Abstract

MATERIALS AND METHODS

RESULTS

Kinetics of IRF-3 activation and deactivation. Indirect immunofluorescence was used to monitor activation and translocation of the transcription factor IRF-3 to the nuclei of A549 cells during infection with wt rRSV, UV-inactivated wt rRSV, and the ΔNS1, ΔNS2, and ΔNS1/2 gene deletion mutants (Fig. 1A and B). As a positive control, cells were transfected in parallel with polypoly(I:C) dsRNA. This positive control induced rapid, efficient translocation of IRF-3 to the nucleus, with peak accumulation at 4 to 6 h posttransfection (Fig. ​(Fig.1A,1A, bottom). This was followed by the rapid loss of nuclear IRF-3, reflecting deactivation of IRF-3 and recycling to the cytoplasm, followed by degradation.

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FIG. 1.

Nuclear localization of IRF-3 in A549 cells in response to infection with wt rRSV or treatment with synthetic polypoly(I:C). (A) A549 cells were mock infected or infected at an MOI of 3 PFU/cell with wt rRSV, ΔNS1, ΔNS2, ΔNS1/2, or the equivalent amount of UV-inactivated wt rRSV or were transfected with polypoly(I:C), as indicated. The cells were fixed at the indicated times postinfection, permeabilized, and analyzed by indirect immunofluorescence with polyclonal antibodies to IRF-3 (green) or a polyclonal antiserum that reacts with both NS1 and NS2 (red). This antiserum detected NS1 more efficiently than NS2 (see the text). (B) The percentage of cells with nuclei positive for IRF-3 or that were positive for NS1/NS2 at each time point and for each treatment was calculated based on multiple fields of view from two independent experiments. The indicated (*) value of IRF-3 for the ΔNS1, ΔNS2, or ΔNS1/2 virus was confirmed to be significantly different from that of wt RSV (P = 9.1 × 10⁻⁸, 1.3 × 10⁻⁷, or 1.6 × 10⁻⁸, respectively; Student's t test). The values of IRF-3 at 14 h versus 10 h for wt RSV also were confirmed to be significantly different (P = 1.1 × 10⁻⁸).

Infection with wt rRSV and the ΔNS1, ΔNS2, and ΔNS1/2 gene deletion mutants efficiently induced nuclear translocation of IRF-3 during the first 10 h p.i. (Fig. 1A and B). Translocation of IRF-3 was not observed when cells were exposed to UV-inactivated wt rRSV, indicating that RSV replication was necessary for IRF-3 activation in A549 cells (Fig. ​(Fig.1A).1A). In cells infected with wt rRSV, the average percentage of nuclei positive for IRF-3 reached a peak of ~26% at 10 h p.i., a value very similar to the peak of 35% observed with polypoly(I:C) (Fig. ​(Fig.1B).1B). One difference was that the peak of nuclear accumulation of IRF-3 in response to wt rRSV occurred somewhat later than in response to polypoly(I:C) (10 h versus 4 to 6 h), which presumably reflects a lag necessary for viral macromolecular synthesis to occur sufficient to activate IRF-3. The percentage of cells with intense nuclear IRF-3 staining then dropped dramatically in wt rRSV-infected cells to ~1% at 14 h p.i., mirroring the export and degradation of IRF-3 as observed following treatment with polypoly(I:C). In cells infected with the NS1/NS2 deletion mutants, the percentage of nuclei positive for IRF-3 reached a maximum of ~35% by 10 h (ΔNS1) or ~30% by 14 h (ΔNS2, ΔNS1/2) p.i. These values did not subsequently decline, indicating that IRF-3 activation continued in cells infected with the gene deletion mutants. Thus, the difference between wt rRSV and the NS1/NS2 gene deletion mutants was not in the ability to activate IRF-3, but rather in the duration of the response. The ability of NS1 and NS2 to individually affect the activation of IRF3 had not been investigated for HRSV or BRSV. Here, deletion of either NS1 or NS2 greatly alleviated the block to activation. This suggested that the cooperative effect of the two proteins in inhibiting IRF-3 activation is much greater than those of the individual proteins.

The expression of the NS1 and NS2 proteins in parallel cultures was visualized with a polyclonal antiserum that recognizes both proteins (Fig. ​(Fig.1A).1A). This showed that the expression of NS1 was readily detectable by 10 h p.i. (illustrated by the ΔNS2 mutant in Fig. ​Fig.1A),1A), whereas NS2 was detected less efficiently (illustrated by the ΔNS1 mutant). The antiserum used here detects NS1 much more efficiently than NS2, as we have observed in previous experiments (unpublished data) and as was confirmed in the present study by Western blot analysis (e.g., see Fig. ​Fig.3B).3B). In wt rRSV-infected cells, the percentage of cells in which expression of NS1 and NS2 was readily detected increased from ~8% to 27% from 10 to 14 h p.i. (Fig. ​(Fig.1B),1B), which coincided with the loss of IRF-3 from the nucleus. This suggests that the de novo synthesis of NS1 and NS2 in wt rRSV-infected cells reached a sufficient level by ~10 h p.i. to block further activation and translocation of IRF-3, so that the normal deactivation pathway led to a rapid depletion of nuclear IRF-3.

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FIG. 3.

Viral protein expression in A549 cells infected with wt rRSV and the NS1/NS2 gene deletion mutants. (A) Wt rRSV and the NS1/NS2 deletion mutants display comparable levels of gene expression, as illustrated by the levels of N and P proteins. A549 cells were infected with the indicated viruses at an MOI of 3 PFU/cell and harvested 6 h, 10 h, or 14 h postinfection. Equivalent amounts of the cell lysates were subjected to SDS-PAGE and Western blot analysis with a polyclonal serum raised against purified RSV virions. The portion of the blot containing the N and P proteins is shown. (B) NS1 and NS2 are located predominantly in the cytosolic fraction of wt-rRSV-infected A549 cells. Cells were either mock infected (lanes 1) or infected with wt rRSV (lanes 2), ΔNS1 (lanes 3), or ΔNS2 (lanes 4) at an MOI of 3. The cells were harvested 20 h postinfection, and nuclear and cytosolic fractions were prepared and analyzed by Western blotting. The nuclear fraction is represented in fourfold-greater relative concentration than the cytosolic fraction. The efficacy of cell fractionation was confirmed using antibodies to USF2 as a marker for nuclear content and α-tubulin as a marker for cytosolic content. The localization of NS1 and NS2 was identified using a polyclonal antiserum that reacts with both proteins but with NS1 more efficiently than NS2.

The effects of NS1 and NS2 on the activation of IRF-3 were also investigated using a reporter plasmid containing the luciferase gene under the control of an IRF-3-responsive promoter. At 8 h p.i., there was significant IRF-3-responsive promoter activity in A549 cells infected with wt rRSV, ΔNS1, ΔNS2, and ΔNS1/2, with little difference between the various viruses (Fig. ​(Fig.2).2). However, there was no increase in IRF-3-responsive promoter activity between 8 h and 14 h p.i. in cells infected with wt rRSV. As luciferase activity is cumulative, this suggests that transcription from the IRF-3-responsive reporter plasmid had ceased. In contrast, cells infected with the NS1/NS2 gene deletion mutants exhibited a continued increase in IRF-3-responsive promoter activity between 8 h and 14 h p.i. This confirmed the results from the immunofluorescence study that wt rRSV and the NS1/NS2 gene deletion mutants each activated IRF-3 efficiently early in infection but that continued activation was observed only in cells infected by the NS1/NS2 gene deletion mutants.

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FIG. 2.

Activation of IRF-3 monitored using an IRF3-responsive reporter gene. A549 cells were transfected with p55CIBLuc, a reporter plasmid containing the luciferase gene under the control of the PRD I element of the IFN-β promoter. Ten hours posttransfection, the cells were mock infected or infected with the indicated viruses at an MOI of 1 PFU/cell. At 8 and 14 h postinfection, cells were harvested and assayed for luciferase activity. Relative light units are given as induction (n-fold) compared to cells that were transfected with the reporter plasmid but not mock infected or infected. The mean values and standard errors of three independent infections are shown. The indicated (*) value for the ΔNS1, ΔNS2, or ΔNS1/2 virus was confirmed to be significantly different from that of wt RSV (P = 1.6 × 10⁻⁵, 0.004, or 2.1 × 10⁻⁸, respectively; Student's t test).

We considered the possibility that the continued activation of IRF-3 in cells infected with the NS1/NS2 gene deletion mutants might somehow be due to altered kinetics of viral macromolecular synthesis. However, Western blot analysis of cell lysates harvested at various times following infection with wt rRSV or the gene deletion viruses showed that there was little difference between viruses in the level of accumulation of the N and P proteins, as markers of RNA replication and gene expression (Fig. ​(Fig.3A).3A). Furthermore, it was demonstrated in a previous study (31) that the intracellular accumulations of viral genomic RNA were similar for wt rRSV and the NS1/2 mutants.

The patterns of immunofluorescence of NS1 and NS2 in infected cells indicated that the two proteins accumulated significantly in the vicinity of the nucleus (Fig. ​(Fig.1A),1A), although the staining pattern was not clearly indicative of nuclear accumulation compared to that of IRF-3. If nuclear accumulation of NS1 or NS2 occurred, it raised the possibility that they might directly affect events in the IRF-3 activation-deactivation cycle in that compartment. However, fractionation of A549 cells infected with wt rRSV, ΔNS1, or ΔNS2 showed that in each case NS1 and NS2 accumulated mostly in the cytoplasm (Fig. ​(Fig.3B3B).

The NS2 protein is important for activating NF-κB.

NF-κB can be efficiently activated by a variety of inducers that include IFN-α/β, which strongly up-regulates the expression of the double-stranded-RNA-activated protein kinase PKR (41). Since wt rRSV and the NS1/NS2 gene deletion mutants vary considerably in their abilities to induce IFN-α/β (31), in particular with mutants lacking NS1 being capable of inducing a very high level, we controlled for this variable by investigating the activation of NF-κB in Vero cells, which lack the structural genes for IFN-α/β. Translocation of NF-κB to the nucleus was monitored by indirect immunofluorescence at various intervals p.i. using a mouse monoclonal antibody specific to p65 (Fig. 4A and B), which is the major form activated in response to HRSV (3). The inactive form of NF-κB, which is sequestered in a complex in the cytoplasm, could be detected as a diffuse staining pattern at each time point (Fig. ​(Fig.4A).4A). In cells infected with wt rRSV, ΔNS1, ΔNS2, or ΔNS1/2, nuclear accumulation of NF-κB was evident in each case by 6 h p.i. (Fig. ​(Fig.4A).4A). Unexpectedly, however, the level of nuclear accumulation of NF-κB was significantly lower in cells infected with ΔNS2 or ΔNS1/2, each of which lacks the NS2 gene. Thus, the presence of the NS2 gene, and presumably expression of its protein, appeared to be important in the activation of NF-κB. However, the effect was not absolute, since there was a residual level of activation by the ΔNS2 viruses. This background was dependent on viral macromolecular synthesis, as indicated by the lack of NF-κB activation by UV-inactivated virus.

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FIG. 4.

Nuclear localization of NF-κB in Vero cells in response to infection with wt rRSV or the NS1/NS2 gene deletion mutants. (A) Vero cells were mock infected or infected at an MOI of 3 PFU/cell with either wt rRSV, ΔNS1, ΔNS2, ΔNS1/2, or the equivalent amount of UV-inactivated wt rRSV. The cells were fixed at the indicated times postinfection, permeabilized, and analyzed by immunofluorescence with a monoclonal antibody against NF-κB p65 (green) or a polyclonal antiserum against NS1 and NS2 (red) that reacted more strongly with NS1 than with NS2. (B) The percentage of cells with nuclei positive for NF-κB or that were positive for NS1/NS2 at each time point and for each treatment was calculated based on multiple fields of view from two independent experiments. The indicated value of NF-κB for the ΔNS2 or ΔNS1/2 virus was confirmed to be significantly different from that of wt RSV at 10 h (*, P = 5 × 10⁻⁶ and 5.7 × 10⁻⁶, respectively; Student's t test) and at 14 h (**, P = 3.3 × 10⁻⁶ and 2.9 × 10⁻⁶, respectively). In contrast, the value for the ΔNS1 mutant was not significantly different from that for wt RSV at each time point.

The effect of NS2 on the activation of NF-κB was also investigated using a reporter plasmid containing the luciferase gene under the control of an NF-κB-responsive promoter (Fig. ​(Fig.5).5). This showed that the magnitude of activation of the NF-κB-responsive promoter (measured in this example at 14 h p.i.) was greatest for cells infected with wt rRSV, was somewhat less for cells infected with ΔNS1, and was very low for cells that were mock infected or infected with ΔNS2 or ΔNS1/2.

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FIG. 5.

NF-κB activation monitored with an NF-κB-responsive reporter gene. Vero cells were transfected with a luciferase construct driven by a promoter element responsive to NF-κB. Ten hours posttransfection, the cells were infected with the indicated viruses at an MOI of 1 PFU/cell. At 14 h postinfection, cells were harvested and assayed for luciferase activity. Relative light units are given as induction (n-fold). The results show the mean values and standard errors of three independent infections. The values for ΔNS2 and ΔNS1/2 were significantly different from that for wt RSV (*, P = 6.5 × 10⁻⁹ and 1.2 × 10⁻¹⁰, respectively) and ΔNS1 (**, P = 1.2 × 10⁻⁵ and 1.2 × 10⁻⁷, respectively).

We also prepared nuclear extracts from the infected Vero cells at various times and subjected them to Western blot analysis with an antibody against p65 as an independent method of monitoring NF-κB activation (Fig. ​(Fig.6A).6A). This confirmed that the nuclear accumulation of NF-κB p65 was substantially higher in cells infected with wt rRSV or ΔNS1 than in cells infected with ΔNS2 or ΔNS1/2 and confirmed that some activation occurred in the absence of NS2. Nuclear extracts were also analyzed using an antibody to USF2 to confirm the purity of the extracts (not shown).

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FIG. 6.

Western blot analysis of (A) the nuclear accumulation of NF-κB p65 and (B) the synthesis of RSV N and P proteins in Vero cells infected with wt rRSV or the NS1/NS2 gene deletion mutants. (A) Nuclear accumulation of NF-κB p65. Cells were mock infected or infected at an MOI of 3 PFU/cell with either wt rRSV, ΔNS1, ΔNS2, ΔNS1/2, or UV-inactivated wt rRSV. The nuclear fractions from replicate cultures were harvested at the indicated times, and equivalent amounts of protein were subjected to SDS-PAGE, followed by Western blotting with a monoclonal antibody against NF-κB p65. (B) Expression of RSV N and P proteins. Cells were infected with the indicated viruses and harvested 6 h, 10 h, and 14 h postinfection. Equal amounts of cell extract were subjected to SDS-PAGE and Western blot analysis of RSV N and P proteins using a polyclonal antiserum to purified virions.

In order to rule out the possibility that the low level of NF-κB activation in response to ΔNS2 and ΔNS1/2 might reflect reduced kinetics or magnitude of viral RNA replication and gene expression in Vero cells, Western blot analysis was used to measure the levels of accumulation of the N and P proteins as markers for viral RNA synthesis (Fig. ​(Fig.6B).6B). The accumulation of N protein at 10 h p.i. was somewhat lower for cells infected with ΔNS1 and ΔNS1/2, reflecting experimental variability. By 14 h p.i., the accumulations of both N and P proteins were similar for wt RSV and the gene deletion mutants.

Lack of activation of AP-1 by wt rRSV, ΔNS1, ΔNS2, or ΔNS1/2.

The activation of the transcription factor AP-1 in response to infection with wt rRSV and the NS1/NS2 gene deletion viruses was evaluated in Vero and A549 cells using a reporter plasmid under the control of an AP-1-responsive promoter (Fig. 7A and B, respectively). As a positive control, cells were cotransfected with a plasmid expressing the known activator MEK kinase, which activated AP-1 to a level 120 to 130 times higher than in mock-infected cells, confirming the efficiency of transfection. There was no significant increase in expression of the AP-1-dependent reporter plasmid in A549 or Vero cells infected with wt rRSV, ΔNS1, ΔNS2, or ΔNS1/2 compared to mock infection.

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FIG. 7.

AP-1 activation monitored with an AP-1-responsive reporter gene was not detected within 14 h of infection with RSV. (A) Vero cells or (B) A549 cells were transfected with a luciferase reporter plasmid. Ten hours posttransfection, the cells were mock infected or infected with the indicated viruses at an MOI of 1 PFU/cell. At 14 h postinfection, cells were harvested and assayed for luciferase activity. Relative light units are given as induction (n-fold) compared to cells that were transfected with the reporter plasmid but were not infected or mock infected. Results show the mean values and standard errors of three independent infections.

Secretion of cytokines from A549 cells infected with the NS1/NS2 gene deletion mutants.

Activation of IRF-3 and NF-κB and the production of IFN-α/β led to changes in the expression of a broad array of cellular genes, including ones involved in inflammation and innate and adaptive immunity. The deletion of NS1 and/or NS2 had the potential for unpredictable effects on the epithelial cell response: for example, since IRF-3 can up-regulate transcription of the RANTES chemokine (22), vaccine candidates in which NS1 or NS2 have been deleted might have the potentially undesirable attribute of increased RANTES expression. We therefore investigated the expression of three proinflammatory factors by A549 cells in response to infection with wt rRSV and the NS1/NS2 gene deletion mutants: the chemokine RANTES, the chemokine IL-8, and the cytokine TNF-α. We chose these factors, as they have been suggested to contribute to disease in RSV-infected infants (17, 30). Our goal was not to attempt to dissect the signaling pathways or determine their relative contributions to the expression of each cytokine, but rather to determine the gross effect of the NS1 and NS2 gene deletions on the human epithelial cell response. ELISA of medium supernatants harvested at 16 and 20 h p.i. confirmed the secretion of IL-8, RANTES, and TNF-α in response to wt rRSV (Fig. 8A, B, and C), as has been previously observed (36, 42). This experiment showed that the secretion of IL-8, RANTES, and TNF-α in response to the gene deletion mutants was reduced compared to wt rRSV, providing an indication of safety for these vaccine candidates.

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FIG. 8.

Secretion of (A) IL-8, (B) RANTES, and (C) TNF-α protein from A549 cells infected with wt rRSV, ΔNS1, ΔNS2, or ΔNS1/2; mock infected; or transfected with polypoly(I:C). Expression levels were measured with Human ELISA kits (BioSource) and calibrated using standards for each cytokine supplied with the kits. IL-8 secretion from cells infected with each of the deletion mutants was statistically lower than that from cells infected with wt RSV at both 16 h p.i. (P = 0.007 for ΔNS1 and ΔNS2 and 0.0006 for ΔNS1/2) and 20 h p.i. (P = 0.06 for ΔNS1 and 0.006 for ΔNS2 and ΔNS1/2). RANTES secretion from cells infected with each of the mutants was also statistically lower than secretion from cells infected with wt RSV at both 16 h p.i. (P = 0.007 for ΔNS1, 0.008 for ΔNS2, and 0.0003 for ΔNS1/2) and 20 h p.i. (P = 0.006 for ΔNS1, 0.007 for ΔNS2, and 0.0001 for ΔNS1/2). The secretion levels of TNF-α from cells infected with the mutants and wt RSV were statistically similar at 16 h p.i. (P = 0.50, 0.49, and 0.34 for ΔNS1, ΔNS2, ΔNS1/2, respectively, compared to wt RSV). However, at 20 h p.i., secretion was significantly lower from cells infected with the mutants (P = 0.003 for ΔNS1, 0.002 for ΔNS2, and 0.004 for ΔNS1/2).

DISCUSSION

Infection by viruses triggers the activation of host cell signaling pathways, including gene products that contribute to rapid intracellular defense against infection and influence innate and adaptive immune responses. Type 1 interferons (IFN-α/β) are part of this defense and establish an antiviral state within infected and neighboring cells. The IFN-α/β response in human cells first involves the production of IFN-β and -α1. Transcription of IFN-β and -α1 is mediated by the transcription factors IRF-3, NF-κB, and AP-1. Once secreted, IFN-β and -α1 bind to cell surface receptors in an autocrine and paracrine fashion. This induces the JAK/STAT signaling pathway, which produces the full complement of IFN-αs, additional IFN-β, and a wide array of interferon-stimulated genes, including ones that establish an intracellular antiviral state. IRF-3 and NF-κB also directly mediate the transcription of certain interferon-stimulated genes and other cellular genes with host defense functions. For example, IRF-3 can directly activate transcription of the RANTES chemokine (22), and NF-κB has been shown to mediate antiviral protection against HRSV in an epithelial cell line independently of IFN-α/β (4).

Viruses have evolved mechanisms to counteract the IFN-α/β response that may involve blocking initial induction, blocking JAK/STAT signaling, or inhibiting the antiviral state at some subsequent step (11, 13). We have shown previously (31) that the NS1 and NS2 proteins of HRSV inhibit the induction of IFN-α/β. This had also been shown for the NS1 and NS2 proteins of BRSV, the bovine counterpart of HRSV (5, 6, 28, 37). For each virus, the proteins exerted their inhibitory activities individually, as well as cooperatively, although the systems were not exactly parallel in that NS1 played the greater role for HRSV compared to NS2 for BRSV.

In the present study, we found that wt HRSV resembled the ΔNS1, ΔNS2, and ΔNS1/2 deletion mutants in the ability to activate IRF-3 during the first ~10 h of infection. Thereafter, the activation of IRF-3 was strongly suppressed in wt HRSV-infected cells, whereas a high level of activation continued in cells infected with each of the gene deletion mutants. In normal cells, IRF-3 is present constitutively and is shuttled continually between the cytoplasm and nucleus but is predominantly cytoplasmic. In response to an inducer, such as viral dsRNA, IRF-3 is phosphorylated and nuclear import is increased over nuclear export. However, without continual stimulation, the phosphorylated form is exported from the nucleus and degraded by the proteosome pathway in the cytoplasm (21). This cycle of IRF-3 activation and degradation was evident in the present study using polypoly(I:C), where a strong activation and subsequent reversal were observed (Fig. 1A and B). Our interpretation of the loss of nuclear IRF-3 in cells infected with wt rRSV is that the accumulation of NS1 and NS2 proteins reached sufficient levels by ~10 h p.i. to block further activation, whereas the nuclear export and degradation part of the cycle continued. Studies with BRSV showed that NS1 and NS2 interfere with IRF-3 activation by blocking its phosphorylation, although the roles of the individual proteins were not investigated (6). It is reasonable to anticipate that the same mechanism holds for HRSV. The present study indicates that deletion of either NS1 or NS2 provides substantial activation of IRF-3, indicating that the two proteins function much more efficiently together than either does individually.

The inhibition of IRF-3 activation is employed by many viruses to suppress the induction of IFN-α/β. The V protein of simian virus 5 (SV5) interferes with both IRF-3 and NF-κB activation, resulting in poor IFN-β induction in wt-infected cells (14, 26). However, unlike HRSV, wt SV5 does not activate IRF-3 even in the early stages of infection, prior to viral protein accumulation. HRSV also differs from SV5 in that NF-κB is activated in wt-HRSV-infected cells. This may explain why wt RSV is a better inducer of IFN-α/β than SV5 and certain other paramyxoviruses (40). The Ebola virus nonstructural protein VP35 and the NS3/4A protease of hepatitis C virus also inhibit IFN-α/β production by inhibiting IRF-3 phosphorylation (2, 10). The herpes simplex virus type 1 protein ICP0 blocks the activation of both IRF-3 and IRF-7 (23); however, the mechanism for this remains to be determined.

We also examined the activation of NF-κB in response to wt rRSV and the gene deletion mutants. However, because these viruses differed greatly in their abilities to induce IFN-α/β (31), and because IFN-α/β can augment NF-κB activation, these experiments were performed in Vero cells, which lack the structural genes for IFN-α/β. Unexpectedly, we found that activation of NF-κB was substantially lower in cells infected with ΔNS2 and ΔNS1/2, which each lack the NS2 gene. This effect could not be attributed to an obvious difference in viral gene expression. This strongly suggests that NS2 is involved in NF-κB activation in HRSV-infected cells. The fact that this effect was observed in the context of a permissive HRSV infection, rather than from an individual expressed cDNA, for example, lends authenticity to the observation. Previously, the F glycoprotein was shown to activate NF-κB in human monocytes through the TLR4 cell surface receptor and CD14 (19). In the present study, UV-inactivated virus did not stimulate NF-κB activation, suggesting that binding of the virus to the epithelial cell and fusion with the plasma membrane were not sufficient to significantly activate NF-κB. Expression of the HRSV P protein from a transfected plasmid also was reported to activate NF-κB (3). In the present study, a low level of NF-κB activation did occur in response to HRSV infection in the absence of NS2, and this presumably represents contributions from dsRNA and the F and P proteins, as well as whatever contribution might occur from oxidative stress or other potential inducers. Clearly, however, the NS2 protein appeared to play a major role.

In the present study we show that AP-1 is not significantly activated in response to RSV infection in either Vero or A549 cells. The limited responsiveness of AP-1 to viral infection has also been demonstrated for SV5 (26) and BRSV (6). The activation of AP-1 in HRSV-infected A549 cells has been shown to be sensitive to the redox state (24), and it might be that variability in the reported results represents differences in the conditions for cell culture. In any event, the constitutive level of AP-1 activation in our experiments was ample for a very robust IFN-α/β response (31).

There is presently no available licensed HRSV vaccine. However, live attenuated viruses containing deletion of the NS1 and/or NS2 genes, among other attenuating mutations, are under development and appear to be promising candidates (34, 38). One of the advantages of the NS1/NS2 deletion mutants as vaccine candidates is the promotion of IFN-α/β expression, which can enhance both innate and adaptive immune responses (31). However, a number of proinflammatory cytokines and chemokines contain NF-κB or IRF-3 binding sites in their promoters (17, 22) and can also be upregulated indirectly by IFN-α/β.

The recent use of cDNA and oligonucleotide microarrays has demonstrated that airway epithelial cells (A549 and primary small airway epithelial cells) infected with RSV show increased expression of a number of chemokines, including I-309, Exodus-1, TARC, RANTES, MCP-1, MDC, MIP-1α/β, IL-8, GRO α/β/γ, ENA-78, I-TAC, and fractalkine (25, 36, 42). It has also been demonstrated that human infants with RSV infection and bronchiolitis have increased secretion of IL-8, MCP-1, TNF-α, eotaxin, MIP-1α, and RANTES (17, 30). Vaccine candidate viruses that stimulate sustained IRF-3 activation, elevated NF-κB levels, and/or elevated IFN-α/β production have the potential to stimulate increased production of proinflammatory chemokines and cytokines that have been hypothesized to contribute to bronchiolitis and airway hyperreactivity. However, in the present study, the NS1/NS2 deletion mutants were found to be associated with reduced, rather than augmented, production of IL-8, RANTES, and TNF-α from A549 cells. This provides additional support for the development of viruses lacking NS1 and/or NS2 as safe live attenuated vaccines.

Acknowledgments

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