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* Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029; and
Immunobiology Center, Mount Sinai School of Medicine, New York, NY 10029
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Introduction |
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and -
plays an essential role in the innate control of virus growth and spreading (8). Most viruses are capable of inducing the maturation of conventional DCs and the expression of type I IFNs by at least two distinct mechanisms. The first mechanism, which is independent of virus replication, is initiated with the recognition of viral components by TLRs localized on the cell membrane or in endosomal compartments (9, 10, 11, 12). Signaling by TLRs leads to the expression of type I IFNs as well as the induction of genes involved in DC maturation (13). The second mechanism is based on the intracellular recognition of a viral component such as dsRNA, requires viral replication, is independent of TLR signaling, and does not rely on type I IFN signaling (5, 6, 14). This TLR-independent mechanism is sufficient for the efficient maturation of DCs and the subsequent initiation of immunity (6).
Sendai virus (SeV) Cantell (C) is a paramyxovirus strain known to potently induce type I IFN synthesis. Although its early history is unclear, this strain has been used extensively in type I IFN research (15, 16, 17, 18) and for the production of purified IFN- (19). Investigations from our laboratory demonstrated that SeV-C is a much stronger inducer of both DC maturation and type I IFN production than SeV-52 or influenza virus (5).
These observations revealed a high correlation between the ability of a virus to induce the maturation of conventional DCs and the strength by which it triggers the synthesis of type I IFNs, supporting the hypothesis that the pathways leading to type I IFN induction and DC maturation after infection with live viruses share common molecular elements. The potent induction of DC maturation by SeV-C does not depend on secreted type I IFNs or TLR signaling but does require intracellular virus replication (5, 6). These characteristics make SeV-C especially valuable for the study of the host molecules and viral elements necessary for the efficient triggering of the TLR-independent induction of DC maturation by live viruses.
Using SeV strains C and 52, we investigated the elements that endow viruses with a potent ability to stimulate DCs. We demonstrate that viral defective-interfering (DI) particles contribute to the superior DC maturation ability of SeV-C as compared with SeV-52. These data reveal a novel role for DI particles as enhancers of the TLR-independent pathway for DC maturation in addition to their reported effect in improving type I IFN production in infected cells.
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Materials and Methods |
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Influenza PR8 virus and standard SeV stocks were grown from a 1/103 dilution of the stock virus in 10-day-old chicken-embryonated eggs at 37°C for 40 h. Subsequent SeV-C and SeV-52 passages at dilutions of 0, 1/107, or 1/106 were grown similarly for 40 h. The influenza 
NS1 strain was grown in 6 day-old-embryonated eggs for 40 h. Allantoic fluid containing virus was harvested, snap frozen using a dry ice/ethanol bath, and stored at –80°C. Viruses were tested for bacterial contamination by inoculation on blood agar plates.
DC2.4, NIH-3T3, 293T, and LLCMK2 cells were grown in tissue culture medium consisting of DMEM (Invitrogen Life Technologies), 10% FBS (heat-inactivated, endotoxin level 0.25 EU/ml; HyClone), 1 mM sodium pyruvate, 2 mM L-glutamine (Invitrogen Life Technologies), and 50 mg/ml gentamicin (Boehringer Mannheim).
C57BL/6 mice and OT-I OVA TCR transgenic mice were purchased from Taconic Farms. The animals were housed in pathogen-free conditions.
Reporter gene assays
NIH-3T3 cells were transiently transfected with 2 µg of IFN- promoter reporter construct (20) (provided by Dr. D. Thanos, Columbia University, New York, NY), driving firefly luciferase production together with 0.2 µg of pRL-TK (Promega) constitutively expressing Renilla luciferase for normalization. To examine the role of retinoic acid-inducible gene I (RIG-I), 250 ng of empty pCAGGS or pCAGGS expressing either RIG-I or the dominant-negative RIG-IC (21) (obtained from Drs. W. B. Cardenas and C. F. Basler, Mount Sinai School of Medicine, New York, NY) were added to the transfection mixture. Transfection was performed using Lipofectamine/Lipofectamine Plus (Invitrogen Life Technologies) according to the manufacturer’s instructions. Twenty-four hours later, cells were infected with SeV-C or SeV-C low DI or were mock infected. Cell extracts were obtained 24 h postinfection and examined for expression of firefly and Renilla luciferase using a dual luciferase assay (Promega).
To test the ability of the influenza NS1 protein to block SeV activation of the IFN- promoter, 293T cells were transiently transfected with 1.0 µg of IFN- promoter reporter construct, 0.1 µg of pRL-TK, and 0.25 or 0.5 µg of pCAGGS empty plasmid or pCAGGS expressing the NS1 1-73 RNA binding domain (provided by Dr. A. García-Sastre, Mount Sinai School of Medicine, New York, NY). Transfections were performed using TransIT-TKO transfection reagent according to the manufacturer’s instructions for DNA plasmids (Mirus Bio). Total DNA transfected was equalized using empty pCAGGS vector. Dual luciferase assay was performed.
Quantitative RT-PCR (qRT-PCR)
RNA was extracted from DC2.4 or bone marrow-derived (BM)-DCs at various time points after virus infection following the manufacturer’s instructions for the High Pure RNA Isolation Kit (Roche). qRT-PCR was performed similarly to a previously published protocol (22). In short, each sample was assayed in triplicate, and the fold change values and control changes for each gene were calculated using the median threshold cycle. Primers for housekeeping genes used for normalization (rps11, GAPDH, tubulin, and -actin) were previously described (23, 24). Copy number was determined using 2500 as an empirical estimate of the number of -actin mRNA molecules/cell (24). Primer sequences used for murine IFN and IL-12p35 genes are as follows: IFN-, 5'-TCCTGAGCCAAAGTGTAGAG-3' and 5'-GAGAACAAGTGCCTTTACAG-3'; IFN-, 5'-AGATGTCCTCAACTGCTCTC-3' and 5'-AGATTCACTACCAGTCCCAG-3'; and IL-12p35, 5'-ACAGCTACCTCAGCATGGTC-3 and 5'-GACGTCTTCGCCCCTTAACA-3'. Primer sequences for the SeV nucleoprotein (NP) gene are as follows: 5'-TGCCCTGGAAGATGAGTTAG-3' and 5'-GCCTGTTGGTTTGTGGTAAG-3'.
Western blots
Abs against STAT1 (E-23) and phosphorylated-STAT1 (A-2) were obtained from Santa Cruz Biotechnology and used according to the manufacturer’s instructions. Total protein extracts were prepared in whole cell extract buffer (50 mM Tris (pH 8.0), 280 mM NaCl, 0.5% IGEPAL, 0.2 mM EDTA, 2 mM EGTA, 10% glycerol, and 1 mM DTT) containing the Complete protease inhibitor mixture (Roche). Protein content was quantified using a standard Bradford assay. Proteins were separated by SDS-PAGE and transferred to nitrocellulose filters, immunoblotted by standard procedures, and prepared for chemiluminescent detection using Renaissance reagents according to the manufacturer’s protocol (PerkinElmer Life Sciences).
Hemagglutination (HA) and infectivity assays
Viruses diluted 1/2 in 0.5% chicken RBCs were incubated for 30 min at 4°C. HA of RBCs indicates the presence of virus particles. The presence of infectious particles was evaluated by infecting LLCMK2 cells with serial 1/10 dilutions of the virus at 37°C. After 1 h of infection, 175 µl of medium containing 2 µg/ml trypsin was added, and the cells were further incubated for 72 h at 37°C. Fifty microliters of medium was then removed from the plate and tested by HA for the presence of virus particles.
Cytokine detection
Supernatants from infected DCs were collected 24 h after infection. IFN- secretion was measured by capture ELISA (PBL Biomedical Laboratories). IL-6 and TNF- detection kits (DuoSet ELISA Development Systems) were purchased from R&D Systems. Assays were performed according to the manufacturer’s protocol. The IL-12p40 ELISA was performed using capture and secondary Abs (C15.6 and C17.8; BD Pharmingen) according to the manufacturer’s protocol. In some experiments, supernatants from cell cultures were collected and analyzed using a multiplex bead assay for cytokines (Upstate Biotechnology).
Generation of BM-DCs and flow cytometry
DCs were prepared according to a standard protocol ensuring the production of immature DCs (5). Briefly, bone marrow precursors were depleted of cells expressing CD4, CD8, B220, and MHC II by magnetic bead separation and cultured in 25 U/ml GM-CSF at a density of 7 x 105 cells/well in a 24-well plate. After 4 days of incubation, all of the cells in the culture expressed the CD11b marker, and 
30% of them corresponded to CD11c+DEC205+GR1– immature DCs (these cells expressed undetectable to low levels of MHC II and costimulatory molecules). The remaining cells were DC precursors (CD11b+CD11c–GR1+). Immature DCs were infected in their original wells to minimize maturation due to manipulation of the cells. Infection with different virus stocks was equalized using 200 HA units or multiplicity of infections (MOIs) of 0.5, 2, or 10. Cells were collected 24 h postinfection and stained with FITC-conjugated Abs to CD80, CD86, or MHC II (BD Pharmingen). Flow cytometry was performed on a Cytomics FC 500 machine (Beckman Coulter) and analyzed using FlowJo software (Tree Star).
Viral RNA isolation and DI particle detection by PCR
RNA was directly extracted from virus in allantoic fluid using TRIzol reagent according to the manufacturer’s instructions (Invitrogen Life Technologies). For detection of DI particle genomes, viral RNA was reverse transcribed using a primer specific for the SeV antigenomic promoter with an added SapI restriction site (5'-CCGGGCTCTTCGGCCACCAGACAAGAGTTTAAGAGATATTTATTC-3'). PCR was performed as described above, using this single primer that can amplify in forward and reverse directions on a copy-back DI particle. As a control for the presence of standard virus genomes, a PCR was performed using the primer designed for the antigenomic promoter as well as a second primer with an added NheI restriction site (5'-GCGCGCTAGCTGTCGGTCTAAGGCAGAAAATGTGG-3') expected to amplify 3400 bp of the L gene.
DI particle purification
DI particles were purified as described previously (25). In short, SeV-C was grown at the standard 1/103 dilution in 100 eggs for 40 h. Allantoic fluid was pooled and concentrated by high-speed centrifugation. Pellets were resuspended and incubated overnight at 4°C in 0.5 ml of PBS/2 mM EDTA. The virus suspension was then added to a 5–45% sucrose gradient and centrifuged at 4°C for 1.5 h at 28,000 rpm. A pellet described to contain viral aggregates was visible as were bands representing high- and low-density viral particles (25). The fractions containing low-density viral particles were collected and pelleted. This was resuspended in PBS/2 mM EDTA and applied to a second 5–45% sucrose gradient and centrifuged at 4°C for 1.5 h at 28,000 rpm. Fractions were collected and analyzed by HA assay and for replication ability. The fractionated DI particles were characterized as described in Results.
Electron microscopy
SeV-C and purified DI (pDI) particles were adsorbed onto Formvar-coated copper grids and negatively stained with 10 g/L phosphotungstic acid at pH 7.0. Viral particles were visualized using a Hitachi H-7000 transmission electron microscope.
In vitro stimulation of OT-I, OVA-specific TCR transgenic cells
CD8+ T cells were purified from the spleens of OT-I mice by negative selection. Briefly, the cells were incubated with Abs against CD4, MHC II, and B220 (BD Biosciences) followed by goat anti-rat-coated magnetic beads (Polysciences) and magnetic separation. Enriched OT-I cells were labeled with CFSE for analysis of proliferation. In short, 107 cells/ml were incubated with CFSE (Molecular Probes) at 5 µM for 10 min at 37°C, extra CFSE was neutralized with an equal volume of FCS, and the cells were washed in PBS.
To establish the cocultures, BM-DCs were infected in the presence of OVA SIINFEKL peptide (10 µM final concentration) for 1 h and incubated in a 1:10 or 1:20 ratio with labeled OT-I cells for 3 days. T cells were stained with anti-CD25 and anti-CD69 Abs (BD Biosciences), and their activation and proliferation was determined by flow cytometry measuring the dilution of the CFSE dye.
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Results |
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In agreement with our previous report documenting the remarkable ability of SeV-C to induce IFN- secretion by BM-DCs (5), SeV-C induces IFNs and mRNA transcription earlier and at significantly higher levels than SeV strain 52 (Fig. 1a). Concurrently, proinflammatory cytokines indicative of DC maturation are secreted at higher concentrations by DCs infected with SeV-C than by those infected with SeV-52 (Fig. 1b and Ref. 5). To gain insight into the mechanism by which these viruses differentially trigger TLR-independent DC maturation, we focused on identifying the viral elements responsible for the different abilities of the SeV strains C and 52 to induce type I IFNs and DC maturation.
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In addition to interfering with type I IFN signaling, many ssRNA viruses encode antagonists able to block type I IFN synthesis. The NS1 protein of influenza virus and the V protein of SeV inhibit the induction of type I IFNs by blocking intracellular dsRNA signaling (20, 31, 32, 33, 34). The influenza virus NS1 protein has also been shown to inhibit viral induction of DC maturation (5, 35). Thus, we hypothesized that SeV-C may encode an impaired type I IFN antagonist allowing stronger DC stimulation by viral dsRNA than SeV-52. Although divergence was found in sequences of other viral proteins, there is 100% amino acid identity between the V proteins from the 52 and C strains (GenBank accession nos. AY909550 and AY909543). Moreover, coinfection of the DC line DC2.4 with SeV-C and 52 did not reduce the ability of SeV-C to induce the production of IFN- mRNA, even when high MOIs of SeV-52 were used (Fig. 1d). To provide sufficient time for the synthesis of the SeV-52 type I IFN antagonist, we preinfected DCs with SeV-52 for 18 h before the infection with SeV-C. SeV-52 was still unable to inhibit IFN- induction by SeV-C under these conditions (Fig. 1e). In a similar coinfection experiment, influenza virus strain PR8, coding for the type I IFN antagonist NS1, inhibited the production of type I IFNs triggered by the antagonist deleted influenza virus NS1 (Fig. 1f). Thus, the difference between SeV-52 and SeV-C’s ability to induce type I IFN and DC maturation is not explained by a mutation in the SeV-C V protein or the absence of a type I IFN antagonist similar to the influenza virus NS1.
SeV-C produces more activating stimulus than SeV-52
The virus replication intermediary dsRNA has been shown to induce type I IFN synthesis through the direct or indirect activation of cellular proteins such as the dsRNA-dependent protein kinase (PKR) (36), the RIG-I (21), the melanoma differentiation-associated gene 5 (mda-5) (34), the TANK binding kinase 1, and the I
B kinase 
(37, 38) that subsequently lead to activation of IFN regulatory factor-3 and other transcription factors. Because the intracellular triggering of DC maturation by viruses requires virus replication (5, 6), we evaluated whether an increased amount of dsRNA produced during viral replication might account for the potent induction of type I IFNs by SeV-C. To do this, we took advantage of the ability of the influenza NS1 protein to bind dsRNA thereby blocking the induction of type I IFN expression (39). NS1 contains an N-terminal dsRNA binding domain between aas 1–73 and an effector domain at residues 74–237. Transfection of the NS1 dsRNA binding domain inhibited the induction of IFN- by both SeV-52 and SeV-C. However, SeV-C required a higher amount of NS1 to reduce the IFN- promoter induction to a level similar to that of SeV-52 (Fig. 2a). These data suggest that the disparity between SeV-52 and SeV-C may be explained by differences in the activating dsRNA produced by the viruses.
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SeV-C stocks have a higher proportion of DI particles than SeV-52
Viral DI particles contain incomplete genomes that replicate only in the presence of standard virus. DI particles are enriched in coinfected cells as they replicate more efficiently than standard virus genomes due to their smaller length and differential promoter sensitivities (41, 42). They, thereby, interfere with standard virus protein production by monopolizing the viral replication machinery. Undiluted passages of SeV can result in enhanced DI particle production, and their presence has been shown to increase the induction of type I IFNs (20, 25). Based upon our observations that SeV-C produces proteins less efficiently than SeV-52 and has potentiated type I IFN induction abilities, we hypothesized that SeV-C stocks have high levels of DI particles responsible for their superior DC-maturing ability (Figs. 1a and 2b and Ref. 5).
Presence of DI particles can be detected using a well-characterized method of calculating the ratio between infectious viral particles and total viral particles (DI particles plus standard infectious virus) (25). SeV-52 and SeV-C stocks were analyzed for their total particle titers using a HA assay based on the binding of chicken RBC to the external HN protein of SeV particles. Infectious particle quantifications (tissue culture infectious dose (TCID)50) were obtained by titration in LLCMK2 cells. As shown in Table I, SeV-52 and SeV-C stocks have equivalent TCID50 values. However, the SeV-C stock contained twice as many HA particles as SeV-52. This lower infectivity to total HA particle (I:HA) ratio of SeV-C compared with SeV-52 (Table I) suggests a higher content of DI particles in SeV-C.
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To test the hypothesis that DI particles are responsible for the strong DC maturation ability of SeV-C, we manipulated its DI particle content by modifying the virus growth conditions. To obtain standard virus depleted of DI particles, SeV-C was inoculated into eggs at a dilution of 1/107. At this dilution, only 33% of infected eggs tested positive for virus at 40 hpi (data not shown), indicating that the average viral inoculum was <1 infectious dose per egg. By pooling the positive eggs, a viral stock was created that was decreased in its DI particle content, as indicated by its raised I:HA ratio compared with the original SeV-C stock (Table I). This stock was then further passaged at a 1/106 dilution, creating the stock herein referred to as SeV-C low DI (Table I).
Virus preparations with different DI particle content were tested for their abilities to induce maturation of mouse BM-DCs. SeV-C low DI had a significantly reduced ability to mature DCs as measured by the up-regulation of costimulatory molecules (Fig. 3a), cytokine secretion (Fig. 3b), and IL-12p35 mRNA production (Fig. 3c). Measurement of viral NP mRNA in cells infected with equal MOIs indicated that all viruses tested were actively replicating (Fig. 3c), although, as seen previously, levels of viral mRNA production did not correlate with DC maturation ability. Additionally, equalizing HA titers (200 HA units) of the various virus preparations used to infect BM-DCs demonstrated that the total number of particles is not the factor responsible for the potent induction of DC maturation by SeV-C (Fig. 3, a and b). Rather, the DC maturation ability of SeV-C is directly influenced by its DI particle to standard virus ratio. DI particles contribute a unique stimulus for DC maturation that is not mimicked by equilibrating the total number of viral particles.
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RIG-I is a dsRNA binding protein known to be involved in the innate immune response to virus infection (21). Taking into consideration the potentially increased dsRNA production of viruses high in DI particle content as compared with standard virus (Fig. 2a), we tested RIG-I for its involvement in the activated signaling by viruses high in DI particle content. As shown in Fig. 4a, overexpression of RIG-I results in superior IFN- promoter induction by SeV-C, whereas the presence of RIG-IC (21), a dominant-negative form of RIG-I, eliminates IFN- promoter induction. Comparison of SeV-C and SeV-C low DI further illustrates the requirement of RIG-I signaling for the enhanced activation ability of the DI particle-rich viruses. Both SeV-C and SeV-C low DI lose their type I IFN induction ability in the presence of a RIG-I dominant-negative protein (Fig. 4b). This suggests that the enhanced production of dsRNA by virus in the presence of DI particles results in a heightened activation of the RIG-I signaling pathway with the consequent increase in the expression of type I IFN.
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As previously described, SeV-52 is both a weak inducer of type I IFN and of DC maturation. Two undiluted passages of this virus significantly decrease its I:HA ratio, reflecting an increased proportion of DI particles in this preparation (Table I). It has been reported that DI particles with genomes flanked by the viral antigenomic promoter are responsible for enhancing type I IFN induction in virus infection (43). A standard RT-PCR assay can be performed to specifically amplify this type of DI species, using only one primer complementary to the antigenomic promoter of the virus. Using this assay, four distinct bands can be detected from amplified SeV-52 RNA (Fig. 5a). When an equivalent amount of SeV-52 high DI RNA was reverse transcribed and amplified, bands representing DI particle species are increased in intensity with a single DI species predominating (Fig. 5a). Thus, confirming the increase in DI particle content indicated by its I:HA ratio, SeV-52 high DI contains higher levels of antigenomic promoter-flanked DI species than SeV-52.
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pDI particles enhance the DC maturation ability of SeVs
pDI particles were isolated from SeV-C using sucrose gradients as described previously (25). SeV-C and pDI particles were then visualized by electron microscopy. SeV-C stocks were found to contain a polymorphic population of particles ranging in size from 50 to 250 nm in diameter, whereas pDI particles are at the smaller end of this size range with the majority having a diameter <100 nm (Fig. 6a). The pDI particle preparation had an I:HA ratio of 80, indicating a dramatic reduction in the proportion of infectious particles compared with SeV-C (Table I). The pDI particles were assessed by three criteria: 1) inability to replicate in the absence of standard virus; 2) interference with viral protein production; and 3) enhancement of type I IFN induction by standard virus. BM-DCs were treated with increasing doses of pDI particles from 5 to 500 HA units and even at the highest dose tested, no significant viral replication was detected as measured by qRT-PCR of NP mRNA transcripts (Fig. 6b). In contrast, infection with increasing doses of SeV-C, the parental virus, shows an increase in NP mRNA correlating with the dose. Likewise, infection with SeV-C low DI results in robust transcription of NP mRNA. Conversely, NP mRNA production by SeV-C low DI is inhibited when cells are coinfected with pDI particles. As expected, type I IFNs were not induced by the pDI particles alone, but coinfection with standard replicative virus allowed high induction of IFN- similar to levels induced by SeV-C (Fig. 6c). Thus, the pDI particles conform to the criteria for DI particles.
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, and IL-12 p40 (Fig. 6d), as well as the surface expression of CD80 and MHC II (Fig. 6e), in BM-DCs coinfected with standard virus. Similar results were obtained using SeV-52 as the standard virus (Fig. 6e and data not shown), demonstrating that a virus that weakly stimulates DCs can gain potent DC maturation ability with the addition of pDI particles. Furthermore, DCs matured by SeV in the presence of pDI particles are better able to activate naive TCR transgenic OT-I cells (Fig. 7). An enhanced expression of the T cell activation markers CD69 and CD25 (Fig. 7b), as well as secretion of IFN-
, IL-2, and TNF- (Fig. 7c) were observed when OT-I cells were primed by DCs activated in the presence of pDI particles. The activation ability of pDI particles was hindered by their UV inactivation (Fig. 7, b and c), showing that the generation of DI particle replication intermediaries is essential for their activity. Fig. 7a shows a control experiment demonstrating that DCs presenting the OVA SIINFEKL peptide specifically stimulated T cell proliferation. These experiments demonstrated that DCs matured in the presence of DI particles, show an enhanced ability to prime naive CD8+ T cells.
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Discussion |
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promoter induction more efficiently than SeV-52 (Fig. 2a). These studies have revealed that DI particles present in the virus stocks are capable of enhancing DC maturation by both SeV-C and SeV-52. SeVs are known to produce several species of DI particles. Internal deletion DI particles are missing internal sequences, making them replication incompetent even though they contain both genomic and antigenomic promoters allowing transcription and replication. Copy-back DI particles lack the genomic promoter that is replaced by the antigenomic promoter preventing transcription but allowing replication. Copy-back DI particles replicate up to 20 times faster than species having both genomic and antigenomic promoters, due to specific sequences within their promoters that confer higher replication abilities (41, 42, 44). Our studies show that viruses with enhanced DC activation abilities have increased levels of antigenomic promoter-flanked copy-back DI particles (Fig. 5a). The increased replication of DI particle genomes compared with standard virus genomes provides a mechanism by which DI particles supply higher levels of a DC-activating virus replication intermediate, most probably dsRNA, that triggers type I IFN and DC maturation induction pathways. This is consistent with previous reports that replication is required for induction of DC maturation by SeV-C (5, 6), and supported by the observation that pDI particles are inert in the absence of standard virus replication machinery yet significantly enhance DC maturation when standard virus is present (Fig. 6).
Although the DC maturation ability of SeV-52 is improved by the presence of DI particles, the levels of cytokine secretion and costimulatory molecule up-regulation are still below that of SeV-C (Figs. 3 and 5). Thus, there may be additional differences between the two viruses. SeV-C may have a genetic predisposition for the production of larger quantities of DI particles, thus enhancing its DC maturation abilities compared with SeV-52. Alternatively, SeV-C stocks may contain a specific DI particle species that more efficiently triggers DC maturation than those present in SeV-52. Regardless of these possible differences, our results clearly demonstrate that DI particles contribute to the DC maturation abilities of each virus.
The findings of these studies confirm our previously reported correlation between type I IFN induction and DC maturation even in the absence of secreted type I IFN signaling (5) and TLR signaling (6), providing evidence that the type I IFN and DC maturation pathways induced by intracellular viral replication share common molecules. Experiments using the NS1 protein from influenza virus suggest that DI particles increase levels of dsRNA in infected cells (Fig. 2a). Recent studies using overexpression of the SeV V protein further support a role for dsRNA in the enhancement of type I IFN induction by DI particles (45). The protein RIG-I has been shown to directly interact with dsRNA leading to induction of type I IFNs (21) and is a likely candidate molecule for involvement in DC maturation in response to virus as other known dsRNA binding proteins, TLR3 and PKR, have been shown to be dispensable for DC maturation (5, 6, 14, 46). In fact, signaling initiated by RIG-I has been shown to be the primary pathway for induction of type I IFNs in conventional DCs (47) and is necessary for efficient type I IFN induction by DI particle-rich viruses (Fig. 4).
Our demonstration that addition of DI particles to a virus that weakly activates DCs dramatically enhances its DC maturation ability and concomitantly the priming of CD8+ T cells could lead to considerable advances in vaccine development. Particular to this study, SeV has recently been shown to be a promising vaccine virus candidate because it is generally well tolerated in humans and provides cross-reactive immunity against human parainfluenza virus type I (48). Addition of DI particles to these live-virus vaccine preparations could enhance their immunogenic potential. Vesicular stomatitis virus has also been shown to gain type I IFN induction ability specifically with the presence of copy-back DI particles, suggesting that this mechanism is applicable to other pathogenic viruses (43). Similar effects remain to be studied in other paramyxoviruses of medical concern such as measles and respiratory syncytial virus, which also naturally produce DI particles (49, 50). Thus, knowledge of DI particle enhancement of virus-induced DC maturation should be considered in the rational design of viral vaccines.
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Acknowledgments |
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Disclosures |
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Footnotes |
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1 This work was supported by Grants 1R01AI41111 and U19AI062623-01 (to T.M.M.), and AI48722 (to C.M.H.) from the National Institute of Allergy and Infectious Diseases, and by funds granted by the Charles H. Revson Foundation (to C.B.L.). The statements made and views expressed, however, are solely the responsibility of the authors. Electron microscopy was performed at the Mount Sinai School of Medicine Microscopy Shared Resource Facility supported with funding from National Institutes of Health-National Cancer Institute shared resources (1 R24 CA095823-01) and National Science Foundation major research instrumentation (DBI-9724504) grants. 
2 Current address: Department of Medicine and Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University; Department of Medicine, Evanston Northwestern, Evanston, IL 60208.
3 Address correspondence and reprint requests to Dr. Carolina B. López, Department of Microbiology, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1124, New York, NY 10029. E-mail address: Carolina.Lopez{at}mssm.edu
4 Abbreviations used in this paper: DC, dendritic cell; SeV, Sendai virus; C, Cantell; DI, defective interfering; RIG-I, retinoic acid-inducible gene I; qRT-PCR, quantitative RT-PCR; BM-DC, bone marrow-derived DC; NP, nucleoprotein; HA, hemagglutination; MOI, multiplicity of infection; pDI, purified DI; PKR, dsRNA-dependent protein kinase; TCID, tissue culture infectious dose; I:HA, infectivity to total HA particles.
Received for publication August 30, 2005. Accepted for publication July 11, 2006.
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References |
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M. Sgarbanti, A. L. Remoli, G. Marsili, B. Ridolfi, A. Borsetti, E. Perrotti, R. Orsatti, R. Ilari, L. Sernicola, E. Stellacci, et al. IRF-1 Is Required for Full NF-{kappa}B Transcriptional Activity at the Human Immunodeficiency Virus Type 1 Long Terminal Repeat Enhancer J. Virol., April 1, 2008; 82(7): 3632 - 3641. [Abstract] [Full Text] [PDF]
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J. S. Yount, L. Gitlin, T. M. Moran, and C. B. Lopez MDA5 Participates in the Detection of Paramyxovirus Infection and Is Essential for the Early Activation of Dendritic Cells in Response to Sendai Virus Defective Interfering Particles J. Immunol., April 1, 2008; 180(7): 4910 - 4918. [Abstract] [Full Text] [PDF]
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M. D. Gainey, P. J. Dillon, K. M. Clark, M. J. Manuse, and G. D. Parks Paramyxovirus-Induced Shutoff of Host and Viral Protein Synthesis: Role of the P and V Proteins in Limiting PKR Activation J. Virol., January 15, 2008; 82(2): 828 - 839. [Abstract] [Full Text] [PDF]
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M. Shingai, T. Ebihara, N. A. Begum, A. Kato, T. Honma, K. Matsumoto, H. Saito, H. Ogura, M. Matsumoto, and T. Seya Differential Type I IFN-Inducing Abilities of Wild-Type versus Vaccine Strains of Measles Virus J. Immunol., November 1, 2007; 179(9): 6123 - 6133. [Abstract] [Full Text] [PDF]
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J. S. Yount, T. M. Moran, and C. B. Lopez Cytokine-Independent Upregulation of MDA5 in Viral Infection J. Virol., July 1, 2007; 81(13): 7316 - 7319. [Abstract] [Full Text] [PDF]
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