Riplet/RNF135, a RING Finger Protein, Ubiquitinates RIG-I to Promote Interferon-β Induction during the Early Phase of Viral Infection*

RIG-I (retinoic acid-inducible gene-I), a cytoplasmic RNA helicase, interacts with IPS-1/MAVS/Cardif/VISA, a protein on the outer membrane of mitochondria, to signal the presence of virus-derived RNA and induce type I interferon production. Activation of RIG-I requires the ubiquitin ligase, TRIM25, which mediates lysine 63-linked polyubiquitination of the RIG-I N-terminal CARD-like region. However, how this modification proceeds for activation of IPS-1 by RIG-I remains unclear. Here we identify an alternative factor, Riplet/RNF135, that promotes RIG-I activation independent of TRIM25. The Riplet/RNF135 protein consists of an N-terminal RING finger domain, C-terminal SPRY and PRY motifs, and shows sequence similarity to TRIM25. Immunoprecipitation analyses demonstrated that the C-terminal helicase and repressor domains of RIG-I interact with the Riplet/RNF135 C-terminal region, whereas the CARD-like region of RIG-I is dispensable for this interaction. Riplet/RNF135 promotes lysine 63-linked polyubiquitination of the C-terminal region of RIG-I, modification of which differs from the N-terminal ubiquitination by TRIM25. Overexpression and knockdown analyses revealed that Riplet/RNF135 promotes RIG-I-mediated interferon-β promoter activation and inhibits propagation of the negative-strand RNA virus, vesicular stomatitis virus. Our data suggest that Riplet/RNF135 is a novel factor of the RIG-I pathway that is involved in the evoking of human innate immunity against RNA virus infection, and activates RIG-I through ubiquitination of its C-terminal region. We infer that a variety of RIG-I-ubiquitinating molecular complexes sustain RIG-I activation to modulate RNA virus replication in the cytoplasm.

RIG-I-like receptors (RLRs) of RIG-I, MDA5, and LGP2, belong to the DEA(D/H) box RNA helicase family (3)(4)(5)(6). RIG-I recognizes the 5Ј end triphosphate of the virus RNA genome or double-stranded RNA (6 -8) to sense infection by various RNA viruses (3,5). The RIG-I protein consists of two N-terminal CARD-like domains, an RNA helicase region and a repressor domain (RD) (9). After recognition of positive or negative single-stranded viral RNA, RIG-I interacts with its adaptor molecule IPS-1/MAVS/Cardif/VISA leading to type I IFN production, thereby protecting host cells from amplified viral replication (10 -13). However, only a few copies of viral RNAs usually penetrate the cell membrane to enter the cell at an early infection, and these RLRs are barely present in intact as well as early virus-infected cells (6). The early viral RNA recognition facility should be different from that of the late phase when RIG-I protein is abundant in the cytoplasm and easily re-organizes the virus RNAs. What molecular mechanism is responsible for initial sensing of viral RNA thus remains unknown.
Other RLRs, MDA5 and LGP2, are structurally similar to RIG-I in their having the helicase domain (5,14). However, MDA5 lacks the RD domain although it possesses CARD-like region at the N terminus like RIG-I. LGP2 does not have a CARD-like region but possesses RD at its C terminus (9). RIG-I and MDA5 recognize different kinds of RNA viruses and in some cases play a redundant role in sensing virus infection, such as influenza B (15). In contrast, LGP2 rather negatively regulates virus replication. LGP2 expression suppressed RIG-I or MDA5 signaling (14,16), and lgp2 gene disruption conferred high susceptibility to virus infection on mice (4).
Recently, the majority of proteins involved in the type I IFNinducing system were found ubiquitinated. For example, the tumor necrosis factor receptor-associated family members, TRAF3 and TRAF6, are ubiquitin ligases to induce ubiquitination of proteins and implicated in activation of IFN regulatory factor (IRF) 3 or nuclear factor (NF) B (13,(17)(18)(19). In contrast, a deubiquitinating enzyme, DUBA or A20, suppresses these signals (19,20). In addition to ubiquitin, ubiquitin-like protein, ISG15, is also conjugated to proteins involved in the IFN-inducing pathway (21,22). Recent studies have revealed that viral RNA sensors are also ubiquitinated. TRIM25 (ZNF147 or EFP), a member of the ubiquitin-protein isopeptide ligase family, which possesses a RING finger domain, ubiquitinates the CARD-like domains of RIG-I thereby facilitating the RIG-Imediated activation of type I IFN signaling (23,24), although Shimotohno and co-workers (25) previously reported that TRIM25 (EFP) does not polyubiquitinate the RIG-I CARD-like region as far under their conditions. Expression of TRIM25 increases RIG-I CARD-like region-mediated signaling; however, it remains to be determined whether the activation of full-length RIG-I requires other ubiquitin ligase (23). Another ubiquitin ligase RNF125 mediates lysine 48-linked polyubiquitination of RIG-I, which leads to degradation of RIG-I through the proteasome (25).
Here we examined what molecular complex participates in an early RIG-I-mediated RNA recognition and IFN signaling by yeast two-hybrid screening. Here we detected two novel RING finger proteins that bound to RIG-I, and we found that one, RNF135, facilitated RIG-I-mediated type I IFN induction via ubiquitinating RIG-I. RNF135 plays a crucial role in the RIG-I response to minimal copies of viral RNA, and by binding to the C-terminal helicase and RD regions of RIG-I, RNF135 facilitates RIG-I C-terminal ubiquitination to up-regulate RIG-Imediated IFN signaling and suppress viral replication. Hence, we renamed it as RNF135 Riplet (RING finger protein leading to RIG-I activation). To our knowledge, this is the first study demonstrating that C-terminal ubiquitination of RIG-I is important for full IFN induction by RIG-I.

EXPERIMENTAL PROCEDURES
Cell Cultures-HEK293 and Vero cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum (Invitrogen), and HeLa cells were in minimum Eagle's medium with 2 mM L-glutamine and 10% fetal calf serum (JRH Biosciences). HEK293FT cells were maintained in Dulbecco's modified Eagle's high glucose medium containing 10% heatinactivated fetal calf serum (Invitrogen).
Plasmids-cDNA fragment encoding a C-terminal region of Riplet was isolated by yeast two-hybrid screening using human lung cDNA library. The 5Ј region encoding the remaining N-terminal region was amplified by PCR using primers Riplet-F1 and Riplet-R1, and human lung cDNA library was used for its template. Two cDNA fragments, which cover the entire ORF of Riplet, were joined by PCR using primers Riplet-F1, R1, F2, and R2 and then inserted into pCR-blunt vector (Invitrogen). The primers sequences are as follows: F1, GCCTCGAG-GCCACCATGGCGGGCCTGGGCCTGGG; R1, CGGCCAG-GTCCTGCAGTAGC; F2, GCACCTGCGGAAGAACACGC; and R2, GGGGATCCCACCTTTACTTGCTTTATTATC-AGG. The obtained cDNA was cloned into XhoI-NotI restriction sites of pEF-BOS expression vector, and the HA tag was fused at the C-terminal end of Riplet. Riplet-DN (dominant negative) expression vector was constructed by amplifying the relevant Riplet cDNA fragment using the primers Riplet-X-F-C and Riplet-R2 and subcloned into pEF-BOS. The primer sequence of Riplet-X-F-C was as follows: GCTCGAGGCCAC-CATGCCGCACCTGCGGAAGAACACGC. Riplet-L248fs expression vector was made by deleting 1 base at position 742 by standard PCR-mediated site-directed mutagenesis methods with primers Riplet-L248fs-F and Riplet-L248fs-R as follows: Riplet-L248fs-F, CCAGAGCCACCCTGCATCAGGAGAGC-TTCTCGG, and Riplet-L248fs-R, CCGAGAAGCTCTCCTG-ATGCAGGGTGGCTCTGG. All cloned RIPLET cDNA fragments were sequenced, and it was confirmed that there were no mutations. Full-length RIG-I expressing vector, Gal4-IRF-3, Gal4-DBD, and p55 UASG-Luc reporter plasmids were gifts from Dr. T. Fujita (Kyoto University, Kyoto, Japan). p125 luc reporter plasmid was a gift from Dr. T. Taniguchi (University of Tokyo, Tokyo, Japan). RIG-I RD expressing vector was made with primers RIG-I RD-F and RIG-I RD-R; the RIG-I dRD cDNA fragment, which encodes ORF of RIG-I from the 1-to 754-amino acid region, was made by using primers RIG-I-(1-754)F and RIG-I-(1-754)R. The obtained cDNA fragments were sequenced, and it was confirmed that there were no mutations caused by PCR. The primers sequences are as follows: Northern Blotting-Human RIPLET 1092-bp cDNA fragment (208 -1299) was used for the probe for Northern blotting. The Northern blot membranes, human 12-lane MTN blot and MTN blot III, were purchased from Clontech. The homology of human RIPLET and TRIM25 in the probe region was 46%. We used a stringent condition for Northern blotting to exclude the cross-hybridization between the RIPLET and TRIM25 genes. Briefly, the probe was labeled with [␣-32 P]dCTP using Rediprime II Random Prime labeling system (GE Healthcare). The labeled probe was hybridized to the membrane with ExpressHyb hybridization solution (Clontech) at 68°C for 1 h. The membrane was washed with washing solution I (2ϫ SSC, 0.05% SDS) for 40 min, and then washed with washing solution II (0.1ϫ SSC, 0.1% SDS) for 40 min. Riplet mRNA bands were detected with x-ray film.
Reporter Gene Analysis-HEK293 cells were transiently transfected in 24-well plates using FuGENE HD (Roche Applied Science) with expression vectors, reporter plasmids, and internal control plasmid coding Renilla luciferase. The total amounts of plasmids were normalized with empty vector. For poly(I-C) stimulation, 24 h after transfection, cells were stimulated with medium containing poly(I-C) (50 g/ml) and DEAE-dextran (0.5 mg/ml) for 1 h, and then the medium was exchanged with normal medium and incubated for an additional 3 h. Cells were lysed with lysis buffer (Promega) and luciferase, and Renilla luciferase activities were measured by the dual luciferase assay kit (Promega). Relative luciferase activities were calculated by normalizing luciferase activity by Renilla luciferase activity, and dividing the normalized value by control in which only empty vector, reporter, and internal control plasmid were transfected. Values are expressed as mean relative stimulations Ϯ S.D. for a representative experiment, and each was performed three times in duplicate (unless otherwise indicated in the legends).
RNA Interference-Reporter and siRNA (20 nM final concentration) for Riplet or control were transfected into HEK293 cells with Lipofectamine 2000 (Invitrogen) by the standard method described in the manufacturer's protocol. Empty vec-tor was added to normalize the final plasmid amount. 48 h after transfection, cells were stimulated with poly(I-C) for 4 h. For VSV infection, 24 h after transfection, cells were infected with VSV at m.o.i. ϭ 1, and cell lysate was prepared after 12 h for reporter gene assays. The degree of gene silencing was confirmed by RT-PCR using RNA extracted from cells 24 h after transfection. PCR primers used for the RT-PCR were Riplet-F3 (ACTGGGAAGTGGACACTAGG) and Riplet-R3 (ACTCAT-ACAGAAGCTTCTCC). siRNAs were purchased from Funakoshi Co., Ltd. (Tokyo Japan), and the siRNA sequences of Riplet siRNA were GACUAUGGACUCUUGUUGUGU (sense) and ACAACAAGAGUCCAUAGUCCU (antisense). Control siRNA sequences were CUGUUGGUUUAGUAAGCCUGU (sense) and AGGCUUACUAAACCAACAGUC (antisense). Another siRNA, Riplet si-1, and control negative siRNA (silencer negative control 1 siRNA, AM4611) were purchased from Applied Biosystems. siRNA sequences were Riplet si-1 GGGAAAGCU-UGCCUUCUAUdTdT (sense) and AUAGAAGGCAAGCUUUCCCd-TdC (antisense).
Virus Preparation and Infection-VSV Indiana strain and poliovirus were amplified using Vero cells. HEK293 cells were transfected in 24-well plates with plasmid encoding RIG-I, Riplet, or no insert. 24 h after transfection, cells were infected with viruses for 24 h, and the titers of virus in culture supernatant were measured by plaque assay using Vero cells. For RNA interference assay, cells were transfected with siRNA with Lipofectamine 2000. 24 h after transfection, cells were infected with viruses at m.o.i. ϭ 0.001 for 18 h, and the titer in culture supernatant were determined by plaque assay.
Immunoprecipitation-HEK293FT cells were transfected in 6-well plates with plasmids encoding FLAGtagged RIG-I and/or HA-tagged Riplet. The plasmid amounts were normalized by the addition of empty plasmid. 24 h after transfection, cells were lysed with lysis buffer (20 mM Tris-HCl (pH 7.5), 125 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Nonidet P-40, 30 mM NaF, 5 mM Na 3 VO 4 , 20 mM iodoacetamide, and 2 mM phenylmethylsulfonyl fluoride), and then proteins were immunoprecipitated with rabbit anti-HA polyclonal (Sigma) or anti-FLAG M2 monoclonal antibody (Sigma). The precipitated samples were analyzed by SDS-PAGE and stained with anti-HA (HA1.1) (Covance) or anti-FLAG M2 monoclonal antibody. For ubiquitination assay of RIG-I, the plasmid encoding two multiple HA-tagged ubiquitins was used. HEK293FT cells were transfected with plasmids encoding FLAGtagged RIG-I, Riplet, or 2ϫ HA-tagged ubiquitin. 24 h after transfection, cells were lysed, and then RIG-I was immunoprecipitated as described above. The samples were analyzed by SDS-PAGE and stained with anti-HA polyclonal antibody (for detection of ubiquitination) or anti-FLAG monoclonal antibody (for detection of RIG-I). Reproducibility was confirmed with additional experiments (see supplemental figures).
Construction of RIG-I 3KA and 5KA Mutant Genes-The C-terminal three or five lysine residues were mutated into alanines (designated as 3KA and 5KA). RIG-I 3KA has K888A, K907A, and K909A, whereas RIG-I 5KA has K849A, K851A, FIGURE 1. Isolation of Riplet by yeast two-hybrid screening. A, yeast cells carrying both RIG-I and Riplet can grow in selective media (SD-WLH, SD-WLHA), whereas yeast cells carrying RIG-I alone only grow in nonselective media (SD-WL), indicating the physical interaction of RIG-I with Riplet. B, human Riplet protein sequence is 60.8% identical to human TRIM25. The RING finger domains and SPRY motifs show higher sequence similarities between the two proteins. aa, amino acids. C, phylogenetic tree constructed by the Neighbor-Joining method shows that Riplet is similar to TRIM25. h, m, r, or zf represent human, mouse, rat, or zebrafish, respectively. The numbers on the node are bootstrap probabilities (n ϭ 1000). D, HeLa cell, human primary-cultured fibroblast cell, MRC5, or bone marrow-derived mouse dendritic cell (BM-DC) were stimulated with poly(I-C) (50 g/ml) for indicated hours. Total RNA was extracted with TRIzol reagent, and then RT-PCR was carried out using primers shown under "Experimental Procedures." GAPDH, glyceraldehyde-3-phosphate dehydrogenase. E, Northern blot membranes containing 1 g of poly(A) ϩ RNA per lane from human tissues were blotted with human Riplet probe.
K888A, K907A, and K909A. The mutant rig-I genes were made by PCR-mediated site-directed mutagenesis. The primers used for the PCR were as follows: K907-909A-forward, GTT CAG  ACA CTG TAC TCG GCG TGG GCG GAC TTT CAT TTT  GAG AAG, and K907-909A-reverse, CTT CTC AAA ATG  AAA GTC CGC CCA CGC CGA GTA CAG TGT CTG AAC;  K888A-forward, GAC ATT TGA GAT TCC AGT TAT AGC  AAT TGA AAG TTT TGT GGT GGA GG, and K888A-reverse, CCT CCA CCA CAA AAC  TTT CAA TTG CTA TAA CTG  GAA TCT CAA ATG TC; K849 -851A-forward, GAG TAG ACC ACA TCC CGC CCA GCG CAG TTT TCA AGT TTT G, and K849 -851A-reverse, CAA AAC TTG AAA ACT GCG CTG GCG CGG GAT GTG GTC TAC TC. PCR was carried with Pyrobest Taq polymerase, and the obtained clones were sequenced to exclude the clones harboring PCR error. To construct the plasmid-expressing mutant RIG-I protein, the wild-type RIG-I gene on pEF-BOS vector was replaced with the mutant rig-I gene.
Real Time PCR-Quantitative PCR analyses were carried out using iCycler iQ real time detection system with Platinum SYBR Green qPCR SuperMix-UDG reagent (Invitrogen). Primer sequences for qPCR were as follows: hGAPDH-qF, GAG TCA ACG GAT TTG GTC GT, and hGAPDH-qR, TTG ATT TTG GAG GGA TCT CG; hIFN-␤-qF, TGG GAG GAT TCT GCA TTA CC, and hIFN-␤-qR, CAG CAT CTG CTG GTT GAA GA; hMx1-qF, ACC ACA GAG GCT CTC AGC AT, and hMx1-qR, CTC AGC TGG TCC TGG ATC TC; and hIFIT-1-qF, GCA GCC AAG TTT TAC CGA AG, and hIFIT-1-qR, CAC CTC AAA TGT GGG CTT TT. Values were expressed as mean relative stimulations, and for a representative experiment from a minimum of three separate experiments, each was performed in triplicate.

RIG-I-binding
Proteins-To isolate the proteins that bind to RIG-I, we performed yeast two-hybrid screening using a human lung cDNA library. Using the RIG-I central region (213-601 amino acids), we isolated a clone that encoded a partial ORF of a gene expressed in a dendritic cell line, DC12, whereas the C-terminal region of RIG-I (557-925 amino acids) resulted in the isolation of two cDNA clones, which encoded partial C-terminal regions of ZNF598 and RNF135 ( Fig. 1A and data not shown). Preliminary expression studies showed that the RNF135 segment affected the RIG-I IFN-␤ inducing activity, whereas the other two proteins had no effect (data not shown). We confirmed the interaction of RIG-I with ZNF598 or RNF135 in HEK293FT cells by immunoprecipitation (data not shown). RNF135 was previously annotated by the genome project and was recently found to be a cause of a genetic disease, neurofibromatosis, although its protein function was unknown. We renamed the protein Riplet (RING finger protein leading to RIG-I activation) based on the following functional analyses. Riplet was most similar to TRIM25 (60.8% sequence homology), in particular between their RING finger domains PRY or SPRY (Fig. 1B). Phylogenetic analysis also supported the notion that Riplet was similar to TRIM25 (Fig. 1C). Thus, we hypothesized that, like TRIM25, Riplet is a ubiquitin ligase.
Expression of Riplet-RIG-I mRNA is induced by type I IFN or poly(I-C) stimulation in mammalian cells. Unlike RIG-I, however, Riplet mRNA was basally expressed in HeLa and primary-cultured MRC-5 cells irrespective of stimulation ( Fig. 1D and data not shown). On the other hand, when we treated bone marrow-derived dendritic cells with poly(I-C), the basal level of Riplet mRNA was increased by the stimulation (Fig. 1D), suggesting that the regulatory mechanism of Riplet expression somewhat differs among cell types, and that Riplet is expressed before virus infection in some cell types. Next we performed Northern blotting of human tissue RNA. Riplet mRNA was detected as a single band of 2.4 kbp, which is slightly longer than the RNF135 cDNA sequence deposited in GenBank TM (accession number AB470605). Human RIPLET is expressed in human skeletal muscle, spleen, kidney, placenta, prostate, stomach, thyroid, and tongue and also weakly expressed in heart thymus, liver, and lung (Fig. 1E).
Riplet Enhances RIG-I-mediated IFN-␤ Induction-At first we characterized the role of Riplet in RIG-I-mediated IFN inducing signaling by reporter gene analyses. When RIG-I was expressed in HEK293 cells, reporter auto-activation was observed even in the absence of exogenous stimulation ( Fig.  2A) as reported previously (25,26). Stimulation with poly(I-C) further enhanced the promoter. Co-expression of Riplet with RIG-I potentiated activation of the IFN-␤ promoter, whereas expression of Riplet alone resulted in only marginal activation ( Fig. 2A). Detection of endogenous IFN-␤ mRNA confirmed that Riplet enhanced RIG-I-mediated activation of IFN-␤ transcription (supplemental Fig. S1). The enhancing role of Riplet in IFN-␤ promoter activation was also supported by activation of IRF-3 and NF-B by Riplet (Fig. 2, B and C). In contrast, expression of a Riplet partial fragment (Riplet-DN) (70 -432 amino acids) that lacked the N-terminal RING finger domain reduced promoter activation (Fig. 2E). The Riplet-L249fs mutant protein, which was isolated from neurofibromatosis patients (27), did not increase the RIG-I-mediated promoter activation (Fig. 2D). These data indicate that Riplet augments RIG-I-mediated IFN-␤ promoter activation, and that both the RING finger domain and the C-terminal region encoding the SPRY and PRY motifs are important for its function. Riplet (residues 70 -432) acted as a dominant-negative form (hereafter called Riplet-DN) (Fig. 2, E and F, left panel). This functional feature of Riplet-DN was confirmed in Fig. 2, B and C, and was later confirmed through RIG-I co-precipitation and ubiquitination analyses (see Fig. 5C and supplemental Fig. S4C). Expression of Riplet-DN did not reduce TLR3 or MDA5 signaling (Fig.  2E), suggesting that Riplet-DN is specific for RIG-I signaling.
Interestingly, the Riplet-DN only partially suppressed the function of the C-terminal deleted RIG-I (dRD), which is a constitutively active form (Fig. 2F, right panel), and RIG-I CARD-like region (dRIG-I)-mediated signaling in high or low dose transfection of dRIG-I was barely inhibited by overexpression of Riplet-DN (Fig. 2F, center panel). These data suggest that Riplet requires the RIG-I C-terminal domain (RD) and partial helicase region to activate RIG-I signaling.
Endogenous Riplet Promotes the RIG-I Signaling-We performed Riplet knockdown by siRNA Riplet using Lipofectamine 2000 reagents, instead of FuGENE HD, to reveal the function of endogenous Riplet. Two siRNAs (Riplet siRNA and Riplet si-1) that target different sites of the Riplet mRNA and two control siRNAs were used for knockdown analyses. The two siRNA or control siRNA were co-transfected with HAtagged Riplet expression vector into HEK293 cells, and after 48 h, cell lysate was prepared and analyzed by Western blotting with anti-HA antibody detecting Riplet. The two siRNAs targeting Riplet abolished exogenously expressed Ripelt-HA, but control siRNA did not (supplemental Fig. S3). Likewise, both Riplet siRNA and Riplet si-1 specifically down-regulate the level of endogenous Riplet mRNA (Fig. 3, A and B).
Using the siRNA, we examined whether Riplet knockdown reduces RIG-I signaling. As expected, RIG-I-mediated IFN-␤ promoter activation was reduced by Riplet siRNA or Riplet si-1 compared with control siRNA (Fig. 3, A and B), indicating that Riplet is required for full activation of the RIG-I signaling. Vesicular stomatitis virus (VSV) is a negative-stranded RNA virus that induces IFN-␤ production via RIG-I (3). Although the IFN-␤ promoter was only minimally activated by RIG-I in response to VSV (m.o.i. ϭ 1) during the early phase of infection (Ͻ12 h), the activity was increased by RIG-I and Riplet (Fig. 3C).
Riplet was silenced by siRNA and then VSV infected the cells. VSVderived up-regulation of IFN-␤ mRNA was started around 6 h postinfection, and Riplet siRNA significantly suppressed the increase of IFN-␤ mRNA at 6 h (Fig. 3D). Because VSV infection is mainly sensed by RIG-I, this is consistent with the notion that Riplet promotes the RIG-I signaling. Other IFN-inducible genes, IFIT1 and MX1, were expressed Ͼ8 h post-infection, and their expressions were also suppressed by Riplet siRNA (Fig. 3, E and F).
Riplet Exerts Protective Activity against Viral Infection-Next we examined the role of Riplet during viral infection. Riplet and/or RIG-I were transiently expressed in the human cells by FuGENE HD reagents, and then the cells were infected with VSV or poliovirus (a positive-stranded RNA virus). The viral titer of the supernatant was determined 24 h post-infection. Under our conditions, expression of RIG-I weakly inhibited VSV propagation. Co-expression of Riplet with RIG-I significantly suppressed VSV replication especially at low m.o.i., whereas Riplet alone did not suppress VSV (Fig. 4, A and B, upper  panel). Therefore, a sufficient amount of RIG-I protein is required for Riplet to exert antivirus activity. This requirement of RIG-I is also observed in reporter gene analyses (Fig. 2). Under a similar setting, the antiviral effect of Riplet was marginally observed against poliovirus, which induces IFN-␤ largely via MDA5 (Fig. 4B, lower panel). To assess the importance of endogenous Riplet for antivirus effect of human cells, Riplet knockdown cells were infected with viruses. In Riplet knockdown cells, the VSV titer was consistently increased compared with the control (p Ͻ 0.05) (Fig. 4C,  left panel). In addition, infection of Riplet knockdown cells with poliovirus resulted in only a slight increase in the poliovirus titer compared with the control (p Ͼ 0.05) (Fig. 4C, right panel). Because poliovirus is mainly recognized by MDA5 but not RIG-I, this marginal effect of Riplet on poliovirus infection was within expectation (3, 28).  1 g), RIG-I expressing vector (0.1 g), and Riplet siRNA or control siRNA (10 pmol), which were purchased from Funakoshi Co. Ltd., were transfected into HEK293 cells in a 24-well plate with Lipofectamine 2000, and 48 h after transfection, the cells were stimulated with poly(I-C) for 6 h, and the cell lysate was prepared, and luciferase activities were measured. RT-PCR was carried out using total RNA extracted from cells 48 h after transfection. B, p125 luc reporter plasmid (0.1 g), RIG-I expressing vector (0.1 g), and siRNA, Riplet si-1, or control si-1 (10 pmol), which were purchased from Applied Biosystems, were transfected into HEK293 cells with Lipofectamine 2000. 48 h after transfection, the cells were stimulated with poly(I-C) for 6 h. The cell lysate was prepared, and luciferase activities were measured. RT-PCR was carried out using total RNA extracted from cells 48 h after transfection. C, HEK293 cells were transfected with the plasmids expressing RIG-I (0.1 g) and/or Riplet (0.1 g) with p125 luc reporter plasmid (0.1 g) in 24-well plates. After 24 h, the cells were infected with VSV (m.o.i. ϭ 1) for 12 h. The luciferase activities of the cell lysates were measured. Expression of Riplet strongly enhanced IFN-␤ promoter activation by VSV through RIG-I. D-F, siRNA (control si-or Riplet si-1) were transfected into HEK293 cells, and after 48 h, the cells were infected with VSV at m.o.i. ϭ 1. RNA was extracted at the indicated hours, and the quantitative PCR were carried out to detect the expression of IFN-␤ (D), IFIT-1 (E), or Mx1 (F) mRNA. *, p Ͻ 0.05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Fig.  S2). To further confirm the physical binding of Riplet to RIG-I in human cells, we carried out immunoprecipitation analyses. Full-length Riplet was co-immunoprecipitated with RIG-I (Fig.  5B), indicating that Riplet binds directly to RIG-I in human cells.

Riplet and Riplet-DN Bind the Helicase and RD Regions of RIG-I-Yeast two-hybrid analysis showed that a C-terminal region of Riplet bound to the C-terminal region of RIG-I. This cytoplasmic interaction between Riplet and RIG-I was confirmed by confocal microscopy in HeLa cells (supplemental
To determine the region responsible for the RIG-I-Riplet interaction, we constructed a RIG-I and Riplet deletion series as shown in Fig. 5A. Riplet-DN also bound to RIG-I (Fig. 5, B and  C), indicating that the RING finger domain is dispensable for the RIG-I-Riplet interaction. This is consistent with the notion that the RING finger domain in ubiquitin ligase proteins is required for their interactions with ubiquitin-conjugating enzymes (29). Unlike TRIM25, Riplet and Riplet-DN failed to co-precipitate the two CARD domains of RIG-I (dRIG-I) (Fig.  5D). However, co-precipitation of the RIG-IC or RIG-RD fragments was observed (Fig. 5, E and F). RD-deleted RIG-I (RIG-I dRD) weakly associated with Riplet (Fig. 5G). Taken together, Riplet preferentially binds the RD and also weakly associates with the helicase region of RIG-I with its C terminus. Reporter gene analyses show that Riplet-DN only weakly suppresses RIG-I signaling and barely suppresses dRIG-I, which contains neither helicase nor RD region. Therefore, the physical interaction is correlated with the results of reporter activity.

Riplet Promotes Ubiquitination of RIG-I-Because
Riplet shares 60% sequence similarity with TRIM25, we hypothesized that Riplet ubiquitinates RIG-I and that this modification leads to activation of RIG-I signaling. To test this hypothesis, we examined RIG-I ubiquitination. As expected, ubiquitination of RIG-I was increased by co-expression of Riplet under two different conditions (Fig. 6, A and B). The quantity of RIG-I ubiquitination was significantly high in the presence of Riplet (Fig.  6C). RIG-I ubiquitination was suppressed if Riplet was replaced with Riplet-DN ( Fig. 6D and supplemental Fig. S4C). However, unlike TRIM25, Riplet binds to the C-terminal region of RIG-I. Therefore, we examined whether Riplet ubiquitinates the C-terminal region. We found that ubiquitination of RIG-IC was enhanced by Riplet expression (Fig. 6E). Both RIG-I dRD and RIG-I RD were also ubiquitinated by expression of Riplet ( Fig.  6F; supplemental Fig. S4A and S5), suggesting that Riplet promotes ubiquitination of the helicase and RD domains of RIG-I in a manner distinct from TRIM25.
Ubiquitin is polymerized through its lysine residue. Lys-63linked polyubiquitination is frequently observed in signal transduction pathways (30). In contrast, Lys-48-linked polyubiquitination usually leads to the degradation of protein through the proteasome. Indeed, TRIM25-mediated Lys-63-linked polyubiquitination activates the CARD-like region of RIG-I, and RNF125-mediated Lys-48-linked polyubiquitination leads to the degradation of RIG-I (23,25). We used K48R or K63R mutated ubiquitin and found that K48R was incorporated normally into RIG-IC, whereas polyubiquitination was decreased by K63R (supplemental Fig. S4B). K63R mutation abolished RIG-I RD polyubiquitination by Riplet (Fig. 6F). These data indicates that Riplet mediates Lys-63-linked polyubiquitination of the RIG-I C-terminal helicase and RD region. Because Riplet-DN reduced the RIG-I-mediated signaling, we examined whether Riplet-DN reduced the RIG-I ubiquitination.
As expected, Riplet-DN reduced RIG-I ubiquitination (Fig. 6D and  supplemental Fig. S4C). These ubiquitination assay data are consistent with the notion that Riplet-mediated Lys-63-linked polyubiquitination of RIG-I is required for full activation of RIG-I signaling.
We tried to determine the ubiquitination sites of RIG-I using Lysto-Ala (KA)-converting mutants. RIG-I has 25 Lys residues in its C-terminal region. These Lys residues of RIG-I were in turn mutated to Ala, and the degree of ubiquitination and IFN-␤-inducing activity were determined with each mutant. RIG-I-mediated IFN-␤ promoter activation was normally augmented by co-expression of Riplet and 3KA RIG-I. Co-expression of Riplet and 5KA, however, and the ubiquitination level of RIG-I and IFN-␤-inducing activity were simultaneously decreased (Fig. 7, A and C). Ripletdependent augmentation of IFN-␤ promoter activation was largely suppressed when RIG-I was replaced with 5KA RIG-I (Fig. 7B). Therefore, Lys-849 and Lys-851 of RIG-I were crucial for RIG-I ubiquitination by Riplet. The results confirmed the importance of ubiquitination of specific Lys residues in the C-terminal region of RIG-I and for RIG-I-mediated IFN-␤ induction.

DISCUSSION
RIG-I plays a central role in the recognition of cytoplasmic viral RNA and is regulated by modification by small modifier ubiquitin or ubiquitin-like protein, ISG15. TRIM25 mediates Lys-63-linked polyubiquitination, which is essential for RIG-I activation (23), and RNF125 mediates Lys-48-linked polyubiquitination (25). RIG-I also harbors ISG15 modification, although the role of ISG15 modification in vivo remains to be deter- mined (21,22,31). Although Riplet and TRIM25 share 60% sequence similarity, the ubiquitination of RIG-I by Riplet is distinct from that by TRIM25; Riplet ubiquitinates the C-terminal region of RIG-I, whereas TRIM25 ubiquitinates its CARDlike region. These findings are also supported by the fact that neither Riplet nor Riplet-DN promoted or inhibited the activation of the IFN-␤ promoter by expression of the RIG-I CARD-like region (data not shown). It has been reported that ubiquitination of the CARD-like region of RIG-I by TRIM25 is critical for RIG-I-IPS-1 signaling (23). However, how this CARD ubiquitination is essential for activation of IPS-1 by RIG-I remains undetermined. Here we emphasize the importance of RIG-I C-terminal ubiquitination for IFN-␤ induction and the antiviral response. Because the C-terminal RD region inhibits the IFN inducing activity of the CARD-like region of RIG-I, it is reasonable that RIG-I C-terminal ubiquitination by Riplet inhibits the conversion from the active to inactive form of RIG-I protein after binding to viral RNA. This initial stabilization of RIG-I via ubiquitination by Riplet would provide a sufficient structure for RIG-I to maintain the accessibility to TRIM25 and facilitate TRIM25-mediated ubiquitination of the CARDlike region of RIG-I, which may lead to potential activation of IPS-1.
RIG-I is an IFN-inducible RNA helicase that is expressed at extremely low levels in resting cells (6). Initial penetration of viruses allows generation of 5Ј-triphosphate RNA and/or double strand RNA followed by induction of IFN-␤ production. This early response to viral infections triggers up-regulation of RIG-I/MDA5 and TLR3, leading to robust IFN-␤ production (3,32,33). We favor the interpretation of our present findings that during the early stages of viral infection with trace amounts of RIG-I and viral RNAs, Riplet helps host cells rearrange RIG-I conformation to activate IPS-1. This issue will need further proof because it is difficult to Riplet was co-immunoprecipitated with FLAG-tagged RIG-I in A but could not co-immunoprecipitate in B because of high salt condition. Expression of Riplet enhanced the ubiquitination of RIG-I. Different gel conditions were employed in A and B. C, ubiquitinated RIG-I was quantitated with NIH image software. **, p Ͻ 0.01. D, FLAG-tagged RIG-I (0.4 g) was transfected into HEK293 FT cells in a 6-well plate with HA-tagged Riplet (0.4 g) or Riplet-DN (0.4 g) and HA-tagged ubiquitin, and immunoprecipitation was carried out with anti-FLAG antibody. The total amount of transfected DNA (2 g/well) was kept constant by adding empty vector (pEF-BOS). The samples were analyzed with 10% acrylamide gel to clearly separate Riplet from Riplet-DN and stained by Western blotting. E, ubiquitination of RIG-IC was also promoted by Riplet expression. HEK293FT cells were transfected with the plasmids encoding RIG-IC (0.4 g), Riplet (0.4 g), and/or HA-tagged ubiquitin (0.4 g) in a 6-well plate, and 24 h after transfection, cell lysates were prepared. The total amount of transfected DNA (2 g/well) was kept constant by adding empty vector (pEF-BOS). FLAG-tagged RIG-ICs were immunoprecipitated with anti-FLAG antibodies, and the proteins were analyzed by Western blotting. F, Ub-K63R are HA-tagged ubiquitin in which the lysine 3 residues were substituted with arginine. The HA-tagged Ub-K63 expressing vectors (1.2 g), FLAG-tagged RIG-IC (0.4 g), and/or Riplet (0.4 g) were transfected into HEK293FT cells in 6-well plates and analyzed as shown in A-D. The total amount of transfected DNA (2 g/well) was kept constant by adding empty vector (pEF-BOS). Ub-K63R was not incorporated into polyubiquitin chain of RIG-I RD. WCE, whole cell extract.
visualize RNRs and viral RNAs in the early infection stage and to understand the mechanisms that allow viruses to uncoat into naked viral RNA and to replicate.
We have provided several lines of evidence indicating that Riplet complements RIG-I-mediated IFN-␤ induction upon viral infection by both Riplet siRNA and overexpression analyses. The C-terminal lysines (849 and 851) of RIG-I are critical for Riplet-mediated RIG-I ubiquitination. However, our data indicate that Riplet alone was unable to induce IFN-␤ production and essentially required RIG-I to confer IFN-␤ induction. Furthermore, Riplet is not ubiquitously distributed over the organs tested. Ubiquitination of RIG-I induced by poly(I-C) or viruses was accelerated in cells pre-transfected with Riplet. Hence, Riplet works case-sensitive to upregulate RIG-I antiviral activity predominantly in some organs. The physiological meaning of this response will be clarified by knockout study.
Unexpectedly, the siRNA experiments were not robust with regard to VSV replication. Possible explanations for this are as follows: 1) the degree of gene silencing is not so profound that the proteins remain in the cells; 2) there are a number of virus-mediated IFN-inducing pathways capable of compensating each other, so that disruption of one factor does not cause a profound effect on VSV replication. Furthermore, in VSV-infected Riplet-knockdown cells, IFN-␤ levels were reduced even at m.o.i. ϭ 1 (Fig. 3D), and accordingly, virus susceptibility was increased at m.o.i. ϭ 0.1 (Fig. 4C), whereas in Riplet-overexpressing cells, antiviral activity was observed only at low m.o.i. (Fig. 4B). We used different transfection reagents and cell conditions in the knockdown and overexpression experiments to obtain high transfection efficiency in each. These conditional differences in knockdown and overexpression analyses might cause part of the discrepancy between the two results on Riplet antiviral activity. Another possibility to explain the apparent inconsistencies between overexpression and knockdown analyses is that high amounts of Riplet efficiently activate the RIG-I signaling, but low amounts are insufficient for RIG-I activation in high m.o.i.-infecting human cells. High amounts of Riplet with overexpressed RIG-I would confer the ability on cells to respond to very low amounts of VSV as observed in the low m.o.i. experiments. Again, riplet knock-out mice would reveal whether it is absolutely required for potential RIG-I activation.
How viral RNAs select RIG-I rather than dicers or the translation machinery is also unknown. During natural infection it is likely that the number of the initial invading virions would be at most several copies/cell. Uncoated viral RNA may assemble a complex consisting of viral and host molecules required for replication. We assume that cells are equipped with various FIGURE 7. The C-terminal two lysine residues of RIG-I are important for ubiquitination by Riplet. A, RIG-I C-terminal lysine residues were substituted with alanine. RIG-I 3KA mutant protein harbors the triple mutations, K888A, K907A, and K909A. The five lysine residues, Lys-849, Lys-851, Lys-888, Lys-907, and Lys-909, were replaced with alanine in RIG-I 5KA mutant. The plasmid carrying wild-type (100 ng/well), RIG-I 3KA (100 ng/well), RIG-I 5KA (100 ng), or Riplet (100 ng) were transfected into HEK293 cells in a 24-well plate together with p125 luc reporter plasmid (100 ng/well). The amount of transfected DNA was kept constant by adding empty vector. After 24 h, the luciferase activities were measured. B, wild-type RIG-I (100 ng), RIG-I 5KA mutant (100 ng), or empty vector (100 ng) was transfected into HEK293 cells in a 24-well plate together with p125 luc reporter plasmids and HCV 3Ј-untranslated region poly(U/UC) RNA (25 ng), which is synthesized in in vitro transcription by T7 RNA polymerase. The amount of transfected DNA was kept constant by adding empty vector. 24 h after transfection, luciferase activities were measured. RIG-I 5KA mutant hardly responded to poly(U/UC) RNA. C, to observe the ubiquitinated RIG-I more clearly, we used 800 ng/well of Riplet and HA-Ub expression vector for the following transfection. HEK293FT cells in a 6-well plate were transfected with the plasmids encoding RIG-I (400 ng/well), RIG-I 5KA (400 ng/well), Riplet (800 ng/well), and/or HA-Ub (800 ng/well). The total amount of DNA was kept constant by adding the empty vector. 24 h after the transfection, the cell lysates were prepared, and the immunoprecipitation was carried out using anti-FLAG antibodies. The immunoprecipitates were analyzed by Western blotting with anti-HA or FLAG antibodies. molecular arms to sensitively detect viral RNA. The molecular complexes sensing viral RNA may not be so simple that we will be able to identify more molecules than Riplet as enhancers for integral RNA recognition. In either case, yeast screening will be a good strategy to pick up such proteins in other RNA recognition systems. A molecular switch selecting IFN induction by virus RNA will then be clarified.
We show that the ubiquitination sites targeted by Riplet are the helicase and RD domains of RIG-I but not its CARD-like domains in contrast to TRIM25. Riplet may be a complement factor of the reported TRIM25 function for RIG-I activation (23). A previous report (25) failed to polyubiquitinate the RIG-I protein by TRIM25 alone. If Riplet were added to TRIM25 for RIG-I ubiquitination in the previous study, Riplet would have enabled TRIM25 to polyubiquitinate the RIG-I CARD-like region. Further studies using TRIM25 and Riplet will be required to clarify this point.
Based on our results, we propose that RIG-I-like receptors form a molecular complex that efficiently recognizes low copy numbers of viral RNA. Riplet is implicated in the RIG-I complex to enhance viral RNA response in some organs. In this context, MDA5-associated molecules might also exist in the cytoplasm to augment IFN output. Although MDA5 possesses the RD domain, it fails to recruit Riplet (data not shown) or augment IFN-␤-induction in conjunction with Riplet (Fig. 2E). Because RLR-associated molecules naturally reside in cells and facilitate inhibition of low dose viral infection until RLRs become expressed, they may be useful therapeutic targets for an early phase antiviral immunotherapy.