with activity rank as the dependent variable and pU/UC characteristic (U-core length, pU/UC length, total number of uridines) as the independent variable. Bonferroni correction for multiple tests yielded an␣value of 0.0083.

RESULTS

Poly-U/UC sequences from transmitted/founder viruses andin vitrovariants.A subset of 14 sequences representing T/F viruses from 6 patients acutely infected with HCV involved in a previous study were selected for further investigation into RIG-I recogni-tion and activarecogni-tion (43). The sequences of the pU/UC regions of these genomes are shown inTable 1. Sequences are labeled with patient identifiers (A to F) and with numbers indicating unique pU/UC motifs. The T/F variant genomes within all of the patients were identical except for the pU/UC sequence. As described by Stoddard et al. (44), variations in the length of the U core between

multiple strains from a single patient are attributed to MuLV poly-merase slippage during cDNA synthesis, where the sequence with the longest U core of each set is representative of the true T/F isolate. Therefore, single-patient variants with differential U-core lengths were included in our analysis as internal comparators to determine how U-core length influences the PAMP activity of T/F variants. Those sequences that are representative of the T/F isolate for each patient are denoted by an asterisk inTable 1, with the associated MuLV-derived variant denoted by “-V.” We previously defined the pU/UC motif as containing three distinct regions, the U core and its flanking 5=and 3=arms (2). The 5=and 3=arms contain poly-uridine stretches with interspersed ribocytosine and riboguanosine nucleotides, while the U cores are composed en-tirely of uridine nucleotides and differ in length. In chronic HCV infection, the pU/UC motif has been shown to range from 12 to 96 nt in length among different viral genotypes, although the same qualifications exist with respect to MuLV polymerase slippage in those other studies (2). We found that the pU/UC motifs in our sequences ranged overall from 100 to 178 nt in length, including the 5=and 3=arms and the U-core motif (Table 1andFig. 1A). Among the sequences representing each T/F isolate, the U core ranged in length from 23 to 49 nt, within vitroU-core variants ranging in length from 13 to 18 nucleotides.

[image:5.585.40.542.76.325.2]

Poly-U/UC sequences from T/F viruses differentially bind and activate RIG-I.To determine how variation among T/F ge-nome pU/UC motifs impacts PAMP recognition by RIG-I, we performed EMSAs using purified, recombinant human RIG-I protein to assess binding to each 5=ppp RNA, including T/F pU/UC RNA and controls. Increasing amounts of recombinant RIG-I protein were incubated with purified RNA and subjected to nondenaturing agarose gel electrophoresis. Sybr gold staining was then used to visualize unbound RNA and shifted RIG-I/RNA complexes. As shown inFig. 1B, the positive-control pU/UC RNA

TABLE 1HCV pU/UC RNA sequences from T/F virus genomesa

Founder

sequence Strain designation Genotype pU/UC

length (nt) 5=arm sequence U core 3=arm sequence

HCV Con1 Con1 1b 122 GGCCAUCCUG(U7)CCC(U11)C U34 CUCC(U9)CCUC(U7)CCUUUUCUUUCCUUU

A1* 10021TF.UC1 1a 146 GGCCAUUUCCUG U33 ACCCUUUUUUCUC(U12)CCUUCUUCUUUAAU

A2-V 10021TF.UC2 1a 136 GGCCAUUUCCUG U18 ACCCUUUUUUCUC(U17)CCUUCUUCUUUAAU

B1* 10051TF.UC1 1b 147 GGCCAUCCUG U24 G(U17)CUUUUUCC(U13)AUUUUCUUCUUU

C1-V 9055TF.UC1 3a 141 CCAUUUUUC U13 GUUUG(U16)CUUUCCUUCUUUCCUGACUUU

UAAUUUUCCUUCUUA

C2* 9055TF.UC2 3a 178 CCAUUUUUC U49 GUUUG(U17)CUUUCCUUCUUUCCUGACUUU

UAAUUUUCCUUCUUA

D1* 110069TF1.UC1 1a 157 GGCCAUUUCUG U41 GUUUCCUUCUUUUUCCUUUUC(U11)CUCCC

UUUAAU

D2-V 110069TF1.UC2 1a 132 GGCCAUUUCUG U14 GUUUCCUUCUUUUUCCUUUUC(U13)CUCCC

UUUAAU

E1-V 10025TF.UC1 1a 100 GGCCAUUUUCUG U14 AUUUUCUUUAAU

E2-V 10025TF.UC2 1a 115 GGCCAUUUUC(U10)CUC U18 AUUUUCUUUAAU

E3-V 10025TF.UC3 1a 124 GGCCAUUUUCUG U20 CC(U12)CCUC(U20)AUUUUCUUUAAU

E4* 10025TF.UC4 1a 144 GGCCAUUUUCUG(U12)C U17 CCUUUUUUUUUCUC(U14)AUUUUCUUUAAU

F1-V 105431TF.UC1 4a 121 GGUCCUAAG U13 CUUCCUUCCUUCUUUCCUUUUCUAAUUUUC

CUUCUUU

F2-V 105431TF2.UC1 4a 124 GGUCCUAAGUUG U15 CCUUCCUUCUUUCCCUUUUCUAAUUUUCCU

UCUUU

F3* 105431TF2.UC2 4a 136 GGUCCUAAGUUG U23 CCUUUCCUUCCUUCUUUCCUUUUCUAAUUU

UCCUUCUUU

aAn asterisk (*) denotes anin vivoT/F pU/UC sequence; “-V” denotes anin vitroU-core variant.

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from the HCV Con1 genome, a validated RIG-I PAMP (1,2), formed a stable complex with increasing amounts of RIG-I. In contrast, reaction of RIG-I with 5=ppp RNA representing the 100-nt X region (X RNA) containing the highly conserved 3= non-translated region (NTR) 3-stem-loop structures (49) of the HCV genome did not form a complex with RIG-I even in the presence of a high RIG-I concentration. Importantly, we observed differ-ential binding to RIG-I among the pU/UC RNAs from T/F vari-ants. Whereas all T/F pU/UC sequences bound to RIG-I at the highest protein concentration, binding was variable at a lower RIG-I concentration that more closely approximates intracellular RIG-I levels. We observed variable nanomolar effective concen-tration (EC) levels of RIG-I required to shift 50% (EC50) of the HCV pU/UC RNAs ranging from 6 nM to 503 nM by EMSA (Fig. 1CandD). Thus, while all T/F pU/UC RNAs can be recognized by RIG-I, they exhibit differential levels of RIG-I binding affinity. Notably, those sequences that bound to RIG-I at low concentra-tions similar to that of the Con1 control included C2 and D1, which contain the longest U cores of the T/F RNAs. In contrast, those sequences with limited binding by RIG-I (E4 and F1) con-tain short U cores.

During virus infection, PAMP recognition and binding by RIG-I induce ATPase activity that drives a conformational change mediating downstream signaling (7,50). We therefore assessed pU/UC-induced ATPase activity and conformational changes of RIG-I following binding to pU/UC RNAs. As expected, each T/F pU/UC RNA motif was capable of stimulating RIG-I ATPase ac-tivity at a level in a range between those seen with the Con1 and X RNA controls (Fig. 2). However, we observed marked variations in the ability of the T/F pU/UC sequences to induce ATPase activ-ity, with the differences in the respective picomolar EC50and EC90 values corresponding to the pU/UC RNA required for ATPase activity induction ranging from 3-fold to over 17-fold.

In the resting state, RIG-I is maintained in an auto-repressed, inactive conformation in which the CARDs are bound to the C-terminal repressor domain (RD) and the helicase insertion do-main (Hel2i) through inhibitory interaction (8,50, 51). Upon recognition of and binding to RNA, RIG-I undergoes an ATPase-driven conformational change to bring the RNA into close contact with the helicase domains and to release the CARDs for interac-tion with MAVS, thus initiating downstream innate immune sig-naling (7,52–54). To assess the RIG-I conformational change in the presence of T/F pU/UC RNA, we conducted limited trypsin proteolysis of RIG-I/RNA complexes (Fig. 3). In the absence of bound RNA, RIG-I exists in a “signaling-off” conformation that is sensitive to trypsin proteolysis, but upon binding to PAMP RNA and ATP hydrolysis, RIG-I changes to a “signaling-on” conforma-tion that protects the 17-kDa RD from trypsin digesconforma-tion and that can be visualized by protein separation by SDS-PAGE (Fig. 3A, Con1 RNA) (1,2,50). We found that the RIG-I signaling confor-mational changes were differentially induced by T/F pU/UC RNA (Fig. 3). At lower and physiological levels (3 pM) of RNA, 4 of the 14 pU/UC sequence variants induced a conformational change that protected the RD at a level equal to that seen with the Con1 pU/UC control, while at saturating levels of RNA, all T/F RNAs induced RD protection, albeit to various degrees (Fig. 3). Because maximal RD accumulation is not quantifiable for this assay, we were unable to quantify the kinetics of RIG-I conformational change. However, a qualitative assessment of RIG-I RD accumu-lation with increased RNA concentrations supports the idea of

differential recognition and binding of RIG-I to the T/F pU/UC motifs, consistent with data quantified as described above. This observation indicates that RIG-I is capable of recognizing all pU/UC RNA motifs but does so with RNA binding affinity that promotes differential ATPase activity and a change to the signal-ing-on conformation. Thus, RIG-I binding and recognition of T/F pU/UC RNA motif are not uniform such that differential recog-nition of HCV T/F RNA could result in innate immune stimula-tion with differential abilities to control infecstimula-tion.

T/F pU/UC RNA sequences differentially trigger RIG-I-de-pendent innate immune signalingin vitro.To determine how the variable RIG-I binding and activation properties of the T/F pU/UC PAMP RNA impose innate immune signaling actions of RIG-I, we transfected each RNA into Huh7 hepatoma cells and assessed their ability to trigger innate immune activation, includ-ing the activation/phosphorylation of IRF3 and induction of IRF3 target genes (IFIT1 and IFN-␤), ISGs (IFIT1, MX1, and RIG-I), and proinflammatory cytokines (TNF-␣and IL-6). Phosphoryla-tion of IRF3 was observed following transfecPhosphoryla-tion with Con1 pU/UC and pU/UC regions from six different T/F variants, A1, B1, C2, D1, E4, and F3. These same RNA constructs induced the protein expression of IFIT1, MX1, and RIG-I (Fig. 4A). We found that IFN-␤mRNA was strongly induced following transfection with a subset of these stimulatory T/F RNAs (Fig. 4C). Notably, sequences C2 and D1 strongly induced IFN-␤transcription as expected based on their ability to bind RIG-I and induce RIG-I ATPase activity and conformational change (Fig. 1to3). In con-trast, transfection with other T/F pU/UC RNAs resulted in low levels of ISG protein and gene expression above those seen with X RNA but below those seen with Con1 pU/UC or other stimulatory T/F RNAs. This lack of innate immune signaling following trans-fection correlated with low levels of RIG-I binding and activation of these RNAs, as shown in the previous assays. In summary, pU/UC sequences of T/F isolates from acutein vivoinfections are efficiently recognized as PAMP by RIG-I to elicit robust innate immune signaling.

It remains possible that some level of innate immune signaling could occur through pathways other than RIG-I, such as Toll-like receptor 2 (TLR3), TLR7, or melanoma differentiation-Associ-ated protein 5 (MDA5) pathways (55–58). Thus, we assessed sig-naling of T/F pU/UC RNA in Huh7.5 cells which lack functional RIG-I but retain TLR and MDA5 signaling (59). As shown inFig. 4BandD, these cells were refractory to innate immune signaling induction by the Con1 pU/UC control PAMP RNA and by the T/F pU/UC RNAs. Thus, HCV pU/UC PAMP motif recognition and innate immune signaling are mediated by RIG-I.

Innate immune response from T/F pU/UC RNA differen-tially restricts HCV replicationin vitro.RIG-I signaling imparts an innate immune response that can effectively restrict HCV in-fection such that viral targeting of MAVS to abrogate the RIG-I pathway is essential for supporting HCV persistence (10,35,36,

60). However, it is not known how differences in PAMP recogni-tion by RIG-I may impart the control of acute HCV infecrecogni-tion, especially prior to accumulation of the viral protease that cleaves MAVS to inactivate the RIG-I pathway. To determine how differ-ential RIG-I signaling by the pU/UC PAMP motifs impacts the outcome of acute HCV infection, we assessed acute infection of HCV 2a after transfection of pU/UC RNAs to differentially trigger RIG-I signaling in Huh7 cells. Equal molar amounts of each T/F pU/UC variant, Con1 pU/UC, and the X RNA control were

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FIG 2Differential ATPase activity of RIG-I bound to T/F PAMP RNA. Graphs show the ATPase activity of 5 pmol purified RIG-I protein incubated with increasing amounts of RNA. Levels of free phosphate released upon incubation with Con1 pU/UC RNA (blue circles), X RNA (red squares), and individual T/F pU/UC RNA (black triangles) are shown as averages of the results of three or more experiments⫾standard deviations (SD). EC50and EC90values for each RNA

were calculated and are shown in the adjacent table.

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fected into cells and followed 18 h later by HCV infection (JFH1 strain; multiplicity of infection, 1.0). At 48 h postinfection, we measured cell-associated viral RNA levels and quantified infec-tious virus released into the supernatant using a sensitive focus-forming-unit assay (Fig. 5AandB, respectively). Cells treated with Con1 pU/UC but not X RNA suppressed HCV replication and virus production. However, we observed differential control of HCV among cells transfected with the T/F pU/UC RNAs such that those RNAs exhibiting the highest innate immune signaling/ PAMP activity (A1, B1, C2, D1, E4, and F3) effectively suppressed HCV infection. The pU/UC RNAs with no or low signaling activ-ity restricted only partially or failed to restrict HCV infection (compareFig. 4and5). Interestingly, modest inhibition of repli-cation was observed in cells treated with T/F pU/UC variants shown to be mildly stimulatory (A2, D2, E2, and F2) in theFig. 4

data sets. Thus, evasion of RIG-I recognition through variation in the pU/UC could effectively allow acute HCV to avoid triggering innate immune signaling prior to the production and activity of

the viral protease, thereby allowing establishment of productive infection of hepatocytes.

RIG-I stimulatory activity of pU/UC sequences correlates with U composition and the length of the U core.To define the characteristics of the pU/UC PAMPs that impart recognition and innate immune signaling activation by RIG-I, we analyzed the T/F sequences for correlation with RIG-I binding, ATPase activity, conformational change, and signaling activities. The level of stim-ulatory activity of an RNA (activity rank) was determined by the sum of the assigned activity ranks from all of the assays presented in this study, with 1 being the highest stimulatory activity and 15 being the lowest (Table 2). We then conducted statistical analysis based on two-tailed Spearman nonparametric correlation to iden-tify determinants of PAMP activity among the pU/UC variants.

Figure 6shows the correlation between the PAMP activity score and the total length of the pU/UC RNA, the ratio of uridine to nonuridine nucleotides, the total number of uridine nucleotides, and the length of the U core. The total length of the polyU/UC

FIG 3Limited trypsin proteolysis of 30 pmol purified RIG-I protein with increasing amounts (1 to 5 pmol) of RNA from T/F pU/UC regions. Following 15 min of protein digestion, products were run on a 12% SDS-PAGE gel and stained with colloidal Coomassie blue. (A) RIG-I (30 pmol) was incubated with increasing amounts of RNA (1 to 5 pmol). FL, full length. (B) RIG-I (30 pmol) was incubated with 3 pmol RNA for densitometric analysis of the quantity of RIG-I RD and CARD-containing helicase (heli-card) accumulation. The results of densitometric analysis of RD accumulation as a percentage of accumulation following incubation with Con1 pU/UC RNA are shown in the table at the bottom of the figure.

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motif itself is not correlative with PAMP activity (Fig. 6A). Impor-tantly, however, the ratio of uridine nucleotides to nonuridine nucleotides present in the pU/UC (Fig. 6B), the number of uridine nucleotides (Fig. 6C), and the length of the U core (Fig. 6D) sig-nificantly correlated with PAMP activity to bind RIG-I and trigger innate immune signaling that restricts HCV infection. Interest-ingly, in examination of the innate immune activation elicited by pU/UC transfection, the only characteristic correlated with activ-ity was U-core length, suggesting that the U core is a defining feature recognized by RIG-I to drive innate immune responses (summarized inFig. 7).

DISCUSSION

The pU/UC motif is a feature of all HCV genotypes and variants and is essential for replication viability of HCV (37). This motif represents a major PAMP of RIG-I recognition in conjunction with 5=ppp of the HCV RNA (1,2,61). In this study, we demon-strated that HCV variants responsible for establishing acute infec-tions contain pU/UC PAMP motifs that can indeed be recognized by RIG-I and thereby trigger robust innate immune responses in the host cell, whilein vitrovariants of each with shorter U-core motifs can escape RIG-I recognition. The correlation of RIG-I

FIG 4T/F HCV pU/UC sequences differentially activate RIG-I signaling. Huh7 cells (A and C) or Huh7.5 cells (B and D) were transfected with purified pU/UC RNA from the indicated T/F genomes, JFH1, or X RNA, and cells were harvested 18 h later for immunoblot analysis (A and B) or RT-PCR assay (C and D).

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activity with U-core composition and length is in support of pre-vious work demonstrating that the presence of a 34-nt poly-U stretch on an otherwise nonstimulatory RNA results in PAMP activity to activate RIG-I and induce innate immune signaling (2).

Through molecular and biochemical studies of RNA/RIG-I in-teractions designed to measure activities of each PAMP RNA and RIG-I, we observed that the pU/UC RNA motifs from the T/F variants differed in their binding, stimulation of ATPase activity, conformational changes, and signaling actions of RIG-I. The var-ious binding properties of the T/F sequences linked to the stimu-lation of differential RIG-I ATPase activity and conformational change to show that only a subset of T/F pU/UC RNAs and vari-ants were actively engaged by RIG-I to promote its activation to a signaling-on conformation. To this end, only those pU/UC RNAs which induced RIG-I to undergo the signaling-on conformational change induced innate immune signaling and IFN induction in cultured hepatoma cellsin vitro. Among these PAMP sequences, the level of RIG-I activity induced correlated with the level of innate immune signaling and the degree of protection from sub-sequent HCV challenge. Since all PAMP sequences had a 5=ppp, a major feature of RIG-I recognition of nonself RNA (3), we con-clude that differences in the pU/UC motifs are responsible for the differential RIG-I recognition results that we observed.

Previous studies using synthetic pU/UC sequences showed that a poly-U-core length of 17 nucleotides is required to elicit a robust RIG-I-dependent signaling cascade in hepatocytes (1,2). Using T/F HCV sequence from acute-infection patients, we now show that pU/UC sequence variants containing fewer than 20 uri-dine nucleotides within the U-core region were capable of detec-tion avoidance to evade RIG-I. Of note is that we did measure a level of innate immune signaling following transfection per-formed with three pU/UC RNA variants containing U cores with fewer than 20 uridines (A2, E2 and E4; seeFig. 4). Sequences E2 and E4 contain poly-U stretches of 10 and 12 uridines immedi-ately preceding the canonical U core (Table 1). These additional poly-U stretches may therefore facilitate RIG-I recognition and activation beyond the U core. However, this explanation does not apply to sequence A2, which exhibited only minimal PAMP activ-ity in our assays, nor does it hold true for sequence E3, which consists of a U core 20 uridines in length and two subsequent poly-U stretches but exhibited little or no PAMP activity. RIG-I

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FIG 5The innate immune response to pU/UC motifs restricts HCV replica-tion. (A) Huh7 cells were transfected with 15 pmol RNA from the indicated T/F genome pU/UC regions or the X region or were mock transfected in duplicate. At 18 h later, the cells were infected with HCV JFH1 (MOI⫽1). Cells were harvested 48 h postinfection, and viral RNA was quantified by a RT-quantitative (qPCR) assay. Data are shown as means of the results of two independent experiments (⫾SD). (B) Quantification of infectious virus from supernatants. Supernatant from infected cells was isolated and infectious virus measured by focus-forming-unit (ffu) assay.

TABLE 2Summary of results

RNA U-core length (nt)

RIG-I scorea

Binding Conformational change ATPase activity Innate immune signaling Immunity Activation rank

Con1 34 1 1 1 4 2 1

A1* 33 6 3 11 6 4 5

A2-V 18 14 2 6 12 6 8

B1* 24 9 12 12 7 5 10

C1-V 13 5 5 9 11 9 7

C2* 49 3 8 7 3 1 3

D1* 41 2 6 2 3 3 2

D2-V 14 12 9 8 13 10 14

E1-V 14 8 11 15 14 11 15

E2-V 18 10 10 13 5 8 11

E3-V 20 4 7 10 10 10 9

E4* 17 15 4 4 1 3 4

F1-V 13 13 13 5 9 11 13

F2-V 15 7 14 14 8 7 12

F3* 23 10 15 3 2 4 6

aScores for RIG-I binding and activation parameters of T/F pU/UC RNAs are listed. Rankings were determined by EC

50value for binding and ATPase activity, RD accumulation following trypsin digestion, densitometric analysis of protein expression, and restriction of virus replication. Activation rank values were determined by the sum of rankings from all assays and used to determine pU/UC sequence-specific correlates of RIG-I activation. Low numerical values indicate high RIG-I stimulatory activity for the respective pU/UC RNA.

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makes contact with PAMP RNA at several amino acid residues within the RD and helicase domains (7). We propose that differ-ences in PAMP sequence composition that mediate differential contacts with RIG-I binding sites would impact the stability of RNA ligand binding to render differential ATPase activity and conformational change results, thus giving various levels of RIG-I signaling activation. In this sense, alterations in pU/UC motif se-quence within the U core and 5=and 3=arms would impact overall RIG-I activation for variable restriction of virus infection.

The pU/UC region in the HCV genome has been previously demonstrated to be essential for genome replication (37). In their study, You and Rice demonstrated a minimum of 27 to 33 uridine nucleotides to be essential for efficient genome replication (37). A long poly-U tract was found to be required for stable interactions with the viral 5=UTR to maintain the kissing-loop formation of 5=

and 3=RNA ends important for HCV replication. Thus, it is likely that U-core length is evolutionarily constrained even during transmission, when pU/UC variation could provide an advantage through innate immune evasion. The variant sequences tested

here may harbor changes in the pU/UC region that disrupt dsRNA regions that are important for RIG-I recognition and/or essential for formation of the kissing loop in replication of viral genomes.

Due to the difficulty in culturing HCV strains other than JFH1 or its derivatives, these T/F viruses have not been successfully cul-tured (44,62,63). However, others have recently demonstrated that transfection of full-length T/F genomic RNA into human hepatocytes, nonparenchymal cells, and liver sinusoidal endothe-lial cells can trigger various innate immune signaling results (62,

63). Thus, full-length HCV T/F genomes retain PAMP activity in host cells. Our studies linked this PAMP activity to the pU/UC motif of the T/F genome. Additionally, other HCV RNA interac-tions may create a proinflammatory state within infected cells and could play a role in regulating inflammatory signaling indepen-dently of RIG-I. Further elucidation of the subcellular localization of RIG-I–PAMP interactions and the RNA species important for RIG-I recognition during productive infection will be key to de-fining the innate immune response to HCV.

Through the work presented here, we concluded that T/F vi-ruses that can establish acute HCV infectionin vivodo contain pU/UC motifs that are efficiently recognized by RIG-I to trigger robust innate immune signaling. While PAMP variations in U-core length can clearly allow escape of detection by RIG-I, differ-ential triggering of RIG-I by T/F genomes during acute infection may not be a dominant feature in driving an outcome of sponta-neous clearance versus progression to chronic infection. Instead, a dominant innate immune feature of progression to chronic infec-tion could be the targeted proteolysis of MAVS by the viral NS3/4A protease to abrogate the RIG-I pathway and allow persis-tent viral replication (16,18,34). Models to define the dynamics of the viral replication kinetics of acute infection and the temporal nature of MAVS cleavage by the NS3/4A protease in determining the outcome of acute HCV infection will aid in our understanding

FIG 6RIG-I stimulatory activity of pU/UC sequences correlates with U composition and the length of the U core. Each T/F pU/UC RNA was assigned an activity rank based on the level of RIG-I activation measured in biochemical assays and the innate immune response elicitedin vitro. Two-tailed Spearman nonparametric correlation was then calculated for all RNAs with an activity score based on the variable characteristics.␣ ⫽0.0083.

FIG 7Diagram (1) representing the HCV genome 3=UTR. Determinants of HCV PAMP activity are indicated. The sequence shown is that of the Con1 pU/UC region. The underlined and bolded region indicates a statistically sig-nificant correlation with RIG-I activity as shown inFig. 6. The underlined sections were shown to correlate with the overall RIG-I activation rank as mea-sured by all assays performed; the section in bold correlates with innate immune signaling and innate immune priming in Huh7 cell transfection assays.

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of virus and host control over acute-stage to chronic-stage virus infection transition.

ACKNOWLEDGMENTS

M. Gale, Jr., and A. Kell are supported by grants AI104002-01, AI88778, and AI083019 and G. M. Shaw by grant AI106000, all from the National Institutes of Health.

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Kell et al.

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Figure

FIG 1 RIG-I binds T/F pU/UC RNA. (A) T/F pU/UC RNA on denaturing agarose gel. (B) Electrophoretic mobility shift assays using 6 pmol RNA incubated withrecombinant RIG-I protein
TABLE 1 HCV pU/UC RNA sequences from T/F virus genomesa
FIG 2 Differential ATPase activity of RIG-I bound to T/F PAMP RNA. Graphs show the ATPase activity of 5 pmol purified RIG-I protein incubated withincreasing amounts of RNA
FIG 3 Limited trypsin proteolysis of 30 pmol purified RIG-I protein with increasing amounts (1 to 5 pmol) of RNA from T/F pU/UC regions
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