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The Journal of Immunology, 2007, 179, 6123-6133
Copyright © 2007 by The American Association of Immunologists, Inc.
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Differential Type I IFN-Inducing Abilities of Wild-Type versus Vaccine Strains of Measles Virus1

Masashi Shingai2,*, Takashi Ebihara*, Nasim A. Begum3,{dagger}, Atsushi Kato{ddagger}, Toshiki Honma{ddagger}, Kenji Matsumoto{ddagger}, Hirohisa Saito{ddagger}, Hisashi Ogura§, Misako Matsumoto*,{dagger} and Tsukasa Seya4,*,{dagger}

* Department of Microbiology and Immunology, Graduate School of Medicine, Hokkaido University, Sapporo, Japan; {dagger} Department of Immunology, Osaka Medical Center for Cancer, Osaka, Japan; {ddagger} Department of Allergy and Immunology, National Research Institute for Child Health and Development, Tokyo, Japan; and § Department of Virology, Osaka City University, Osaka, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Laboratory adapted and vaccine strains of measles virus (MV) induced type I IFN in infected cells. The wild-type strains in contrast induced it to a far lesser extent. We have investigated the mechanism for this differential type I IFN induction in monocyte-derived dendritic cells infected with representative MV strains. Laboratory adapted strains Nagahata and Edmonston infected monocyte-derived dendritic cells and activated IRF-3 followed by IFN-beta production, while wild-type MS failed to activate IRF-3. The viral IRF-3 activation is induced within 2 h, an early response occurring before protein synthesis. Receptor usage of CD46 or CD150 and nucleocapsid (N) protein variations barely affected the strain-to-strain difference in IFN-inducing abilities. Strikingly, most of the IFN-inducing strains possessed defective interference (DI) RNAs of varying sizes. In addition, an artificially produced DI RNA consisting of stem (the leader and trailer of MV) and loop (the GFP sequence) exhibited potential IFN-inducing ability. In this case, however, cytoplasmic introduction was needed for DI RNA to induce type I IFN in target cells. By gene-silencing analysis, DI RNA activated the RIG-I/MDA5-mitochondria antiviral signaling pathway, but not the TLR3-TICAM-1 pathway. DI RNA-containing strains induced IFN-beta mRNA within 2 h while the same recombinant strains with no DI RNA required >12 h postinfection to attain similar levels of IFN-beta mRNA. Thus, the stem-loop structure, rather than full genome replication or specific internal sequences of the MV genome, is required for an early phase of type I IFN induction by MV in host cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The type I IFNs represent a family of soluble cytokines with biological and antiviral activity (1, 2). In acute-phase viral infection, IFNs are secreted from affected cells via activation of IFN-inducing signal pathways. Recently, how virus induces type I IFNs is a focus for investigation and it was found that virus nucleotides trigger activation of the intracellular molecular cascades for the IFN production. In vertebrates, nucleic acid-sensing receptors recognize viral nucleotides to induce the production of type I IFNs. Endosomal TLRs, TLR3 (3, 4), TLR7 (5, 6), and TLR9 (7), cytoplasmic PKR (8), and retinoic acid-inducible gene I (RIG-I)5-like RNA-recognition receptors (9, 10), function as viral RNA sensors and individually induce type I IFNs through their pathways. These pathways involve activation of IRF-3 transcription factor, which is crucial for promoter activation of IFN-beta (1). Type I IFNs act in either an autocrine or paracrine fashion by inducing the expression of hundreds of genes that together establish an antiviral state, which restricts the spread of virus around neighboring cells. In addition, type I IFNs enhance the function of NK cells (11, 12) and the differentiation of virus-specific CTL (13). Although the exact mechanism by which these effectors are induced in virus-infected hosts remains unknown, this innate response to viral patterns triggers a various array of antiviral immunity and eliminate virus-infected cells.

Many viruses have developed mechanisms to circumvent the host antiviral responses, thus potentially augmenting early spread of virus (14, 15). Viral strategies include inhibition of cytoplasmic RNA sensors RIG-I or melanoma differentiation-associated gene 5 (MDA5), blocking IFN regulatory factor-3 (IRF-3)-activating signals of the IFN system, and interfering with IFNR response by modulating the JAK-STAT-1 pathway (14, 15). Viral proteins are known to associate these IFN-inhibitory modes (15). Some virus species may harbor other inhibitory modes for IFN production. In contrast, there appears a strain-to-strain difference in virus-mediated IFN-{alpha}/IFN-beta induction. Low IFN-inducing viruses can actively suppress the IFN production of the high IFN-inducing strains. These findings have barely been analyzed in conjunction with the various inhibitory modes of virus molecules and host receptors for IFN signaling.

In measles virus (MV), wild-type strains barely induce production of significant quantities of IFN-{alpha}/IFN-beta (16). In contrast, measles vaccine strains, including the attenuated Edmonston (ED) strain, are efficient IFN inducers that cause part of the attenuation of virulence. The wild-type MV strains actually suppress IFN production induced by coinfected MV ED infection (16). Furthermore, 10 passages of the wild-type MVs on Vero cells are sufficient to transform their phenotype from an IFN suppressor to an IFN inducer (16). These earlier studies stressed the total output of IFN production, measuring the IFN-{alpha}/IFN-beta content >24 h postinfection (p.i.; late phase), thus the event involving replication and translation of viral genome RNA. Yet, the possible participation of viral V and C proteins in the IFN production has not been sufficiently considered under the contemporary knowledge (15). The molecular basis of strain-specific IFN-inducing ability, therefore, remains largely undetermined.

We have compared the IFN-{alpha}/IFN-beta induction and sensitivity of the laboratory passaged attenuated MV strains with those of recent wild-type viruses isolated and passaged on human PBMC, the B95a marmoset B cell line, or early passage lots of Vero cells. We found that the majority of vaccine and laboratory adapted MV strains rapidly induced the IFN-beta mRNA within 12 h p.i. (early phase) independent of viral protein translation. A further finding was that the robust production of IFN-beta in human myeloid dendritic cells (mDCs) and epithelial cells paralleled the elevating of the level of virus-specific defective interfering RNA (DI RNA). The DI RNAs are subviral replicons originating from the viral genome and are associated with many RNA viruses (17). Wild-type MV isolates induced significantly lower production of IFN in mDCs and they contained undetectable levels of DI RNA. Thus, we speculate that DI RNA in MV isolates is a crucial determinant for high IFN induction in MV laboratory adapted and vaccine strains.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell culture and reagents

The human lung epithelial cell line (A549), A549/CD150, African green monkey kidney cell line (Vero), Vero/CD150, HEK293, and 293-3-46 cells (kindly provided by M. A. Billeter, University of Zurich, Zurich, Switzerland) were maintained in DMEM supplemented with 10% heat-inactivated FCS (JRH Biosciences) and antibiotics. Chinese hamster ovary (CHO), CHO/CD46, and CHO/CD150 (18) were cultured in Eagle’s MEM with 5% heat-inactivated FCS. B95a cells were grown in RPMI 1640 supplemented with 5% heat-inactivated FCS and antibiotics. Cells were kept in 5% CO2 at 37°C incubator. Vero/CD150 was established according to the method by Ono et al. (19). For establishing CD150-expressing A549 and Vero sublines, pCNX2-huCD150, an expression plasmid for human CD150 bearing a neomycin-resistance gene was introduced into A549 cells using Lipofectamine 2000 (Invitrogen Life Technologies) according to the manufacturer’s recommendation. Two days after transfection, the neomycin analog G418 (Sigma-Aldrich) was added to the medium at the final concentration of 1 or 2 mg/ml for A549 or Vero cells. During the selection, G418-containing medium was changed once every 4 days. G418-resistant, stably transfected clones were propagated for the study of surface expression of CD150 by FACS. Poly I:C was purchased from Amersham. Mouse mAbs against human CD150 (IPO-3) was purchased from eBioscience and mAbs against human CD46 (M75 and M177) were produced in our laboratory (20). Rabbit polyclonal Ab (pAb) against human CD150 or human CD46 was produced in our laboratory (21). HRP-conjugated goat anti-rabbit Igs were obtained from American Qualex Manufacturers.

Virus preparation and titration

Nagahata (NV) and Edmonston (ED) strains were obtained from the Research Institute for Microbial Diseases (Osaka University, Osaka, Japan) and University of Washington (Seattle, WA), respectively. Ichinose (IC)-B and IC-V were provided from Drs. F. Kobune (National Institutes of Health, Tokyo, Japan) and K. Takeuchi (Tsukuba University, Tsukuba, Japan) (22). Wild-type MV strains were from Osaka University, Osaka City University, and Osaka Prefectural Research Institute. Masusako (MS) and other strains of MV were propagated in our laboratory (21, 23, 24). Vaccine strains of MV were purchased from Takeda, Tanabe, and Banyuu (Osaka, Japan). ED, NV, IC-V, MS, Yokota, CAM, AIK-C, Schwarz, and Tanabe strains were maintained in Vero cells in our laboratory. MS and Yokota strains are early passage lots (<10 passages). IC-B, Na-PBMC, Suz-PBMC, MOE, HY, Moka, and Kishida strains were maintained in B95a cells as reported (22). Virus titer was determined as PFUs on Vero/CD150 and the multiplicity of the infection (MOI) of each experiment was calculated based on this titer (19). For Ab blocking of measles virus entry receptors, cells were pretreated with pAb (35 µg/ml) against human CD46 or human CD150, or mAb (5 µg/ml) against human CD150 or human CD46 for 1 h before the virus infection (25).

Preparation of human monocyte-derived immature dendritic cells and macrophages

Human PBMC were isolated from buffy coat of normal healthy donors by methylcellulose sedimentation followed by standard density gradient centrifugation with Ficoll-Hypaque (Amersham Bioscience) (26). For human immature mDC and macrophage preparations, CD14+ monocytes were obtained from human PBMC by using MACS system (Miltenyi Biotec) with anti-human CD14 mAb-conjugated microbeads. For mDCs, CD14+ monocytes were kept in RPMI 1640 containing 10% FCS, 500 IU/ml human GM-CSF, 100 IU/ml human IL-4 (PeproTech) and antibiotics for 6 days. For macrophages, CD14+ monocytes were kept in RPMI 1640 containing 10% FCS, 500 IU/ml human GM-CSF, and antibiotics for 9 days. Morphological changes were examined by phase contrast microscopy (Olympus IX-70).

Determination of human IFN-beta level

For the estimation of secreted IFN-beta, culture medium were centrifuged to remove cell debris and the supernatants were stored at –80°C until the assay. The level of secreted human IFN-beta in the culture medium was determined according to the manufacturer’s protocol using ELISA kit (Fujirebio).

FACS cytometric analysis of cell surface Ags

Methods for FACS analyses were described previously (26). Briefly, cells were suspended in PBS containing 0.1% sodium azide and 1% BSA (FACS buffer) and incubated for 30 min at 4°C with anti-CD46, CD150, or isotype control, washed, followed by FITC-labeled anti-mouse IgG F(ab')2. Cells were washed and fluorescence intensity was measured by FACS.

Expression profile analysis with GeneChip

All microarray experiments and their data were analyzed according to Minimum Information About a Microarray Experiment (MIAME) guidelines. Total RNA from mDCs was extracted by the RNeasy kit (Qiagen) according to the manufacturer’s instructions. Total RNA (10 µg) from each sample was used to prepare cRNA. Gene expression was analyzed with GeneChip Human Genome U133A probe array (Affymetrix), which contains ~22,000 gene probe sets. Data analysis was performed with Affymetrix microarray suite software version 5 (MAS 5.0; Affymetrix) and GeneSpring software version 6.1 (Silicon Genetics). The default settings of MAS 5.0 were used to calculate scaled gene expression values for each sample.

Native PAGE, SDS-PAGE, and Western blotting

Whole cell extracts were prepared in 20 mM HEPES containing 25% (v/v) glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT, 30 mM NaF, and 1 mM Na3VO4. Protein concentration of the lysate was measured by Bio-Rad protein assay kit. For detection of phosphorylated IRF-3, whole cell extracts (60 µg) were subjected to SDS-PAGE in a 7.5% polyacrylamide gel. Proteins were electrophoretically transferred to Immobilon-P (Millipore) and probed with 1/500 diluted anti-IRF-3 pAb (FL-425; Santa Cruz Biotechnology). Dimer formation of IRF-3 was observed by separating the cell extracts (20 µg) on 7.5% polyacrylamide gel native PAGE (Dai-ichi Pure Chemicals), transferred to membrane, and probed with anti-IRF-3 pAb (1/100 diluted; IBL), washed with PBS containing 0.5% Tween 20 three times and incubated with HRP-conjugated goat anti-rabbit Ig pAb for 1 h at 37°C. Following second incubation, the membranes were washed three times with PBS-Tween 20 and proteins were detected with an ECL chemiluminescence kit (Amersham Biosciences) (26).

RT-PCR and quantitative PCR (Q-PCR)

To remove the viruses attached on cell surface, cells were treated with trypsin, neutralized with serum containing medium, and washed three times with ice-cold PBS. Total RNA was extracted with RNeasy mini kit (Qiagen), 2 µg of total RNA was incubated at 70°C for 5 min, kept on ice for 2 min, and reverse transcription was performed with Moloney murine leukemia virus-reverse transcriptase (Promega) at 37°C for 90 min followed by PCR or Q-PCR. For PCR, ex-TaqDNA polymerase (Takara) was used. Sequences of the primers used for RT-PCR were reported earlier (27). Reaction for Q-PCR was performed with iQ SYBER Green Supermix and amplified PCR products were measured by iCycler iQ real-time PCR analyzing system (Bio-Rad). Primers for PCR were designed using Primer Express software (PerkinElmer/Applied Biosystems). The following primers were used for Q-PCR: human beta-actin forward, 5'-CCTGGCACCCAGCACAAT-3'; reverse, 5'-GCCGATCCACACGGAGTACT-3'; human IFN-beta forward, 5'-CAACTTGCTTGGATTCCTACAAAG-3'; reverse, 5'-TATTCAAGCCTCCCATTCAATTG-3'; MV-H-forward 5'-CCCTTATCAACGGATGATCC-3'; reverse-5'-GTGATCAATGGCCCGAATCC-3'. Normalized value for each mRNA expression was calculated as relative quantity of mRNA divided by the relative quantity of human beta-actin.

RNA interference

The method for gene-silencing using small interference RNA (siRNA) oligonucleotides was described previously (28). The method for transient gene-silencing using siRNA oligonucleotides was described previously (29). The sequences of the siRNA for TICAM-1 (29), TANK-binding kinase 1 (TBK1), I{kappa}B kinase-related kinase {epsilon} (IKK{epsilon}) (30), RIG-I, MDA5, and mitochondria antiviral signaling (MAVS) (IPS-1/Cardif/VISA) (31) were reported previously. The method for establishing stable gene-silenced HeLa cells was reported in a preceding report (28). Knockdown status was analyzed by RT-PCR.

Reverse genetics to produce recombinant MV

MV323 (wild-type Ichinose strain) and MV2A (ED strain) were recovered from p+MV323 and p+MV2A, respectively, as previously described (27, 32). Briefly, 293-3-46 cells were transfected with p+MV323 and p+MV2A, plus L gene-expressing plasmid. Two days later, B95a cells were overlaid. Recovered viruses were amplified with Vero or Vero/CD150 cells.

p(–) MV minigenome replicon GFP and in vitro transcription of the RNA replicon

The vector p(–)MV minireplicon GFP was constructed for transcribing the MV minireplicon GFP RNA(–) as a MV short genome. MV minireplicon GFP RNA contains the leader sequence, noncoding region of the N gene, GFP gene, noncoding region of the L gene, and the trailer sequence. The cDNA of MV minireplicon GFP was inserted into pT7 MV vector (containing T7 promoter, T7 terminator, genomic hepatitis {delta} virus ribozyme) which was constructed by modifying p+MV323 (provided Dr. K. Takeuchi, Tsukuba University) (32). MV minireplicon GFP RNA– was transcribed from p–MV minireplicon GFP RNA+ using MEGAscript T7 kit (Ambion) in vitro. Calf intestine alkaline phosphatase (CIAP; Takara) treatment of the transcribed RNA was performed according to the manufacturer’s protocol.

Plasmid transfection and luciferase assay

A luciferase reporter gene plasmid, pISRE-Luc (firefly luciferase, experimental reporter), was purchased from Stratagene, pRL-TK vector (Renilla luciferase for internal control) was obtained from Promega. All transfections were conducted on HEK293 cells growing in 24-well plates. Usually, 100 ng of pISRE-Luc and 3 ng of pRL-TK vector were introduced into cells according to the manufacturer’s procedure (Qiagen). At 24 h posttransfection, synthetic MV minireplicon RNA or poly I:C was introduced into cells by Lipofectamine 2000 (Invitrogen Life Technologies). Six hours later, cells were harvested with trypsin, washed with PBS, and treated with 20 µl of passive lysis buffer (Promega) and the assay was performed using dual luciferase reporter assay system (26). Fold induction against the control medium is indicated.

RT-PCR amplification of cDNA from 5' copy-back DI RNAs

We modified the RT-PCR amplification protocol of Calain (33), where the copy-back DI RNAs were amplified using two sets of MV primers (for 5' copy-back DIs, JM396; 5'-TATAAGCTTACCAGACAAAGCTGGGAATAGAAACTTCG-3'/JM403; 5'-CGAAGATATTCTGGTGTAAGTCTAGTA-3', and for standard genome, JM396/JM402; 5'-TTTATCCAGAATCTCAARTCCGG-3') (34, 35). Viral RNA from the culture supernatant was extracted with QIAamp Viral RNA Mini kit (Qiagen). Total RNA from viral-infected cells was extracted with the RNeasy mini kit (Qiagen). Two micrograms of total RNA was incubated at 70°C for 5 min, kept on ice for 2 min, and reverse transcription was performed with Moloney murine leukemia virus-reverse transcriptase (Promega) at 37°C for 90 min followed by PCR. The PCR-amplified products were cloned into pCR blunt vector (Invitrogen Life Technologies) and sequencing was performed using the BigDye Terminator version 3.1 Cycle Sequencing kit (Applied Biosystem).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
MV attenuated strains induce IFN-beta in mDC infection

Type I IFN-inducing activity of various MV strains was tested using mDCs by ELISA (Table I). Laboratory adapted strains ED, NV, and IC-V, and vaccine strains CAM, AIK-C, Schwarz, and Tanabe, all produced IFN-beta in infected mDCs. These stains were propagated through Vero cells. CHO cells expressing either CD46 or CD150 formed syncytia when infected with these strains (Table I). In contrast, wild-type isolates largely established in Osaka were mostly passaged with B95a cells and barely produced IFN-beta. Only CHO cells expressing CD150 formed syncytia by wild-type MV infection. Because the results on the receptor usage of these strains are consistent with ability of type I IFN induction, MV adaptation to the CD46 receptor might have linked the high IFN-inducing phenotype.


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  		Table I. Cellular responses to various strains of MV

 
Defective IRF-3 activation in low IFN inducers of wild-type strains

Activation of IRF-3 was tested with human mDCs using representative laboratory adapted (NV, ED) and a Vero-adapted wild-type (MS) strain. NV and ED but not wild-type strain MS induced dimerization (Fig. 1A) and phosphorylation (Fig. 1B) of IRF-3. The results were confirmed with other laboratory adapted, vaccine, and wild-type strains (data not shown). Thus, strain-specific IRF-3 activation determines IFN-beta induction in mDCs. NV and vaccine strains induced IFN-inducible genes, IP-10, IFIT1, and IFN-beta, but the wild-type-strain including MS did not (Fig. 1C). An example of our comprehensive approaches regarding type I IFN-inducible genes are shown by microarrays using mRNAs from immature mDCs infected with representative strains (Fig. 1D). NV and ED strains, but not the MS strain, up-regulated IFN-inducible genes, supporting their high vs low IFN-beta-inducing properties.


Figure 1
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  		FIGURE 1. Laboratory adapted and vaccine strains highly induce type I IFN and IFN-inducible genes in monocyte-derived mDCs. Monocyte-derived mDCs were stimulated with medium alone (cont), poly I:C (10 µg/ml for 1 h), or MV strains (MOI = 1 typically for 8 h). At timed intervals, cells were lysed for IRF-3 analysis. CD46-adapted strain (NV or ED), CD46 nonadapted strain (MS), or vaccine strains (CAM, AIK-C, Schwarz, or Tanabe). A, Native gel analysis for IRF-3 dimerization; or B, SDS-PAGE for IRF-3 phosphorylation. C, At indicated intervals, MV-H, IP-10, IFIT1, IFN-beta, and GAPDH mRNAs were detected using RT-PCR. D, Three major clusters of MV-regulated genes in mDCs infected with various MV strains. mDCs were infected with the indicated MV strains at MOI = 1. 8 h later, RNAs were extracted from the mDCs and analyzed by GeneChip. Data were analyzed by applying a hierarchical-tree algorithm to the normalized intensity. Induced genes are indicated by shades of red; repressed genes are indicated by shades of blue. Gene symbols are shown in the right column.

 
What factor is responsible for high IFN-beta-inducing properties in MV?

The findings in Fig. 1 allowed us to surmise that NV and ED contain a factor that is responsible for higher induction of IFN-beta and MS and other wild-type strains lack it. Then, the question is what factor participates in high IFN-beta induction in MV laboratory adapted strains. NV and ED, but not MS, use CD46 as an entry receptor (18, 21). Both stimulation and mAb-blocking studies on the receptor CD46 suggested no direct participation of CD46 in IFN-beta induction (M. Taniguchi and T. Seya, unpublished data). A previous report suggested the involvement of nucleocapsid (N) protein of MV in IFN induction (36, 37). Transfectants with N protein, however, did not induce IFN promoter activation (M. Shingai and T. Seya, unpublished data). N protein-expressing cells further transfected with MV RNA elevated IFN promoter activation compared with mock transfection with RNA alone (data not shown). Thus, these previous findings did not explain the differential induction of IFN-beta in wild-type vs vaccine strains upon infected cells. Because the IFN-inducing ability of NV was dominant in cells coinfected with MS in an early phase, IFN-inducing rather than IFN-inhibitory factors (38) govern the IFN-inducing phenotype of MV in the early phase.

Our repetitive trials suggested that 5' copy-back DI RNA has the ability to induce IFN-beta. 5' copy-back DI RNA was present in most of the laboratory adapted and vaccine strains (Fig. 2A). By using the method of Calain and others (33, 34, 35), a unique primer set enabled us to detect 5' copy-back DI RNA by RT-PCR (Fig. 2, A and B). Standard genome-specific primers are primer A (JM396) and B (JM402). DI RNA-specific primers are: primer A (JM396) and C (JM403). The standard genome RNAs were detected in the culture supernatants from all MV strains using RT-PCR amplified with primers A and B (Fig. 2C, upper panel). In contrast, the 5' copy-back DI RNAs were detected in the culture supernatants of the laboratory adapted strains (NV, ED, and IC-V) and vaccine strains (Schwarz and Tanabe) using RT-PCR amplified with primers A and C (Fig. 2C, lower panel). The 5' copy-back DI RNAs were not detected by this method in any wild-type strain or in CAM and AIK-C vaccine strains (Fig. 2C, lower panel). Amplified fragment sizes of the 5' copy-back DI RNAs were variable depending on the strains cells were infected with. In Tanabe strains, multiple fragments were detected. These fragments were cloned into the plasmid vector and sequenced. Their sizes and predicted structures are shown in Table II and Fig. 2B, where the sites of the primers are indicated for detection of DI RNA. Their predicted structures consisted of the stem and loop of various sizes in these DI RNAs (Fig. 2B, Table II).


Figure 2
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  		FIGURE 2. Laboratory adapted and vaccine strains contain 5' copy-back DI RNA. A, Model for the origin of 5' copy-back DI RNA. Two RNA polymerase complexes synthesized a negative-strand RNA from the positive-strand RNA template. The polymerase complex switches the template to a negative-strand RNA synthesized by a front RNA complex. B, The product perfectly matches with a part of the terminal and RNA duplex (stem). C, RT-PCR amplification of 5' copy-back DI RNA from various strains of MV culture supernatants. RT-PCR was performed using standard genome-specific primers (primer A, JM396; and primer B, JM402, in the upper panel) or DI-specific primers (primer A, JM396; and C, JM403, in the lower panel).

 

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  		Table II. Properties of DI RNAs of MV strains

 
The stem-loop RNA duplex induces IFN-stimulated response element (ISRE) promoter activation

We next examined whether the stem-loop structure of MV is crucial for type I IFN induction. The GFP sequence was inserted into the leader and trailer of MV (Fig. 3A). This synthetic RNA (named MV minireplicon RNA) has highly complementary sequence elements in 3' and 5' regions (black and gray boxes), which are needed for replication, transcription, and encapsidation. The synthetic MV minireplicon RNA forms the stem-loop containing RNA duplex by these complementary sequences (Fig. 3A). IFN-beta-inducing activity of MV minireplicon RNA was examined by reporter assay in HEK293 cells. MV minireplicon RNA was either simply added to the cells or transfected by lipofection. When intact MV minireplicon RNA was transfected, luciferase activity with the ISRE promoter showed ~20-fold against that of medium control (Fig. 3B). Detectable levels of the ISRE promoter activation were seen only 6 h after the RNA administration (data not shown). Transfection of denatured MV minireplicon RNA resulted in the loss of IFN-inducing function (Fig. 3B). Thus, the stem-loop structure rather than the MV-specific RNA sequence may be essential for type I IFN elevated by MV DI RNA.


Figure 3
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  		FIGURE 3. Viral minireplicon RNA induces IFN signaling. A, Schematic representation of vector p(–)MV minireplicon GFP and transcribed synthetic MV minireplicon GFP RNA– (linear and stem loop forms). MV minireplicon GFP RNA(–) was transcribed using T7 in vitro transcription. Black boxes and gray boxes were highly complementary to a sequence element, which is present in 3' and 5' regions of the MV genome, suggesting that the synthetic MV minireplicon consists of stem and loop. B, Synthetic minireplicon RNA activates the ISRE promoter. HEK293 cells were transfected with pISRE-Luc and phRL-TK. Twenty-four hours later, cells were transfected with in vitro-transcribed MV minireplicon RNA, or heat-treated denatured MV minireplicon RNA. Six hours later, the cells were lysed and luciferase reporter assay was performed. The experiments were performed at least three times and a representative one is shown.

 
Measles virus ED strain with 5' copy-back DI RNA highly induce IFN-beta

Because 5' copy-back DI RNAs forming the stem-loop structures with double helix induce IFN-beta, we examined whether type I IFN induction by MV strains correlated with the amplitude of accompanied DI RNA. mDCs were infected with ED strain with DI RNA and rED strain (MV2A) without DI RNA at MOI = 0.1. The mRNA levels of IFN-beta and MV-H in mDCs were determined by Q-PCR 12, 24, and 48 h p.i. (Fig. 4A). IFN-inducing activity of ED (with DI RNA) was high in an early phase (<12 h) of infection while that of MV2A was low. The IFN-beta mRNA levels of cells infected with rMV2A (ED), MV323 (IC), and wild-type MS strain, all lacking DI RNAs, were low at 12 h p.i. and then increased in a time-dependent manner (Fig. 4A). Viral growth was monitored with the MV-H mRNA level. MV strains without DI RNAs efficiently replicated in mDCs in comparison with those with DI RNAs (Fig. 4B). Presence of DI RNAs in the lots of these MV strains was confirmed using RT-PCR (Fig. 4C). Similar results were obtained with A549/CD150 cells (data not shown). Thus, the induction of IFN-beta is closely associated with coexisting DI RNAs in an early phase of MV infection.


Figure 4
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  		FIGURE 4. MV-containing DI RNA induces robust IFN-beta in mDCs in an early phase. IFN-beta induction and MV replication profiles in monocyte-derived mDCs. Monocyte-derived mDCs were infected with MV2A (rMV ED strain), MV323 (rMV IC-B strain), NV (laboratory adapted strain with DI RNA), MS (wild-type strain without DI RNA), or ED (laboratory adapted strain with DI RNA) at MOI = 0.1, or treated with medium or poly I:C (20 µg/ml). A, Twelve, 24, or 48 h after infection, the mRNA levels of human IFN-beta in mDCs infected with each strain were measured by Q-PCR. The values were normalized to that of beta-actin mRNA. Fold induction against medium at 12 h is shown. B, MV-H mRNA level was also measured by Q-PCR. The value for MV-H mRNA expression was normalized to that of beta-actin mRNA. Relative fold induction against infection of each strain at 0 h is shown. Experiments were performed at least three times and representative results are shown. C, RT-PCR amplification of 5' copy-back DI RNA from culture supernatants of rMVs (ReMVs; MV2A and MV323) or MVs (NV, MS, and ED). RT-PCR was performed as described in Fig. 2C using standard genome-specific primers (primers A and B, upper panel) or DI-specific primers (primers A and C, lower panel).

 
In other experiments using A549/CD150 cells, parallelism between the levels of IFN-beta mRNA (Fig. 5A) and the amplitude of DI RNA (Fig. 5B) was analyzed. DI RNA was detected over 30 min, then increased in a time-dependent manner (Fig. 5B). Thus, the initial source of DI RNA should be in the virion. Viral DI RNA was increased 2 and 6 h p.i., suggesting that a replication-dependent increase of DI RNA proceeds after the uptake of virion. The IFN induction and the level of DI RNA were almost parallel (Fig. 5, A vs B).


Figure 5
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  		FIGURE 5. MV strain-specific IFN-beta mRNA induction in infected cells. A, A549/CD150 cells without TLR3 were infected with mock, MV2A (rMV ED strain), MV323 (rMV IC-B strain), NV (laboratory adapted strain with DI RNA), MS (wild-type strain without DI RNA), or ED (laboratory adapted strain with DI RNA) at MOI = 0.1. At timed intervals, RNA samples were recovered and mRNAs of IFN-beta and beta-actin were measured by Q-PCR. The value for IFN-beta mRNA expression was normalized to that of beta-actin mRNA. Fold induction against control medium is shown. B, RT-PCR for DI RNA and the standard genome of MV was performed as in Fig. 2C using genome-specific primers (primers A and B, upper panel) or DI-specific primers (primers A and C, lower panel). Experiments were performed at least three times and representative results are shown.

 
MV induces IFN via IKK{epsilon}, TBK-1, and NAK-associated protein 1 (NAP1), but not TICAM-1

mDCs express TLR3 in the endosomes (39) and RIG-I/MDA5 in the cytoplasm (9, 10), which recognize dsRNA. TLR3 recruits TICAM-1/TRIF (29, 40) while RIG-I and MDA5 recruit MAVS/IPS-1/Cardif/VISA adaptors (31, 41, 42, 43). They converged upon NAP1, which assembles IKK{epsilon} and TBK1 to follow the IRF-3-dependnet IFN-beta-inducing pathway (28). Gene-silencing analysis was performed to identify the molecules that are involved in MV (ED)-mediated type I IFN induction.

HeLa cells expressing TLR3 responded to poly I:C to induce IFN-beta (Fig. 6A, right panel). The mRNAs of TICAM-1, IKK{epsilon}, and TBK1 were silenced with siRNA in this study. The gene-silenced cells were infected with MV ED and, 24 h later, the IFN-beta mRNA levels were measured with the ED-infected cells. Cells depleted of IKK{epsilon} or TBK1 hampered the high induction of IFN-beta mRNA (Fig. 6A, left panel), suggesting that the two kinases are crucial factors for IFN-beta induction. In contrast, the absence of TICAM-1 barely affected the ED-derived IFN-beta mRNA level (Fig. 6A, left panel). Cells stimulated with poly I:C were used as control for the TLR3 pathway and, as expected, poly I:C-dependent IFN-beta induction depended on TICAM-1, IKK{epsilon}, and TBK1 (Fig. 6A, right panel). Likewise, NAP1 knockdown exhibited down-regulation of the IFN-beta mRNA (Fig. 6B) when cells with stably silenced NAP1 or GFP were used instead of the transient knockdown cells (28). When NAP1-depleted cells were stimulated with poly I:C, 40% of the mRNA level was reduced within 6 h (cells die during long-term incubation with poly I:C). In the HeLa cell system, ED infection resulted in a 50% decrease of the NAP1-associated IFN-beta induction. The incomplete blockade of IFN-beta induction by NAP1 silencing may reflect the presence of an escaping NAP1 moiety due to a high endogenous level of NAP1 or functional compensation of NAP1 with other TANK family proteins (28, 44). Thus, MV ED induces IFN-beta in HeLa cells, which involves NAP1, IKK{epsilon}, and TBK1, but not TICAM-1. Therefore, the trigger for type I IFN induction by DI RNA is not the TLR3-TICAM-1 pathway.


Figure 6
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  		FIGURE 6. MV ED-mediated type I IFN induction depends on IKK{epsilon}, TBK1, and NAP1, but not on TICAM-1. A, HeLa cells with TLR3 were transfected with siRNA targeted at TICAM-1, IKK{epsilon}, or TBK1, and 72 h later cells were treated with Mock, ED, or poly I:C. Twenty-four hours later, RNA samples were recovered and mRNAs of IFN-beta and beta-actin were measured using RT- and Q-PCR. Normalization was performed. Relative fold induction against mock infection in GFP knockdown is shown. B, NAP1 or GFP stable knockdown HeLa cells were treated with Mock, ED (24 h), or poly I:C (2 h), and Q-PCR was performed as in A. Knockdown of each mRNA was confirmed by RT-PCR. Experiments were performed at least three times and representative results are shown.

 
Both RIG-I and MDA5 sense DI RNA

To determine whether RIG-I and/or MDA5 recognize DI RNA, we transfected the MV minireplicon RNA into HEK293/pISRE-luc cells (Fig. 3B) subtly overexpressing RIG-I or MDA5. ISRE promoter activation was 12-~18-fold increased due to the minimal constitutive expression of RIG-1or MDA5 (left control bars in Fig. 7). When DI RNA was added into the cytoplasm, high ISRE promoter activation was induced in these cells (Fig. 7). DI RNA was heated to denature the stem-loop structure or treated with CIAP to remove 5' phosphates. In RIG-I-expressing cells, CIAP treatment of DI RNA more significantly reduced RIG-I-mediated IFN promoter activation than heat denaturation. In contrast, the reverse is true in MDA5-expressing cells, where CIAP treatment had almost no effect on MDA5-mediated IFN promoter activation. Hence, DI RNA enhances both RIG-I- and MDA5-mediated IFN-beta induction. Both sense the stem-loop structure, while only RIG-I senses 5' phosphates under the conditions setting.


Figure 7
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  		FIGURE 7. Phosphorylation and duplex structure of DI RNA differentially contribute to ISRE promoter activation via the cytoplasmic RNA sensors. HEK293 cells with pISRE-luc were prepared as in Fig. 3B. HEK cells were transfected with full-length RIG-I (center panel) or MDA5 (200 µg/5 x 105 cells; right panel) by polyfection. Empty vector was used as a control (left panel). Twenty-four hours later, cells were transfected with GFP minireplicon RNA (8 µg/ml) by Lipofectamine 2000. Six hours later, cells were harvested for reporter assay. Closed bars to the left in each panel represent reporter activity of cells with no stimuli, whereas DI means loading DI RNA. Non, Intact DI RNA; heat, heat-inactivated DI RNA; CIAP, CIAP-treated DI RNA. One of two similar experiments is shown.

 
DI RNA stimulates the cytoplasmic IFN-inducing pathway

We finally examined whether DI RNAs of MV follows the pathway involving RIG-I/MDA5 and MAVS for signaling IFN-beta induction by gene-silencing technology. RIG-I, MDA5, MAVS, and TICAM-1 were silenced with siRNA (Fig. 8A), then MV minireplicon RNA or poly I:C was transfected into HeLa cells. Six hours later, the IFN-beta mRNA levels were measured by Q-PCR (Fig. 8B). The siRNA of GFP was used as control. When MV minireplicon RNA was transfected into HeLa cells, the IFN-beta mRNA levels in the RIG-I- or MAVS-silenced cells were decreased by <20% of the control GFP siRNA cells. In the MDA5- or TBK1-silenced cells, the IFN-beta mRNA levels were decreased by >50% of the control. In TICAM-1 silencing, however, no decrease of IFN-beta mRNA was observed. When poly I:C was transfected, the cells efficiently induced IFN-beta. The level of IFN-beta mRNA was decreased in cells depleted of MDA5, MAVS, or TBK1 to ~25% of the GFP control (Fig. 8B). RIG-I silencing resulted in a 40% decrease of IFN-beta induction in this setting. TICAM-1 silencing minimally affected the IFN-beta mRNA level, suggesting that participation of TLR3 in IFN-beta induction is negligible in HeLa cells. Taken together, these results strongly suggest that DI RNA induces IFN-beta through the RIG-I/MDA5-MAVS pathway in the cytoplasm.


Figure 8
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  		FIGURE 8. The route for type I IFN induction by MV minireplicon RNA and MV strains. HeLa cells were transfected with siRNA targeted at TICAM-1, RIG-I, MDA5, MAVS site A and B, or TBK1. A, Knockdown of each mRNA was confirmed by RT-PCR. B, Seventy-two hours after siRNA transfection, cells were transfected with MV minireplicon or poly I:C; or C, treated with Mock, MV2A (rMV ED strain), ED, or NV (ED and NV were laboratory adapted strains with 5' copy-back DI). Twenty-four hours later, RNA samples were recovered and the mRNA levels of IFN-beta, and beta-actin were measured using RT- and Q-PCR. Normalization was performed. Relative fold induction against medium or mock infection in GFP knockdown is shown. The experiments were performed at least three times and representative results are shown.

 
Similar experiments were performed using HeLa cells depleted of the factors in the IFN-inducing pathways and various MV strains. The rMV2A induced >100-fold less amounts of IFN-beta mRNA compared with MV minireplicon RNA under the same conditions. ED and NV strains having DI RNA efficiently induced IFN-beta mRNA early in infection of HeLa cells (Fig. 8C, right panel). The minute induction of IFN-beta was suppressed by knockdown of RIG-I or MDA5 while MAVS silencing resulted in complete abrogation of IFN-beta induction (Fig. 8C, left panel). Hence, in HeLa cells RIG-I and MDA5 share the IFN-beta-inducing ability which totally relies on MAVS. Thus, the results again support the importance of DI RNA in IFN-beta induction and involvement of the cytoplasmic RIG-I pathway in the induction of IFN-beta in MV infection.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
MV is a negative-strand RNA virus which is believed to generate dsRNA for virus replication. However, a recent report suggested that influenza virus barely produces more than the detection limit of dsRNA by quantitative ELISA (45). Instead, triphosphate ssRNA activates RIG-I and induces IFN-{alpha}/IFN-beta (45, 46). What is responsible for IFN induction in MV-infected cells has remained largely unknown. Here, we demonstrated that DI RNA participates in MV-mediated IRF-3 activation and type I IFN induction, which occur before virus replication (early phase). Infection with wild-type MV would produce triphosphate ssRNA but in the amount less than that required to activate RIG-I.

Naniche et al. and we previously reported that wild-type MV strains barely produce type I IFN during infection (16, 21). We demonstrated that an early induction of type I IFN by vaccine and laboratory adapted strains of MV depends on the coincidental DI RNA that resides in the virus particles. DI RNA rapidly induced type I IFNs, usually within 2 h after the virion fused with host cells. In contrast, effective viral RNA replication started 12 h p.i. Thus, in the context of DI RNA, IFN induction precedes viral replication to prevent the host cells from damage that accompanies viral replication. Furthermore, replication of the viral genome is retarded once DI RNA replicates in the same cells. At least, MV DI RNA inhibits viral amplification by two independent ways: IRF-3-mediated type I IFN induction and blockage of viral replication. Ultimately, virus strains containing DI RNA are attenuated in host cells by multiple means. Vaccine and laboratory adapted strains tend to possess DI RNA in addition to the genome within the particle. This DI RNA may act as a suppressor of virulence for initial step of virus infection.

We have learned from an earlier study on the MV substrains IC-B (84-01 B95a isolate) and IC-V (84-01 Vero isolate) (22) that the MV isolates from a single patient with acute measles gave rise to differential contents of DI RNA. The B95a-propagated substrain (IC-B) was highly pathogenic in experimental infection with cynomolgus monkeys while the Vero-propagated substrain (IC-V) had low pathogenicity in vivo. We have carefully propagated the MV strains IC-V and IC-B from their original lots and reproducibly detected DI RNA in the IC-V but not the IC-B stock. The MV substrains IC-B and IC-V derived from the same clinical material behave like wild-type and laboratory adapted strains, respectively. Their differential infectious properties appear to be rooted in the properties of the cells where the viruses were propagated. B95a cells can take up broad viral populations through the two orthologs of CD46 and CD150. Vero cells are known to have some deficiency in IFN induction (47). Adaptation of MV strains to Vero cells may foster amplification of DI RNA leading to the formation of DI RNA-containing virus particles that possess less toxicity by inducing type I IFN and blocking replication.

We performed PCR assay using the several original lots of the vaccine strains to detect the DI RNA. Unexpectedly, DI RNA was not detected in the CAM and AIK-C vaccine strains at least at the conditions used. It is possible that these two strains actually do not contain DI RNA or the primers chosen do not recognize the putative DI RNA sequences of these strains. As CAM and AIK-C have been established via plaque purification (48, 49), the original plaque may not have contained DI RNA. Alternatively, V or C proteins of these vaccine strains may be less functional for inhibition of IFNR-mediated amplification of type I IFN production (38, 50). The actual mechanism by which type I IFN is induced in CAM- or AIK-C-infected cells needs further investigation.

Previous studies suggested that the MV N-protein acts as a type I IFN inducer (36, 37). We found that N-protein minimally enhances IFN-{alpha}/IFN-beta production and in conjunction with DI RNA far more up-regulates the IFN production than N protein alone (data not shown). Although the mechanism by which N protein potentiates DI RNA-mediated IFN production in the cells expressing the minireplicon RNA is intriguing, the possibility of interaction between the N protein and DI RNA has remained unaddressed. Anyhow, DI RNA rather than N protein is a potential factor affecting the degree of IFN-{alpha}/IFN-beta induction in the infected cells.

IFN-beta was induced in human cells expressing either CD46 or CD150 in response to the infection by laboratory adapted strains. Laboratory and vaccine strains use CD46 in addition to CD150 as an entry receptor (51, 52). Wild-type strains do not enter the cells via CD46 (17, 18). As CD46 is ubiquitously expressed in human cells, including lymphocytes, mDCs, and macrophages, one can predict that CD46 is associated with IFN-beta-inducing signals (53). However, irrespective of several experimental trials, we could not connect CD46 signaling with IFN-beta induction (54). One possibility is that DI RNA production inadvertently coincides with CD46 receptor usage during MV strains passage in Vero cells. Recent reports indicate that CD46 functions as an immune modulator in lymphocytes and dendritic cells/macrophages (55, 56). The possibility that CD46 is involved in DI RNA-mediated immune responses should be revisited. Likewise, further studies on the properties of the RNA polymerase, nucleocapsid, and MV V protein in association with DI RNA production and receptor signal output in MV-infected cells will be required for further elucidation of measles-mediated immune modulation.

Previous reports suggested that the production of DI RNA is associated with viral-persistent infection (34). We considered whether DI RNA has an ability to regulate MV-persistent infection. DI RNA suppresses viral replication and reciprocally accelerates host IFN induction. Under the conditions where DI RNAs are being produced in cells, viruses are difficult to proliferate. Virus must change and adapt to the hostile environment for its survival and persistency of infection. Thus, DI RNA may be a factor to promote virus evolution and selective adaptation of viruses to host cells. However, the question still remains as to what factor is responsible for promoting initial viral persistency. If DI RNA plays a pivotal role in advancing viral adaptation, one could envisage it as a two-edged sword, decreasing pathogenicity and enhancing persistent infection. Additional study is needed to further explore this possibility.

After completing this study, we found two articles that were released relating to Sendai virus (SeV) DI RNA (57, 58). The level of IFN-beta activation is shown to be proportional to that of SeV DI RNA replication and the viral V and C proteins are effective in blocking the copy-back DI RNA-induced activation of the IFN-beta promoter (57). The other investigators state that SeV DI RNA is required for its robust mDC maturation (58). These investigators claim the importance of RIG-I in DI RNA-mediated IFN-beta induction in SeV. In this study, we not only experimentally show that DI RNA activates IRF-3, but also further define the pathway taken by DI RNA to produce robust IFN-beta. In MV ED studies, DI RNA, rather than the formation of the genomic dsRNA, causes IFN-beta induction and mDC maturation in infected cells.

Note added in proof.

Importance of 5'-phosphate-ended RNA in activation of RIG-I-mediated IFN response was also reported in MV infection by Plumet et al. (59).


   Acknowledgments
 
We are grateful to Drs. A. Hirano (University of Washington, Seattle, WA), Y. Yanagi (Kyushu University, Hakata, Japan), F. Kobune (National Institutes of Health, Tokyo, Japan), T. Kimoto (Osaka Prefectural Public Health Institute, Osaka, Japan), A. Ueda (Osaka University, Osaka, Japan), K. Takeuchi (Tsukuba University, Ibaraki, Japan), N. Inoue, T. Akazawa (Osaka Medical Center for Cancer, Osaka, Japan), and M. Ayata (Osaka City University, Osaka, Japan) for providing cells, MV strains, and discussions. Thanks are also due to M. Kurita-Taniguchi (Osaka Medical Center for Cancer, Osaka, Japan) for technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by CREST and Innovation, Japan Science and Technology Corporation, and by Grants-in-Aid from the Ministry of Education, Science, and Culture (Specified Project for Advanced Research), and the Hepatitis C Virus Project in National Institutes of Health of Japan, and by the Takeda Foundation, the Uehara Memorial Foundation, the Mitsubishi Foundation, the Akiyama Foundation, and the NorthTec Foundation. Back

2 Current address: National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892. Back

3 Current address: Department of Molecular Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan. Back

4 Address correspondence and reprint requests to Dr. Tsukasa Seya, Department of Microbiology and Immunology, Graduate School of Medicine, Hokkaido University, Kita-ku, Sapporo 060-8638 Japan. E-mail address: seya-tu{at}pop.med.hokudai.ac.jp Back

5 Abbreviations used in this paper: RIG-I, retinoic acid-inducible gene; MDA5, melanoma differentiation-associated gene 5; IRF, IFN regulatory factor; MV, measles virus; p.i., postinfection; mDC, myeloid DC; DI, defective interference; CHO, Chinese hamster ovary; pAb, polyclonal Ab; MOI, multiplicity of infection; siRNA, small interference RNA; TBK1, TANK-binding kinase 1; IKK{epsilon}, I{kappa}B kinase-related kinase {epsilon}; MAVS, mitochondria antiviral signaling; ISRE, IFN-stimulated response element; Q-PCR, quantitative PCR; NAP1, NAK-associated protein 1; CIAP, calf intestine alkaline phosphatase; SeV, Sendai virus; VISA, virus-induced signaling adaptor. Back

Received for publication February 12, 2007. Accepted for publication July 27, 2007.


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 Materials and Methods
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 Discussion
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