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Previous Article | Next Article 
Journal of Virology, July 2007, p. 7189-7199, Vol. 81, No. 13
0022-538X/07/$08.00+0 doi:10.1128/JVI.00013-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Department of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104,1 Department of Microbiology,2 Emerging Pathogens Institute, Mount Sinai School of Medicine, New York, New York 100293
Received 3 January 2007/ Accepted 12 April 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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/ß) response. While MHV strain A59 appeared to induce IFN-ß gene transcription and low levels of nuclear translocation of the IFN-ß transcription factor interferon regulatory factor 3 (IRF-3), MHV did not induce IFN-ß protein production during the course of infection in L2 mouse fibroblast cells. In addition, MHV was able to significantly decrease the level of IFN-ß protein induced by both Newcastle disease virus (NDV) and Sendai virus infections, without targeting it for proteasomal degradation and without altering the nuclear translocation of IRF-3 or IFN-ß mRNA production or stability. These results indicate that MHV infection causes an inhibition of IFN-ß production at a posttranscriptional level, without altering RNA or protein stability. In contrast, MHV induced IFN-ß mRNA and protein production in the brains of infected animals, suggesting that the inhibitory mechanisms observed in vitro are not enough to prevent IFN-/ß production in vivo. Furthermore, MHV replication is highly resistant to IFN-/ß action, as indicated by unimpaired MHV replication in L2 cells pretreated with IFN-ß. However, when L2 cells were coinfected with MHV and NDV in the presence of IFN-ß, NDV, but not MHV, replication was inhibited. Thus, rather than disarming the antiviral activity induced by IFN-ß pretreatment completely, MHV may be inherently resistant to some aspects of the antiviral state induced by IFN-ß. These findings show that MHV employs unique strategies to circumvent the IFN-/ß response at multiple steps. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mouse hepatitis virus (MHV), a group II coronavirus, has long been used as a tool for studying coronavirus biology and pathogenesis. MHV causes hepatic and central nervous system diseases of varying severity depending on the strain and is therefore used as a model for hepatitis, viral encephalitis, and demyelination (76). While there is much known about the general immune response to MHV (76), there is limited information about the alpha/beta interferon (IFN-/ß) response induced by MHV infection (21, 63, 75, 82).
Often, upon viral infection, the innate immune response is activated, leading to induction of the IFN-/ß response. The IFN-/ß response, involving the expression of one IFN-ß gene and several IFN- genes, represents one of the first lines of defense against viral infection (20). Cellular receptors such as Toll-like receptor 3 in endosomes (37), and retinoic acid-inducible gene I (RIG-I) (14, 30, 79) and Mda5 (melanoma differentiation-associated protein 5) (30, 33) in the cytoplasm recognize viral patterns, resulting in the activation of the transcription factor interferon regulatory factor 3 (IRF-3). Normally found in the cytoplasm of resting cells, activated IRF-3 dimerizes and is translocated to the nucleus, where it drives the transcription of the IFN-ß gene with the help of the transcription factors NF-
B, AP-1, and the coactivator CBP/p300 (20, 28, 53). Once produced, IFN-ß is secreted from the infected cell, where it can induce an antiviral state in neighboring cells to limit viral spread (62). When bound to its receptor, IFN-ß initiates a signaling cascade that results in the transcription of IFN-stimulated genes (20), which have antiviral, antiproliferative, and immunomodulatory properties (13). In addition, IFN-ß primes cells to produce IFN-s after viral infection, thereby amplifying the IFN-/ß response (12, 17, 36). Many viruses have developed a variety of mechanisms to subvert the IFN-/ß response by inhibiting IFN-/ß production, signaling, or the action of one or more of the IFN-stimulated genes (20), making it difficult for the host to fight viral infection.
In this study, we used MHV strains A59, JHM, and MHV-2 to investigate the interaction between MHV and the IFN-/ß response during infection in vitro and in vivo.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Viruses. Recombinant A59 (RA59) (46), recombinant JHM (RJHM) (41), wild-type JHM (MHV-4 or JHMSD) (31), MHV-1 (11), MHV-2 (51), MHV-3 (74), and A59 expressing EGFP (RA59-EGFP) (52) have been described previously and were grown in 17Cl-1 cells. Sendai virus (SeV) strain Cantell (4) and Newcastle disease virus (NDV) expressing GFP (rNDV-GFP) (42) and monomeric red fluorescent protein (rNDV-mRFP) (39) were grown in 10-day-old embryonated eggs. RA59, Sendai virus, rNDV-GFP, and rNDV-mRFP will are to as MHV-A59, SeV, NDV-GFP, and NDV-RFP, respectively, in the remainder of the text. In addition, both RJHM and wild-type JHM are referred to as MHV-JHM, and distinctions between the two are made in the figure legends.
Quantitative real-time PCR.
RNA was isolated from L2 cells infected with different strains of MHV, NDV-RFP, or SeV or coinfected with NDV-GFP and MHV-A59 or SeV and MHV-A59 using an RNeasy mini kit (QIAGEN, Valencia, CA) at the indicated times postinfection. RNA was then DNase treated using the Turbo DNA-free kit (Ambion, Austin, TX) according to the manufacturer's instructions. Real-time PCRs without reverse transcriptase were performed to ensure adequate removal of genomic DNA. cDNA was generated as follows: approximately 350 ng of DNase-treated RNA was heated with 5 nM random hexamers (Invitrogen, Carlsbad, CA) for 3 minutes at 85°C in a PCR thermocycler (PTC-200; MJ Research, Hercules, CA). A 10 mM concentration of deoxynucleoside triphosphate mix (final concentration of 0.25 mM per each reaction mixture; Invitrogen, Carlsbad, CA), first-strand enzyme buffer (Invitrogen, Carlsbad, CA), SuperScript II reverse transcriptase (50 U; Invitrogen, Carlsbad, CA), and RNase inhibitor (30 U; Amersham Biosciences, Piscataway, NJ) were added to each reaction mixture for a total volume of 20 µl per reaction mixture. Reverse transcription was performed in a PCR thermocycler (PTC-200; MJ Research, Hercules, CA) for 50 min at 42°C followed by 5 min at 95°C. Real-time PCR analysis was carried out on 2 µl cDNA using the iQ SYBR Green PCR mix (Bio-Rad, Hercules, CA) and 8 nM of each primer pair listed in Table 1. Real-time PCR was performed on an iQ5 iCycler (Bio-Rad, Hercules, CA). Cycle threshold (CT) values were normalized to 18S rRNA levels, resulting in a 
CT value [CT = CT(IFN-ß/SeV/NDV) – CT(18S)].
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Cytokine ELISAs. L2 cells were infected with MHV-A59, MHV-JHM, MHV-1, MHV-2, NDV-RFP, NDV-GFP, or SeV alone or simultaneously infected with MHV-A59 and either NDV-GFP or SeV at an MOI of 1 PFU/cell for each virus. L2 cells were infected with MHV-3 at an MOI of 0.4 PFU/cell. At 24 h postinfection (p.i.), supernatants were collected, and secreted IFN-ß and interleukin-12 (IL-12) protein levels were quantified using a mouse IFN-ß enzyme-linked immunosorbent assay (ELISA) kit (PBL Biomedical Laboratories, Piscataway, NJ) or mouse interleukin-12 p70 (IL-12p70) Quantikine ELISA kit (R&D Systems, Minneapolis, MN), respectively, per the manufacturer's instructions. Intracellular protein was obtained by lysing cells in lysis buffer (0.15 M NaCl, 5 mM EDTA pH 8, 10 mM Tris-HCl pH 7.6, 1% Triton X-100) at 18 h p.i. Equivalent amounts of total intracellular protein were applied to a mouse IFN-ß ELISA kit (PBL Biomedical Laboratories, Piscataway, NJ), and IFN-ß levels were measured per the manufacturer's instructions.
Northern blot assay. L2 cells were infected with MHV-A59, SeV, or MHV-A59 and SeV at an MOI of 1 PFU/cell, and at 12 h p.i. total RNA was isolated using an RNeasy mini kit (QIAGEN, Valencia, CA). For each sample, 5 µg total RNA was analyzed by Northern blotting (NorthernMax-Gly; Ambion, Austin, TX). The Northern blot was probed with an IFN-ß probe generated using the Strip-EZ RNA system (Ambion, Austin, TX) with PCR primers specific for IFN-ß (Table 1). The blot was exposed to a phosphorimager screen and analyzed with a Storm 850 PhosphorImager and ImageQuant 1.2 software (Molecular Dynamics).
Proteasomal degradation assay.
For IFN-ß protein analysis, L2 cells were infected with MHV-A59 or SeV or coinfected with an MOI of 1 PFU/cell of each virus. At 1 h p.i., cells were treated with 5 µM lactacystin. At 17 h posttreatment, supernatants and cell lysates were collected as described above. Intracellular and released IFN-ß protein concentrations were measured using a mouse IFN-ß ELISA kit (PBL Biomedical Laboratories, Piscataway, NJ). To verify the inhibitory activity of lactacystin on proteasomal degradation of IB, 293T cells were treated with 5 µM lactacystin, and 6 h posttreatment, cells were treated with 10 ng tumor necrosis factor alpha (TNF-). At 30 min post-TNF treatment, cell lysates were collected, as described above, and 67.5 µg total protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. The Western blot was probed with a polyclonal IB antibody (kindly provided by Catherine Wharry, University of Pennsylvania, Philadelphia).
Infections of mice. Virus-free 3- to 4-week-old male C57BL/6 mice were purchased from the National Cancer Institute (Frederick, MD). Mice were anesthetized with isoflurane (IsoFlo; Abott Laboratories) for intracranial (i.c.) inoculations. Virus was diluted in phosphate-buffered saline (PBS) containing 1% bovine serum albumin, and mice were inoculated with 50 PFU of MHV-A59 or MHV-JHM in a total volume of 20 µl in the left cerebral hemisphere. At days 1, 3, 5, and 7 postinfection, animals were sacrificed and brains were obtained. Half of each brain was placed in RNALater (Ambion, Austin, TX) and stored at –20°C for RNA isolation, and the remainder of the brain was flash-frozen on dry ice and stored at –80°C for protein isolation.
In vivo levels of IFN-ß transcription.
Mouse brain tissues were transferred to TRIzol reagent (Invitrogen, Carlsbad, CA) and homogenized using a handheld homogenizer (OMNI TH; OMNI International, Marietta, GA). RNA was isolated as follows: a one-fifth volume of phenol-chloroform-isoamyl alcohol was added to the homogenized tissues to induce phase separation. RNA was precipitated using 1 volume of 70% ethanol and purified using an RNeasy mini kit (QIAGEN, Valencia, CA). RNA was DNase treated using a Turbo DNA-free kit (Ambion, Austin, TX) according to the manufacturer's instructions. Real-time PCRs without reverse transcriptase were performed to ensure adequate removal of genomic DNA. cDNA was generated as described above, using 1,000 ng DNase-treated RNA in a total volume of 20 µl per reaction mixture. IFN-ß mRNA levels were measured on a custom-designed low-density array (LDA) card (Applied Biosystems [ABI], Foster City, CA), a high-throughput version of real-time PCR, according to the manufacturer's instructions. LDA cards were read on a 7900HT sequence detection system (ABI Prism, Foster City, CA) and analyzed using the SDS2.1 software (ABI Prism, Foster City, CA) in collaboration with Centocor Inc. (Radnor, PA). Cycle threshold (CT) values were normalized to 18S rRNA levels, resulting in a CT value [CT = CT(IFN-ß) – CT(18S)].
In vivo levels of IFN-ß protein. Flash-frozen mouse brain tissues were homogenized in cold PBS supplemented with Complete EDTA-free protease inhibitors (Roche Applied Science, Indianapolis, IN) using a handheld homogenizer (OMNI TH; OMNI International, Marietta, GA). Protein samples were diluted to 800 µg/ml total protein, and IFN-ß protein levels were measured using a mouse IFN-ß ELISA kit (PBL Biomedical Laboratories, Piscataway, NJ).
IFN-ß pretreatment. L2 cells seeded on 24-well plates were treated with 0, 100, or 1,000 U/ml recombinant mouse IFN-ß (Calbiochem, La Jolla, CA). At 24 h posttreatment, cells were infected with MHV-A59, MHV-2, MHV-JHM, or NDV-RFP at an MOI of 1 PFU/cell for 1 h. At 10 h p.i., MHV-infected cells and supernatants were collected and stored at –80°C. Prior to determining titers, cells were lysed by freeze-thawing and cellular and released virus titers were determined on L2 cell monolayers by a standard plaque assay as described previously (22). NDV-RFP-infected cells were examined at 24 h p.i. under a Nikon Eclipse 2000E-U fluorescence microscope (Melville, NY). For RA59-GFP and NDV-RFP coinfection experiments, L2 cells were seeded on a 24-well plate and treated with 0 or 1,000 U/ml recombinant mouse IFN-ß. At 24 h posttreatment, cells were simultaneously infected with RA59-GFP and NDV-RFP at an MOI of 1 PFU/cell for each virus, and at 24 h p.i., cultures were examined by fluorescence microscopy.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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/ß response during the course of MHV infection (21, 63, 71, 75, 82). To examine the levels of IFN-ß gene transcription during the course of MHV infection, murine L2 cells were infected with MHV-A59, MHV-JHM, or MHV-2 and RNA was extracted at 6, 8, 12, and 24 h p.i. Quantitative real-time PCR was performed using primers specific to the mouse IFN-ß gene (Fig. 1A). MHV-A59 induces IFN-ß gene transcription starting at around 12 h p.i., continuing through the first 24 h of infection, while MHV strains MHV-JHM and MHV-2 induce undetectable amounts of IFN-ß mRNA. We next compared the kinetics of IFN-ß gene transcription induced by MHV with that of SeV, a virus that induces a potent IFN-/ß response. While SeV induces IFN-ß gene transcription starting at 3 h p.i., MHV-A59 does not induce IFN-ß mRNA production until 12 h p.i. (Fig. 1B). These data indicate that IFN-ß transcription is delayed during MHV infection.
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MHV does not induce IFN-ß protein production. To determine if the delayed transcription of IFN-ß induced by MHV resulted in IFN-ß protein production, we measured IFN-ß protein at 24 h p.i. in supernatants from MHV-infected cells using an ELISA. The ELISA was carried out on supernatants from MHV-A59-, MHV-JHM-, and MHV-2-infected cells, as well as from cells infected with MHV-1, a strain of MHV that results in a respiratory disease similar to SARS (11), and MHV-3, a strain of MHV that induces fulminant hepatitis (74). These ELISA data indicate that none of the strains of MHV examined induced measurable amounts of IFN-ß protein (limit of detection is 15.6 pg/ml, or approximately 0.6 U/ml, as defined by the manufacturer) compared to NDV-RFP and SeV, which induced high levels of IFN-ß protein (Fig. 2A).
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MHV inhibits NDV- and SeV-induced IFN-ß protein production. Since MHV-A59 appears to induce IFN-ß gene transcription but not IFN-ß protein production, we investigated whether MHV is able to inhibit IFN-ß production induced by other viruses. Thus, L2 cells were infected with NDV-GFP and SeV in the absence or presence of MHV-A59. At 24 h p.i., released IFN-ß protein was measured by an ELISA. In the absence of MHV-A59, both NDV-GFP and SeV infection induced IFN-ß production, with SeV infection inducing particularly high levels of IFN-ß protein (Fig. 3A). However, IFN-ß protein levels produced during NDV and SeV infection were decreased by statistically significant amounts (approximately 80% decrease) in the presence of MHV-A59 (Fig. 3A). Again, to rule out the possibility that MHV inhibited the secretion of IFN-ß protein induced by SeV, intracellular levels of IFN-ß protein at 18 h p.i. were analyzed by ELISA. Although the results did not reach statistical significance (P < 0.11), coinfection with MHV-A59 was able to reduce the amount of IFN-ß protein induced by SeV by 46% (Fig. 3A).
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MHV does not inhibit NDV- or SeV-induced IRF-3 nuclear translocation or IFN-ß gene transcription. Many viruses have developed mechanisms to inhibit IFN-ß production and, frequently, viruses that inhibit IFN-ß production do so by inhibiting the activation of IRF-3 and its subsequent movement to the nucleus (20). Since coinfection with MHV was able to reduce the levels of IFN-ß protein induced by NDV-GFP and SeV, we investigated whether MHV could inhibit the level of IFN-ß mRNA transcription induced by these viruses. In order to examine if MHV is able to inhibit IFN-ß gene transcription induced by either NDV-GFP or SeV, RNA was isolated at 1, 3, 6, 8, 12, and 24 h p.i. from cells infected with NDV-GFP (data not shown) or SeV in the absence or presence of MHV-A59, and we measured IFN-ß mRNA by real-time PCR (Fig. 4A). MHV-A59 infection does not alter IFN-ß mRNA levels in cells coinfected with either NDV-GFP (data not shown) or SeV at any of the time points examined. IRF-3 localization was also examined in NDV-RFP- and SeV-infected cells in the presence of MHV-A59. To this end, Vero cells were transfected with pEGFP-hIRF3 (IRF-3-GFP) and pCI-neo 1a[1-4] (MHVR). Transfected cells were coinfected with MHV-A59 and NDV-RFP (Fig. 4B) or MHV-A59 and SeV (Fig. 4C). Using a polyclonal antibody against MHV (Fig. 4B) or a polyclonal antibody against MHV and a monoclonal antibody against SeV (Fig. 4C), we examined the subcellular localization of IRF-3-GFP in coinfected cells. Both NDV-RFP (data not shown) and SeV (Fig. 1D) induced IRF-3 nuclear translocation in the absence of MHV infection. Coinfection with MHV-A59 was unable to prevent the movement of IRF-3-GFP to the nucleus in NDV-RFP-infected (Fig. 4B) or SeV-infected (Fig. 4C) cells.
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MHV-A59 does not inhibit protein synthesis and does not induce proteasomal degradation of the IFN-ß protein. Since MHV appears to significantly alter IFN-ß protein levels induced by NDV and SeV but has no effect on IFN-ß mRNA levels or stability, we wanted to ensure that the decrease in IFN-ß protein levels was not indicative of general cytokine production or secretion inhibition. MHV has been known to decrease host protein synthesis during infection under some conditions (23, 56). To this end, supernatants were collected from cells infected with NDV-GFP or SeV in the absence or presence of MHV-A59, and we measured levels of another cytokine, IL-12, by ELISA. IL-12 is induced during both SeV (47) and NDV (19) infection. The presence of MHV-A59 in NDV-GFP- and SeV-infected cells does not result in decreased levels of IL-12 (Fig. 5A), indicating that MHV-A59 is not inhibiting general host protein synthesis. The fact that IL-12 levels are increased in the presence of MHV-A59 is possibly a result of decreased IFN-ß protein in these cells, since IL-12 is negatively regulated by IFN-ß (6) and MHV-A59 does not induce detectable levels of IL-12 in L2 cells (data not shown). In addition, MHV infection of transfected 293T cells expressing the firefly luciferase gene under the control of the chicken ß-actin promoter did not affect luciferase expression (data not shown). These data indicate that MHV does not inhibit general protein synthesis under these conditions.
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B degradation was followed after a 30-min treatment with TNF in the presence and absence of 5 µM lactacystin. In the presence of TNF, IB is rapidly degraded, while the addition of lactacystin leads to an accumulation of IB. These data indicate that MHV-A59 does not inhibit IFN-ß protein production by NDV and SeV by downregulating general protein synthesis or by targeting IFN-ß protein for proteasomal degradation.
MHV induces IFN-ß gene transcription and protein production in vivo.
Since MHV does not appear to induce detectable IFN-/ß in vitro, we investigated whether MHV is able to induce IFN-ß gene transcription in vivo. To do this, C57BL/6 mice were infected with MHV-A59 or MHV-JHM i.c., and IFN-ß mRNA levels were quantified in the brains of infected mice at days 1, 3, 5, and 7 p.i. using an LDA (Fig. 6A). At day 5 p.i., both MHV-A59 and MHV-JHM induced IFN-ß gene transcription in the brains of infected animals to levels above mock-infected animals. At day 7 p.i., while IFN-ß mRNA levels started to wane in MHV-A59-infected animals, IFN-ß mRNA in MHV-JHM-infected animals continued to increase to levels approximately 140-fold higher than A59-infected animals. At this time postinfection, the T-cell response is peaking in MHV-A59-infected animals, while MHV-JHM-infected animals become moribund (25). These data indicate that MHV-A59 and MHV-JHM are able to induce IFN-ß gene transcription in the brains of infected animals and that MHV-JHM induces higher levels of IFN-ß mRNA. This is consistent with previous studies (48, 49) that demonstrated that both MHV-A59 and MHV-JHM are capable of inducing IFN-ß transcription in brains of infected mice and that MHV-JHM induces prolonged IFN-ß transcription compared to MHV-A59. In addition, both MHV-A59 and MHV-JHM induce IFN-ß protein production at 5 days p.i. (Fig. 6B). This indicates that while MHV appears to inhibit IFN-ß protein production in vitro, and this might contribute to some delay in the IFN response in vivo, it is unable to completely block IFN-ß protein production in vivo.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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/ß response. Transmissible gastroenteritis virus, a group I coronavirus, is known to induce high levels of IFN-, both in vivo and in vitro (5, 50). HCoV-229E, another group I virus, is able to induce IFN-ß transcription in human monocyte-derived macrophages (9), and bovine coronavirus, a group II virus, induces IFN-/ß production in vivo (67). SARS-CoV appears to modulate the IFN-/ß response by multiple mechanisms. SARS-CoV induces IRF-3 nuclear translocation by 8 h p.i., although IRF-3 is found in the cytoplasm by 16 h p.i. without inducing IFN-ß transcription (59). In addition, the Nsp1 protein of SARS promotes host mRNA degradation, thereby inhibiting IFN-ß mRNA accumulation (29). A recent study showed that the proteins encoded by orf3a and orf6 of the SARS-CoV genome inhibit IFN-/ß synthesis and signaling, while the nucleocapsid protein is able to inhibit IFN-/ß synthesis only (32).
We have further investigated the ability of MHV to induce, as well as to respond to, IFN-ß. To do this, we used MHV-A59, MHV-JHM, and MHV-2 because they differ in tissue tropism and virulence in vivo; JHM induces viral encephalitis that is generally lethal, A59 infects both the central nervous system and the liver and often induces a demyelinating disease, and MHV-2 predominantly infects the liver (76). This is the first study to show that these strains of MHV are capable of inducing IRF-3 nuclear translocation in a minority (approximately 15%) of infected Vero cells at late times (24 h) postinfection and that MHV-A59 is able to induce IFN-ß gene transcription late in infection of L2 cells in vitro. While it is not clear why neither MHV-2 nor MHV-JHM is able to induce IFN-ß mRNA production, the slower kinetics of replication as well as the lower final titers achieved by these two strains compared to MHV-A59 may contribute to the inability to induce IFN-ß mRNA. Recent studies report that IRF-3 remains localized to the cytoplasm at 8 h p.i. of MHV-infected 17Cl-1 (81) and L2 cells (71), consistent with our observation of nuclear translocation only later after infection. The delayed activation of IFN-ß transcription seen with MHV is similar to results observed with SARS-CoV. SARS-CoV infection of dendritic cells results in the induction of IFN- transcription starting around 24 h p.i. (60), and infection of nonlymphatic cells results in very low levels of IFN-ß transcription starting at 16 h p.i. (61).
Although MHV-A59 is able to induce some IFN-ß gene transcription, IFN-ß protein levels remain undetectable through the first 24 h of infection as measured by ELISA. These data are consistent with previous studies that showed that MHV does not induce IFN-ß protein production in myeloid dendritic cells (82) or several other cell lines (BHK-21, NCTC 1469, Wira, and 17Cl-1 cells) (21, 81). Consistent with our data of IFN-ß transcription starting at approximately 12 h p.i., a recent transcriptional profiling study (72) showed that MHV does not induce IFN-ß transcription in mouse fibroblast cells early in infection (3 to 6 h p.i.). In fact, our data indicate that MHV appears to induce delayed IFN-ß gene transcription, compared to SeV, a strong inducer of IFN-ß. In another recent study, IFN-ß transcription remained undetectable as late as 24 h p.i. in 17Cl-1 or 293T cells (81). The differences between that study and ours may be the result of the use of different cell types. MHV replicates with slower kinetics in 17Cl-1 cells compared to L2 cells (unpublished data) and may not induce a potent IFN-/ß response in 17Cl-1 cells, and the kinetics of replication have not been described in 293T cells, a cell type not readily infected with MHV.
Since MHV does not induce IFN-ß protein production despite the induction of moderate levels of IFN-ß mRNA, we carried out coinfections of MHV with SeV and NDV, both of which induce high levels of IFN-ß (Fig. 2A), to investigate whether MHV actively inhibits IFN-ß protein production. This is the first study to show that coinfection with MHV significantly reduces the IFN-ß protein production induced by these viruses as measured by ELISA. Since NDV replication was uninhibited and SeV replication was only slightly decreased in the presence of MHV, we conclude that the reduction in IFN-ß protein is specific to MHV replication and not decreased replication of SeV or NDV.
Many viruses have developed mechanisms to inhibit the IFN-/ß response and, frequently, viruses prevent the activation of the IFN-ß transcription factor IRF-3. For example, the NS1 protein of influenza A virus (65), the E3L protein of vaccinia virus (58), the NS3-4a protease of hepatitis C virus (27), and the phosphoprotein (P) of rabies virus (7) inhibit IRF-3 activation by several mechanisms, including sequestration of double-stranded RNA, binding to the cytoplasmic RNA sensor IRG-I, proteolytic activation of the adaptor molecule IFN-ß promoter stimulator 1, or inhibition of IRF-3-activating kinases. To determine if the inhibition of IFN-ß protein induced by SeV or NDV-GFP was due to MHV-induced inhibition of IRF-3 translocation, we performed coinfections and determined the subcellular localization of IRF-3. We found that while IRF-3 undergoes nuclear translocation in SeV-infected (Fig. 3B) and NDV-infected (data not shown) cells, coinfection with MHV is unable to abrogate IRF-3 activation. The SeV data are consistent with recent data from Versteeg et al. (71), who showed that MHV-A59 is unable to abrogate SeV-induced IRF-3 nuclear translocation at 8 h p.i. in L2 cells.
Although MHV is unable to inhibit SeV- or NDV-induced IRF-3 nuclear translocation, there are other possible mechanisms by which MHV could inhibit IFN-ß gene transcription induced by these viruses. For example, bovine viral diarrhea virus (BVDV) induces IRF-3 nuclear translocation, but once IRF-3 is in the nucleus, BVDV is able to block it from binding DNA in the nucleus and can also target cytoplasmic IRF-3 for proteasomal degradation, thereby inhibiting IFN-ß gene transcription (2, 24). In addition, human rhinovirus 14 induces nuclear translocation of IRF-3 but is then able to inhibit IRF-3 homodimerization and subsequent IFN-ß gene transcription (45). However, quantitative real-time PCR analysis of IFN-ß gene transcription induced by SeV and NDV in the absence and presence of MHV indicates that MHV does not affect IFN-ß mRNA production by these viruses, consistent with recent data using SeV reported by Versteeg et al. (71). We also show that the IFN-ß mRNA transcript made upon SeV infection is intact and is not degraded during MHV-A59 coinfection.
Taken together, these data suggest that there is an inhibition of IFN-ß protein production from the mRNA transcribed at late times post-MHV infection. Similarly, coinfection with MHV is able to reduce IFN-ß protein induced by infections with heterologous viruses, such as SeV and NDV. This is the first report of a coronavirus causing inhibition of the IFN-/ß response at the protein level rather than at the transcriptional level; however, there are other examples of this within other virus groups. The 3A protein of many picornaviruses is able to inhibit endoplasmic reticulum-to-Golgi trafficking (16) and significantly inhibit cellular protein secretion in infected cells (10). Using a poliovirus with a mutation in the 3A gene, Dodd et al. (15) showed that the 3A protein is involved in reducing the secretion of several proinflammatory and antiviral cytokines, including IFN-ß. There is no evidence to date that MHV alters host endoplasmic reticulum-to-Golgi trafficking during infection, nor does it inhibit the secretion of IL-12 (Fig. 5). Furthermore, levels of intracellular IFN-ß protein as well as secreted protein remain undetectable during MHV infection (Fig. 2). Therefore, MHV does not appear to specifically inhibit the secretion of IFN-ß protein.
Although MHV does not mount a significant IFN-/ß response in mouse fibroblast cells in vitro, previous studies have shown that MHV infection results in IFN-ß production in several biologically relevant cell types. Both MHV-A59 and MHV-2 have been shown to induce IFN-ß transcription in primary astrocytes (75), and MHV-A59 induces IFN- in plasmacytoid dendritic cells (8), the major IFN-/ß-producing cells in the animals (3, 35). There is evidence that MHV induces IFN-/ß production in vivo as well, since MHV3 can induce IFN-ß protein production in the sera and peritoneal exudate cells of infected animals (54). As shown in Fig. 6, both MHV-A59 and MHV-JHM induce IFN-ß in vivo at both the mRNA and protein level, suggesting that while MHV is unable to induce a substantial IFN-/ß response in a fibroblast cell line, it may be able to induce IFN production in certain cell types found in the brain. Preliminary experiments with primary neuronal cell cultures have indicated that these cells do not produce measurable amounts of IFN-ß protein (data not shown). Future studies will be directed at examining which cells produce IFN-ß in vivo during MHV infection.
MHV is resistant to the antiviral effects of IFN-ß pretreatment during in vitro infection of L2 cells. Even in the presence of 1,000 U/ml of IFN-ß, a dose that completely abrogates NDV replication, MHV replication is reduced by less than 10-fold. However, MHV is not capable of inhibiting the general antiviral activities induced by IFN-ß, as coinfection with MHV was unable to rescue NDV replication in the presence of IFN-ß. Similarly, while BVDV is resistant to posttreatment with IFN-, it is unable to rescue vesicular stomatitis virus or encephalomyocarditis virus replication in the presence of IFN- (55). Therefore, it is possible MHV is capable of protecting itself from the antiviral effects of IFN-ß by evading some specific downstream action induced by IFN-/ß without rescuing NDV replication. In fact, a recent paper showed that the MHV nucleocapsid protein inhibits double-stranded RNA-dependent protein kinase and 2',5'-OAS-RNase L activity, downstream effects of the IFN-/ß response (78). We are currently undertaking studies that will examine IFN-/ß signaling during MHV infection.
Despite the fact that MHV is not sensitive to inhibition by IFN-ß in vitro, the IFN-/ß response induced during MHV infection in vivo appears to play an important role in pathogenesis, since treatment of mice with IFN-/ß prior to infection is able to reduce MHV-induced disease (1, 38, 40, 57, 68, 73) and treatment with anti-IFN antibodies can exacerbate illness (34). In addition, inoculation of IFN receptor –/– mice with a low dose of MHV-A59 results in rapid death, around 2 days p.i. (8). The basis for the difference in sensitivity of MHV infection to IFN-ß in vivo, and not in vitro, is not clear. While it is possible that IFN-/ß plays a direct role in inhibiting viral replication in vivo, the IFN-/ß response also has many immunomodulatory effects, such as inducing NK and T-cell activation and survival (66), which may play a critical role in protection from MHV infection in vivo but would not play a role in inhibiting viral replication in vitro.
Our data demonstrate that MHV employs an unusual combination of mechanisms to modulate the IFN-/ß response, in that MHV is both able to delay IFN-ß transcription and inhibit IFN-ß protein production in vitro. While the mechanism behind the delay in IFN-ß gene transcription during MHV infection is unknown, the data presented here narrow down the possible mechanisms by which MHV inhibits IFN-ß protein production during coinfections with NDV or SeV. Since there is not an accumulation of intracellular IFN-ß protein during MHV infection in vitro, it is unlikely that MHV affects the secretion of the protein. Studies utilizing the proteasomal inhibitor lactacystin showed that the IFN-ß protein is not being specifically targeted for proteasomal degradation in the presence of MHV-A59. In addition, we examined the stability of the IFN-ß mRNA and found that MHV-A59 does not induce degradation of the transcript in coinfected cells. These data indicate that the block of IFN-ß protein production occurs at a step between mRNA synthesis and protein translation. It is possible that the IFN-ß mRNA is not being loaded onto polysomes and therefore not being translated or, alternatively, the mRNA may be loaded onto polysomes but translation of the mRNA may be blocked by micro-RNAs (26). Future experiments will be aimed at elucidating this mechanism. It will also be interesting to investigate whether the effects observed with MHV will be applicable to other coronaviruses, such as human group I and group II coronaviruses and the SARS coronavirus.
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ACKNOWLEDGMENTS |
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and IB antibodies. We also thank Fred DelVecchio and Patrick Branigan at Centocor, Inc., for their help and support with the LDA cards and Richard Cadagan for technical assistance. This work was partly supported by NIH grants NS-54695 and AI-60021 to S.R.W. and CIVIA, an NIH-funded Center for Investigating Viral Immunity and Antagonism (U19AI62623), and NIH grant AI-52106 to A.G.-S. J.K.R.-C. and E.P.S. were partially supported by NIH training grant NS-07180.
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FOOTNOTES |
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Published ahead of print on 25 April 2007.
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and interferon ß gene transcription by interferon regulatory factor 3. Proc. Natl. Acad. Sci. USA 95:9837-9842.
