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Journal of Virology, January 2008, p. 254-267, Vol. 82, No. 1
0022-538X/08/$08.00+0     doi:10.1128/JVI.01384-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Expression of the E3L Gene of Vaccinia Virus in Transgenic Mice Decreases Host Resistance to Vaccinia Virus and Leishmania major Infections{triangledown}

Elena Domingo-Gil, Eva Pérez-Jiménez,{dagger} Iván Ventoso,{ddagger} José L. Nájera, and Mariano Esteban*

Department of Cellular and Molecular Biology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Ciudad Universitaria Cantoblanco, 28049 Madrid, Spain

Received 26 June 2007/ Accepted 17 October 2007


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ABSTRACT
 
The E3L gene of vaccinia virus (VACV) encodes the E3 protein that in cultured cells inhibits the activation of interferon (IFN)-induced proteins, double-stranded RNA-dependent protein kinase (PKR), 2'-5'-oligoadenylate synthetase/RNase L (2-5A system) and adenosine deaminase (ADAR-1), thus helping the virus to evade host responses. Here, we have characterized the in vivo E3 functions in a murine inducible cell culture system (E3L-TetOFF) and in transgenic mice (TgE3L). Inducible E3 expression in cultured cells conferred on cells resistance to the antiviral action of IFN against different viruses, while expression of the E3L gene in TgE3L mice triggered enhanced sensitivity of the animals to pathogens. Virus infection monitored in TgE3L mice by different inoculation routes (intraperitoneal and tail scarification) showed that transgenic mice became more susceptible to VACV infection than control mice. TgE3L mice were also more susceptible to Leishmania major infection, leading to an increase in parasitemia compared to control mice. The enhanced sensitivity of TgE3L mice to VACV and L. major infections occurred together with alterations in the host immune system, as revealed by decreased T-cell responses to viral antigens in the spleen and lymph nodes and by differences in the levels of specific innate cell populations. These results demonstrate that expression of the E3L gene in transgenic mice partly reverses the resistance of the host to viral and parasitic infections and that these effects are associated with immune alterations.


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INTRODUCTION
 
Vaccinia virus (VACV) is a large double-stranded DNA (dsDNA) virus that replicates in the cytoplasm of the cells and encodes a variety of immunomodulatory molecules that antagonize innate and adaptive immune responses of the host, thus providing antiviral escape mechanisms (1, 29). VACV E3 is one of the viral proteins that antagonize the interferon (IFN) system. The first demonstration of the effect of E3 on IFN pathways came from an analysis of a VACV mutant lacking the E3L gene that showed enhanced sensitivity to IFN (2). E3L has been demonstrated to be a host range gene necessary for efficient VACV replication in several cell lines (3) and is required for VACV pathogenesis (8). The E3L gene encodes two proteins of 25 and 20 kDa, which are expressed early during infection. The E3 protein is present in both the nucleus and cytoplasm of infected and transfected cells (11, 51). E3 has two domains, a N-terminal Z-DNA-binding domain (Z{alpha}) and a C-terminal dsRNA-binding domain. Both domains are required for infection and viral pathogenesis in the mouse model (7, 8). The N-terminal domain is highly conserved among poxviruses and is involved in the direct inhibition of the IFN-induced protein kinase PKR, in the nuclear localization of E3, and in Z-DNA binding activity of the protein (23, 24, 26, 39). The role of the E3 N-terminal domain in VACV pathogenesis involves the modulation of host cellular gene expression at the transcriptional level and inhibition of apoptosis of host cells through Z-DNA binding (25). The N-terminal domain is also involved in the inhibition of adenosine deaminase ADAR-1 (28) and is required for neurovirulence and neuroinvasiveness in vivo, but not for induction of a protective immune response (7).

The C-terminal domain contains the dsRNA-binding region required for the IFN resistance and for the broad-host-range phenotype of the virus (3, 10). The binding of E3 to dsRNA inhibits the activation of both protein kinase PKR and 2'-5'-oligoadenylate synthetase (2'-5'OAS), two enzymes induced by IFN and activated in response to dsRNA (12, 13, 27, 37). Activation of PKR by dsRNA results in the phosphorylation of the {alpha} subunit of the eukaryotic translation initiation factor eIF-2 (eIF-2{alpha}), leading to a global inhibition of protein synthesis and virus replication (19, 45). Upon stimulation with dsRNA, 2'-5'OAS activates an endogenous endoribonuclease (RNase L), which cleaves cellular and viral RNAs, thereby producing a general inhibition of protein synthesis and virus replication (15, 16, 22, 49). E3 also blocks the induction of genes, such as alpha/beta interferon (IFN-{alpha}/β) through the inhibition of phosphorylation of the transcription factors IFN regulatory factor 3 (IRF3) and IRF7 (42, 50). Moreover, expression of E3 in NIH 3T3 cells results in inhibition of eIF-2{alpha} phosphorylation and I{kappa}B{alpha} degradation in response to dsRNA. E3 interferes with several cellular pathways, promotes cellular growth, and impairs antiviral activity and resistance to apoptosis (18).

Most of the previous studies were performed in cultured cells and clearly revealed a pleiotropic effect of E3 on host cell functions. To further characterize the biological role of E3 in cells, we have established an E3L-inducible NIH 3T3 cell line and generated transgenic mice expressing the E3L gene. In the inducible NIH 3T3-E3LTetOFF cell culture system, E3 is able to inhibit the phosphorylation of eIF-2{alpha} and partially block the antiviral response induced by IFN. Transgenic mice (TgE3L) are more susceptible to viral and parasitic infections than control animals and show some alterations in cellular immune responses. The TgE3L mice are the first model with a VACV gene able to partly reverse the antiviral host response.


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MATERIALS AND METHODS
 
Cells and viruses. Baby hamster kidney BHK-21 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), L-glutamine (2 mM; Sigma), penicillin (100 U/ml; Sigma), streptomycin (0.1 mg/ml; Sigma), gentamicin (5 µg/ml; Sigma), and amphotericin B (Fungizone) (0.5 µg/ml; Sigma). African green monkey kidney cell line BSC40 was grown in complete DMEM supplemented with 10% newborn calf serum. Lymphoma-derived EL4 cells and primary lymphocytes obtained from the spleens and lymph nodes of mice were grown in RPMI 1640 medium (Gibco BRL), supplemented with 10% FCS, penicillin (100 U/ml), streptomycin (0.1 mg/ml), L-glutamine (2 mM), β-mercaptoethanol (10 µM; Sigma), and HEPES (pH 7.4) (10 mM). The viruses used in this work were: vaccinia virus strain Western Reserve (VACV-WR), the recombinant WRluc expressing luciferase in the thymidine kinase (TK) locus (38), the mutant VACV lacking the E3L gene in the WR strain (vv{Delta}E3L) (11), and the recombinant alphavirus Sindbis virus Totto1101-luc, expressing the luciferase gene as part of the NSP3 protein (35). vv{Delta}E3L was kindly provided by B. L. Jacobs (University of Arizona), and Sindbis virus Totto1101-luc was kindly provided by C. Rice (Rockefeller University, New York, NY). VACV-WR and WRluc were grown and titrated in BSC40 cell monolayers. vv{Delta}E3L and Sindbis viruses were grown and titrated in BHK-21 cell monolayers. All viruses were purified by ultracentrifugation on sucrose cushion.

Generation of the E3L-TetOFF cell line. The E3L gene from VACV-WR was cloned into bidirectional plasmid pBI-EGFP (Clontech) that allows the simultaneous expression of E3L and enhanced green fluorescent protein (EGFP) from the same promoter. pBI-EGFP-E3L and pTK-Hyg plasmids were transfected into NIH 3T3 mouse embryonic fibroblast (MEF) line TetOFF (Clontech), and clones were selected in the presence of hygromycin (Hyg) (50 µg/ml; Sigma). E3L-TetOFF cells were grown in complete DMEM without amphotericin B supplemented with 10% FCS in the presence of tetracycline (Tet) (1 µg/ml; Sigma).

Construction of E3L cassette and generation of TgE3L mice. The E3L gene from VACV-WR was cloned into pcINeO (Promega) to obtain pcINeO-E3L (18). This plasmid was digested with BglII and DraIII, giving a fragment of 2,289 base pairs which contained the E3L gene under the control of the cytomegalovirus promoter, a chimeric intron upstream of the gene, and the simian virus 40 polyadenylation region at the end. This fragment was purified with Geneclean II (XQ-Biogene) with two additional steps of ultrafiltration with 0.22-µm filters (Millipore Millex GV4) and dialysis through microdialysis membranes (Millipore VM; 0.05-µm pore size) for 3 h. DNA was quantified and microinjected into the pronuclei of fertilized oocytes of a C57BL/6 mouse as described previously (21, 32). The microinjected oocytes were implanted into pseudopregnant female mice, resulting in several litters. The mice were bred, and DNA from the tail of the offspring was analyzed by PCR and slot blotting to detect the transgene. Transgenic founders were backcrossed with C57BL/6 mice to obtain transgenic lines. PCR-positive founder animals were confirmed by slot blot analysis, and both the integrity and transmission of the transgene were analyzed in the different generations.

PCR for E3L mouse genotype. Genomic DNA from tail biopsy specimens was extracted using the Easy-DNA kit (Invitrogen). The β-actin gene was amplified with the primer pair Actin-5 (5'-GGCACCACACCTTCTACAATG-3') (forward) and Actin-3 (5'-GTGGTGGTGAAGCTGTAGCC-3') (reverse) that amplified a 340-bp fragment. The E3L gene was amplified with primer E3L-1 (5'-CGCAGAGATTGTGTGTGAGGC-3') (forward) and either primer E3L-2 (5'-CTCTTCCGTCGATGTCTACAC-3') (reverse), which amplified a 440-bp fragment, or primer E3L-3 (5'-GGAGGAATATCGTCGGAGCTG-3') (reverse), which amplified a shorter fragment of 152 bp. Amplifications were made in a total volume of 25 µl containing 0.2 mM of each deoxynucleoside triphosphate, 0.2 µM of each primer, 1.5 mM MgCl2, 75 to 500 ng of DNA template, reaction buffer (1x), and 1 unit of DNA polymerase Platinum Taq (Invitrogen). The PCR protocol consisted of an initial step of 2 min at 94°C, followed by 35 cycles of 15 s at 94°C, 30 s at 58°C, and 1 min at 68°C. The final extension cycle was 7 min at 72°C. PCR products were resolved in 1% agarose gel in Tris-borate-EDTA (TBE) buffer with 0.5 µg/ml ethidium bromide and were visualized using UV light.

RT-PCR analysis. Total RNA was purified from different tissues homogenized in RNA extraction buffer using an Ultraturrax T8 mechanical homogenizer (Janke and Kunkel, Staufen, Germany). RNA was isolated using Ultraspec-II RNA resin purification system (Biotecx) following the manufacturer's instructions. A 1.5-µg sample of total RNA was digested with DNases to avoid genomic DNA contamination (Ambion Turbo kit). PCR with Taq Platinum DNA polymerase (Invitrogen) using primers for β-actin was carried out to ensure there was no DNA contamination. Reverse transcriptase PCR (RT-PCR) was carried out with 0.5 µg of total RNA using Superscript retrotranscriptase (Invitrogen) according to the manufacturer's instructions. After reverse transcription, PCR was performed by adding 4 µl of each sample to the PCR mix containing specific forward E3L-1 primer and reverse E3L-2 or E3L-3 primer. The PCR conditions used and products were resolved as described above.

Western blot. Mouse tissues were resuspended in luciferase buffer (Promega) to 0.1 g/ml supplemented with protease and phosphatase inhibitors (1 mM NaF and 1 mM Na3VO4), disrupted with an Ultraturrax T8 mechanical homogenizer and kept at 4°C for 15 min for lysis. Lysates were then centrifuged at 13,000 rpm for 15 min at 4°C, and supernatants were stored at –70°C until protein quantification by the Bradford method. Proteins were denatured by boiling in Laemmli buffer with β-mercaptoethanol, and cell cultures were lysed directly in Laemmli buffer. For immunoblots, protein samples were fractionated on 12% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad Laboratories) in a semidry blotting apparatus (Bio-Rad) for 45 min at 200 mA. Membranes were blocked for 1 h in phosphate-buffered saline (PBS) containing 5% nonfat dried milk and then probed with polyclonal anti-E3 (1/500), polyclonal anti-VACV-WR (1/2,000) (36), polyclonal anti-Sindbis virus capsid (anti-C; 1/5,000) (46), monoclonal anti-β-actin (Sigma; 1/2,000), polyclonal anti-phospho eIF-2{alpha} (eIF-2{alpha}P) (1/1,000; Biosource), polyclonal anti-eIF-2{alpha} (1/500; Santa Cruz), polyclonal anti-murine PKR (1/200; Santa Cruz), and polyclonal anti-ISG15 (1/1,000; generously provided by Ivan Horak, Germany) antibodies. Proteins were detected using horseradish peroxidase-labeled secondary antibodies and an ECL kit (Amersham Biosciences).

Preparation and culture of primary mouse embryonic fibroblasts derived from TgE3L mice. Homozygous mice were intercrossed, and MEFs were prepared from the resulting embryos at day 12.5 postcoitum. Individual sibling embryos were removed, while maintaining sterility, and transferred to tissue culture dishes containing PBS with penicillin (100 U/ml; Sigma), streptomycin (0.1 mg/ml; Sigma), and gentamicin (5 µg/ml; Sigma) antibiotics in a petri plate. The heads, livers, and hearts were removed from the embryos. The remaining tissue was minced and divided into pieces with the help of a cutter and incubated at 37°C for 45 min with 0.25% trypsin with gentle shaking. The tissue was resuspended in 5 ml of DMEM with 10% FCS followed by vigorous pipetting to break up the tissue into cells and decanted, pieces of the remaining floating tissue were removed with a Pasteur pipette, and the supernatant was centrifuged for 5 min at 800 rpm. The cells were then cultured in fresh DMEM with 10% FCS. Cell lines were grown for a limited number of passages to avoid immortalization. Control MEFs from C57BL/6 mice were prepared by similar procedures.

Virus inoculation of mice and tissue collection. Groups of C57BL/6, TgE3L, and BALB/c mice (male and female) matched by age and weight were used in this work. C57BL/6 and BALB/c mice were purchased from Harlan Iberica S.A. (Barcelona, Spain) and housed in the Animal Facility of the Centro Nacional de Biotecnología-CSIC (Madrid, Spain) under pathogen-free conditions. Mice were always anesthetized with isofluorane before virus inoculation by the intraperitoneal (i.p.) route (200 µl), by tail scarification (t.s.) (10 µl) (7), or by the subcutaneous (s.c.) route (30 µl). Mice were observed daily for body weight loss and signs of illness. The organs removed (liver, spleen, ovaries, and lymph nodes) were stored at –70°C until analysis or kept at 4°C in RPMI 1640 medium for cell extraction. Cells from the peritoneal cavity (PC) were obtained by performing peritoneal washes, flushing 10 ml of cold PBS directly into the PC with a 21-gauge syringe. The fluid was removed, centrifuged at 1,500 rpm for 5 min, and stored at –70°C until luciferase measurement. Serum was obtained from blood extracted from the retro-orbital plexus using a capillary tube and stored at –70°C. All animal manipulations were carried out according to the guidelines of the Swiss Institute for Experimental Cancer Research and Centro Nacional de Biotecnología.

Virus titration in mouse tissues. A 0.1-g/ml homogenate was prepared from each weighed organ by adding complete DMEM with 10 µg/ml gentamicin in an Ultraturrax T8 mechanical homogenizer. All homogenates were subjected to three cycles of freezing (–70°C) and quick thawing (37°C). Samples were sonicated for 30 seconds and centrifuged 10 min at 2,000 rpm at 4°C. Supernatants were collected and used for virus plaque assays. Plaques were stained with crystal violet or by immunostaining, and virus titers are shown as PFU per gram of tissue.

Measurement of luciferase activity. Samples were processed as described above for Western blot assay adding 100 to 200 µl of luciferase assay reagent (Promega Corp., Madison, WI) per tissue. Clarified supernatants were used to measure luciferase activity in the presence of luciferin and ATP according to the manufacturer's instructions, using a Lumat LB 9501 luminometer (Berthold, Nashua, NH). The luciferase activity was expressed as luciferase reference units per milligram of protein (34).

Evaluation of specific T cells by the ELISPOT assay. The enzyme-linked immunospot (ELISPOT) assay to detect antigen-specific T cells was performed as previously described (31). The antibodies used were monoclonal anti-murine IFN-{gamma} antibody (6 µg/ml) (R4-6A2; Pharmingen, San Diego, CA) or monoclonal anti-murine interleukin 2 (IL-2) antibody (6 µg/ml) (MAb JES6-1A12; Pharmingen, San Diego, CA), biotinylated anti-murine IFN-{gamma} monoclonal antibody (2 µg/ml) (XMG1.2; Pharmingen, San Diego, CA), or biotinylated anti-murine IL-2 monoclonal antibody (2 µg/ml) (JES6-5H4; Pharmingen). VACV-infected EL4 cells (5 PFU/cell for 4.5 h) were used as antigen-presenting cells. The spots were counted with an AID ELISPOT reader system (Vitro).

Flow cytometry analysis of cell populations in tissues of transgenic mice. Recipient 12-week-old C57BL/6 and TgE3L mice, uninfected or infected with WRluc for 16 h, were killed, and single-cell suspensions were prepared from the spleen, blood, and peritoneal washes. Blood was obtained from the orbital plexus using the Microvette system for capillary blood collection. Cells from the spleens were extracted and washed several times with PBS, and the erythrocytes were lysed with 0.1 M NH4Cl for 5 min at 4°C. The number of cells in single-cell suspensions was counted, and equal amounts of cells were treated with Fc block (Fc{gamma}III/II 2.462; BD Pharmingen) (1/100) before additional staining. Cells were stained with biotinylated anti-mouse pan-NK cells (DX5) followed by streptavidin labeled with Alexa Fluor 488, anti-CD3e labeled with allophycocyanin, anti-CD11b labeled with phycoerythrin, anti-CD4 labeled with fluorescein isothiocyanate, and anti-CD8a labeled with phycoerythrin. All the antibodies were purchased from BD Pharmingen and added for 30 min at 4°C (1/200). Peripheral blood mononuclear cells (PBMCs) from blood were stained directly in the blood sample (100 µl/sample) and lysed with BD FACS lysing solution (BD Biosciences) after staining. Cells were fixed in 4% paraformaldehyde for 15 min at room temperature, washed with PBS, and analyzed using FACSCalibur Becton Dickinson instruments and ProQuest software.

Leishmania major infection. L. major (WHOM/IR/-173) was a kind gift from Nicholas Glaichenhaus (CNRS, Valbonne, France). Promastigotes were cultured at 27°C in Schneider's medium (Gibco BRL, United Kingdom) with antibiotics. The virulence of the strain was preserved by periodic passages through BALB/c mice. Frozen stocks grown in culture until the stationary phase were used for the experiments. BALB/c, C57BL/6, and TgE3L mice, 10 to 12 weeks of age, were challenged s.c. in the right hind footpad with 5 x 104 live, late-stationary-phase L. major promastigotes. Lesion development at the inoculation site was measured weekly with a digital caliper (Mauser Digital, Switzerland) and expressed as the increase in thickness of an infected hind foot versus an uninfected hind foot. Mice were sacrificed at 8 weeks postchallenge, and serum, lymph nodes, and spleen were harvested for immunological studies. The number of parasites in infected draining lymph nodes was quantified by limiting dilution after 7 and 15 days of incubation at 27°C in Schneider's medium containing 20% FCS and antibiotics (9). The statistical significance (P < 0.05 or P < 0.005) for the values for immunization groups of mice was determined by Student's t test.


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RESULTS
 
Inducible expression of E3 in the TetOFF system reverses the antiviral action of IFN. We have previously described an NIH 3T3 cell line expressing low levels of E3 that showed enhanced resistance to the antiviral action of IFN (18). Thus, to better define the antiviral action of E3, we took advantage of the TetOFF system and generated a cell line that could be induced to express E3. Stable E3L-TetOFF cells containing the E3L gene from VACV-WR were generated as described in Materials and Methods. Two proteins of about 25 and 20 kDa were synthesized by E3L-TetOFF cells upon Tet withdrawal (Fig. 1A, mock-infected cell extracts). Detailed analysis of protein induction showed that E3 was produced to levels in uninfected E3L-TetOFF cells comparable to those produced during virus infection (compare at 24 hours postinfection [hpi] the mock- and VACV-infected cell extracts). To test the activity of E3-expressing cells, a complementation assay using a VACV mutant lacking E3L (vv{Delta}E3L) was performed. Thus, E3L-TetOFF cells induced for 36 h without Tet were mock infected or infected with 0.2 PFU/cell of either vv{Delta}E3L or VACV-WR. At different times postinfection, cells were collected and lysates were analyzed by Western blotting using a specific antibody. In all cases, the main E3 product was the protein of 25 kDa. The synthesis of the smaller product of 20 kDa is likely the result of "leaky scanning" past the first translation initiation codon (47) due to alternative initiation of translation; only minor and variable amounts of the 20-kDa protein have been detected in extracts of VACV-infected cells (12).


Figure 1
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  		FIG. 1. Generation and characterization of an inducible E3L-TetOFF cell line expressing E3. (A) E3 expression was induced in the absence of tetracycline (– Tet) for 36 h; thereafter, cells were mock infected or infected with vv{Delta}E3L or VACV-WR (0.2 PFU/cell) for the indicated times. Cells were lysed at 8, 16, and 24 hpi in Laemmli buffer, and the proteins were resolved on 12% SDS-polyacrylamide gels. E3 and VACV proteins were detected by Western blot analysis. {alpha}-E3, anti-E3 antibody. (B) E3L-TetOFF monolayers induced for 36 h in the absence of tetracycline were incubated with type I murine IFN (IFN-{alpha}/β) 102 U/ml for 16 h. Cells were then mock infected or infected with vv{Delta}E3L or VACV-WR (0.2 PFU/cell) for 6 h. Cells were lysed in Laemmli buffer, and proteins were resolved on 12% SDS-polyacrylamide gels. eIF-2{alpha}P, total eIF-2{alpha}, PKR, and ISG15 proteins were detected by Western blot analysis with specific antibodies. For VACV expression, similar infections were done for 6 and 8 hpi, and the Western blot was reacted with anti-VACV polyclonal antiserum. The positions of molecular mass markers (in kilodaltons) are shown to the right of the blots.

To define the effect of induced E3 on VACV protein synthesis, we performed Western blotting using a polyclonal anti-VACV antibody. As shown in Fig. 1A (bottom blot), in cells infected with vv{Delta}E3L, the induction of E3 rescued viral protein synthesis, as confirmed by the accumulation of viral proteins as the length of time of infection increased (8 to 24 hpi). This finding was also confirmed by the recovery of vv{Delta}E3L virus yields in E3-induced cells (not shown).

Since E3 inhibits activation of the IFN-induced protein kinase PKR and the main substrate of this enzyme is eIF-2{alpha} (12, 17, 26, 27), we next determined whether E3 expression prevented the phosphorylation of eIF-2{alpha}. To do this, E3L-TetOFF cells were induced for 36 h, treated with murine IFN-{alpha}/β in order to induce antiviral proteins as PKR, and later infected with either VACV or its E3L deletion mutant. As shown in Fig. 1B (top blot), the phosphorylation of eIF-2{alpha} was detected only in cells infected with vv{Delta}E3L in the presence of Tet, showing that E3 blocked eIF-2{alpha} phosphorylation. In E3-producing cells, there is expression of IFN-inducible genes, since the levels of PKR and ISG15 were enhanced after IFN treatment (Fig. 1B). Significantly, the antiviral response of IFN was blocked in cells expressing E3, as defined by the rescue of vv{Delta}E3L proteins (Fig. 1B, bottom blot). Clearly, in IFN-treated cells infected with vv{Delta}E3L, there was a severe inhibition of viral protein expression when E3 was not present, while viral proteins accumulated in E3-induced cells (compare the levels of viral proteins in vv{Delta}E3L-infected cells treated with IFN and Tet to those in cells treated with IFN but not with Tet).

To provide additional evidence that E3 was able to reverse the antiviral action of IFN, we carried out a rescue assay of Sindbis virus replication. Recombinant Sindbis virus (Totto1101-luc) was used to infect E3L-TetOFF cells induced for 36 h in the absence of Tet and treated for 16 h with different amounts of murine IFN-{alpha}/β. After 6 hpi, viral translation was measured by luciferase assay and capsid protein levels. As shown in Fig. 2A, in cells expressing E3 without IFN treatment, translation of Sindbis mRNAs was about 1 log unit higher than in noninduced cells (treated with Tet). In IFN-treated cells, luciferase levels remained higher when E3 was produced, indicating that E3 expression inhibited the cellular antiviral mechanisms and thus enhanced translation of Sindbis virus mRNA. This was also confirmed by Western blotting as shown by the levels of the viral capsid protein in the presence of E3 (Fig. 2B). Clearly, Sindbis virus mRNA translation was more resistant to low doses of IFN when E3 was synthesized.


Figure 2
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  		FIG. 2. Expression of E3 protein prevented the antiviral action of IFN against Sindbis virus infection. E3L-TetOFF cells induced for 36 h in the absence of tetracycline (– Tet) were incubated for 16 h with different doses of murine IFN-{alpha}/β and then infected with 10 PFU/cell of Sindbis-luc virus (Totto1101) for 6 h. (A) Cells were lysed in luciferase buffer, and luciferase activity and protein quantity were measured. RLU/mg prot, relative light units per milligram of protein. (B) Fifteen-microgram samples of total protein from the lysates were resolved on 12% SDS-polyacrylamide gels, and the capsid protein from Sindbis virus was detected with specific antibody. The intensity of bands was measured by densitometry.

The findings of Fig. 1 and 2 with the E3L-TetOFF system show that expression of E3 in cultured cells inhibits the antiviral action of IFN for both VACV-WR and Sindbis virus. These effects correlate with E3 inhibition of eIF-2{alpha} phosphorylation and, hence, of PKR activation.

Generation and characterization of E3L transgenic mice. The enhanced resistance of E3-expressing cells to the antiviral action of IFN shown above prompted us to investigate whether similar resistance could be transferred to an organism. To do this, we generated transgenic mice (TgE3L) with a transgene containing the E3L gene under the control of the cytomegalovirus promoter. A schematic diagram of the inserted cassette is shown in Fig. 3A. The insertion of E3L in the mouse genome was followed by hybridization of mouse tail DNA with specific E3L primers (Fig. 3B). Three founders were obtained, but only one had the transgene integrated in the germ line. The homozygous mice contained about six copies of the transgene as determined by slot blot hybridization (not shown).


Figure 3
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  		FIG. 3. Generation of E3 transgenic mice. (A) Map of the E3L transgene microinjected in the pronuclei of fertilized oocytes for the generation of transgenic mice as described in Materials and Methods. The positions of the cytomegalovirus promoter (CMV-promoter) and the simian virus 40 polyadenylation region (SV40polyA) are shown. (B) Genotyping of the founders. Specific primers for E3L gene were used to amplify the transgene (440 bp). pcINeo-E3L was used as a positive control (C+), and genomic DNA extracted from C57BL/6 mice was used as a negative control (C–). Water was used as a blank (b). The β-actin gene was amplified as a control of the quality of DNA extracted from tail biopsy specimens, and genomic DNA extracted from C57BL/6 mice was used as a positive control (C+). The positions of molecular size standards (in base pairs) are shown to the left of the blots.

Expression of the transgene was determined in different tissues by diverse techniques. First, total RNA extracted from different tissues of homozygous animals was used for RT-PCR using specific E3L primers (E3L-1 and E3L-2). Figure 4A shows a representative example in which E3L mRNA was detected in the thymus, testis, bone marrow, spleen, and kidney from a transgenic mouse although at low levels as judged by the low intensities of the bands. We next determined the presence of E3L mRNA in total RNA extracted from the brains of homozygous and heterozygous animals by RT-PCR using different pairs of E3L primers. The two pairs of primers, primers E3L-1/E3L-3 and E3L-1/E3L-2, amplified fragments of 152 bp and 440 bp, respectively, from the mRNA of transgenic mice (Fig. 4B, top blot). E3L mRNA was also detected in the brains of transgenic mice by Northern blotting using a radioactive probe (not shown). To assure the integrity of the mRNAs, RT-PCR of the β-actin gene was performed (Fig. 4A and B, middle blots). RT-PCR of mRNAs extracted from different tissues of a control wild-type mouse resulted in no amplification with any of the E3L set of primers (not shown), and a control RT-PCR without adding RT was done to ensure that no genomic DNA contamination was being amplified (Fig. 4A and B, bottom blots). Expression of the transgene was confirmed by Western blotting using polyclonal anti-E3 antibody. As shown in Fig. 4C, low levels of E3 protein were detected in different tissues, with higher levels found in the liver, lung, and testis. An actin control was included to show the protein concentration loaded on the gel from each sample (Fig. 4C, bottom blot), and tissues from nontransgenic mice were also included to show lack of cross-reactivity of a 25-kDa band with the antibody (Fig. 4D).


Figure 4
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  		FIG. 4. Characterization of E3L expression in transgenic mice. (A) RNA from different tissues of a homozygous mouse was used to detect E3L mRNA by RT-PCR using primers E3L-1 and E3L-2. The tissues analyzed were muscle (M), thymus (Thym), testis (Ts), lung (L), spleen (Sp), bone marrow (BM), stomach (St), kidney (K), and liver (Li). Water was used for a blank (b), and RNA from VACV-infected MEFs was used as a positive control for E3L messenger (C+). – RT, no RT. (B) Detection of E3L mRNA in the brains of control, heterozygous, and homozygous mice. Primers E3L-1 and E3L-2 amplified a 440-bp fragment (top blot, right). Primers E3L-1 and E3L-3 amplified a 152-bp fragment (top blot, left). Genomic DNA from the tails of transgenic mice was used as positive controls for the RT-PCR (C+), and water was used for the blank (b). β-Actin was amplified in both cases as a housekeeping gene (bottom two blots), and RT-PCR in the absence of RT (– RT) using the β-actin primers was performed to confirm lack of genomic DNA contamination in the samples. The positions of molecular size standards (in base pairs) are shown to the left of the blots in panels A and B. (C) Detection of E3 protein in different tissues of transgenic mice. Fifty-microgram samples of total protein extracted from the tissues were resolved on 12% SDS-polyacrylamide gels. Polyclonal antibody against E3 was used to detect endogenous levels of the transgenic protein. A murine cell line infected with VACV was used as a positive control (+). Similar patterns of expression were detected in different transgenic mice analyzed. β-Actin was detected as an internal control. (D) Western blot of tissues from nontransgenic control C57BL/6 mice incubated with polyclonal anti-E3. The positions of molecular mass markers (in kilodaltons) are shown to the left of the blots in panels C and D.

The findings of Fig. 3 and 4 reveal that we have generated C57BL/6 transgenic mice that express the VACV E3L gene.

E3 expression from transgenic MEFs inhibits cellular antiviral responses. To assess whether cells derived from TgE3L mice have a phenotype similar to that of the E3L-inducible cultured cell system, we generated MEFs derived from transgenic and control C57BL/6 mice as described in Materials and Methods. Different clones of MEFs obtained from individual sibling embryos (TgE3L2, TgE3L4, and TgE3L8) as well as from pools of three sibling embryos (TgE3Lp) were analyzed by Western blotting with anti-E3 antiserum. As shown in Fig. 5A, low levels of E3 protein were detected only in the pooled clones (TgE3Lp), although by RT-PCR we found E3 mRNA expression in all clones (not shown). To define to what extent TgE3L MEFs support virus replication and its correlation with eIF-2{alpha} phosphorylation, MEFs from control C57BL/6 and TgE3L mice were infected with Sindbis-luc virus, and eIF-2{alpha}P was analyzed at different times postinfection. eIF-2{alpha}P was detected only in control MEFs after 4 hpi as a result of activation of an antiviral response (Fig. 5B), while TgE3L MEFs did not show phosphorylation of eIF-2{alpha} at any time postinfection. As determined by luciferase expression, the levels of Sindbis virus replication were similar in both cell lines up to 6 hpi, and thereafter the virus replicates better in TgE3L MEFs (Fig. 5C), correlating with the absence of eIF-2{alpha} phosphorylation. These findings reveal that E3 expression in MEFs derived from transgenic mice partly inhibits cellular antiviral responses and this effect occurs with low levels of E3 protein produced.


Figure 5
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  		FIG. 5. Characterization of TgE3L MEFs for antiviral response. (A) Detection of E3 protein in different MEF clones derived from C57BL/6 mice and transgenic TgE3L mice. TgE3L MEF clones 4, 6, and 8 (TgE3L4, -6, and -8) were generated from independent sibling embryos, while TgE3Lp was generated from a pool of three different sibling embryos. Fifty-microgram samples of total protein extracted from the cells were resolved on 12% SDS-polyacrylamide gels. Polyclonal antibody against E3 was used to detect endogenous levels of the transgenic protein. A murine cell line infected with VACV-WR was used as a positive control (+), and MEFs from a nontransgenic mouse served as a negative control. {phi}, empty lane. (B) MEFs from TgE3L and C57BL/6 mice were infected with 10 PFU/cell of Sindbis-luc virus for different times, the cells were lysed in Laemmli buffer, and proteins were resolved on 12% SDS-polyacrylamide gels. eIF-2{alpha}P and eIF-2{alpha} proteins were detected by Western blotting with specific antibodies. (C) Levels of Sindbis-luc virus replication in control and TgE3L mice at different times postinfection. Luciferase (Luc) activity was measured in duplicate samples, and values are referred as relative light units per mg of protein (RLU/MG) as determined by using the Bradford assay kit (Bio-Rad).

TgE3L mice are more susceptible to VACV infection than C57BL/6 mice. To test the functional significance of E3 expression in transgenic mice, both TgE3L and parental C57BL/6 mice were inoculated by t.s. with 8 x 106 PFU/mouse of vv{Delta}E3L or 1 x 106 PFU/mouse of VACV-WR viruses. A higher dose of vv{Delta}E3L virus was used because it is an attenuated virus and has been described as non virulent in the murine model (8). Signs of illness, such as weight loss and tissue injury, were monitored for 16 days after infection. Weight loss has been consistently used to measure pathogenesis and directly correlates with fever in poxvirus infections in animals (8). As shown in Fig. 6A, the parental C57BL/6 mice inoculated with vv{Delta}E3L did not exhibit weight loss or signs of illness. In contrast, TgE3L inoculated with vv{Delta}E3L lost weight mainly after day 12 postinoculation. This was more pronounced in TgE3L mice infected with wild-type VACV than in parental mice (P < 0.05). We noticed that the lesion at the site of virus inoculation in the tail took longer to heal in TgE3L mice than in parental C57BL/6 mice when the mice were infected with vv{Delta}E3L (Fig. 6B). Moreover, when infected with VACV-WR, the lesion did not heal in TgE3L mice during the experiment. While C57BL/6 mice developed an ulcer, TgE3L mice had poor signs of healing even after 16 days postinoculation, showing an erythematous inflammatory ulcer.


Figure 6
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  		FIG. 6. TgE3L mice developed more signs of disease to VACV infection than control animals did. Two groups of 16-week-old male C57BL/6 and TgE3L mice (n = 4) were inoculated by t.s. with 8 x 106 PFU/mouse of vv{Delta}E3L or 1 x 106 PFU/mouse of VACV-WR viruses. (A) Weight loss (determined by averaging the weights of the mice per group at each time point divided by the initial average weight) was monitored daily for 16 days after inoculation (dpi). The asterisk indicates statistical significance (P < 0.05) of the two values indicated by the bracket. (B) Representative photographs of tail lesions taken at days 7 and 12 postinoculation. The results of one representative experiment of three experiments are shown.

To evaluate the extent of VACV replication in mice and the ability of the virus to infect tissues different from the tissue of the inoculation site, we used a recombinant VACV that expresses the luciferase reporter gene under the control of the viral p7.5 early/late promoter (WRluc) (38). Thus, C57BL/6 and TgE3L mice were inoculated by the i.p. route with 2 x 106 PFU/mouse of WRluc. The luciferase activity was determined in different tissues at 48 hpi. As shown in Fig. 7A, luciferase levels in the spleens (P < 0.05), livers, and ovaries of TgE3L mice were higher than those found in the same tissues of infected C57BL/6 mice. Higher virus replication was also observed in cells of the peritoneal cavity by 73 hpi (P < 0.005), where WRluc replicated 4.3 times more in TgE3L mice than in control mice (Fig. 7B). This was consistent with higher virus yields in TgE3L mouse tissues than in control mouse tissues. The virus titer in the spleen was about 8.7-fold higher for VACV-infected TgE3L mice than in VACV-infected C57BL/6 mice, while it was 2.2-fold higher in the liver (not shown). We also wanted to determine whether E3 protein from transgenic mice could rescue the viral activity of the deletion mutant virus vv{Delta}E3L. For this purpose, transgenic and control mice were inoculated by the i.p. route with 6 x 107 PFU/mouse of vv{Delta}E3L, and virus titers were determined from tissue homogenates. Higher viral titers were obtained at 24 hpi in the spleen, ovaries, and liver of transgenic mice (Fig. 7C). The findings of Fig. 6 and 7 reveal that VACV replication is enhanced in TgE3L mice and that these animals develop delayed injury healing.


Figure 7
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  		FIG. 7. Higher viral spread of VACV after intraperitoneal inoculation in TgE3L mice. Two groups of 12-week-old female C57BL/6 and TgE3L mice (n = 4) were inoculated i.p. with 2 x 106 PFU of WRluc per mouse. At 48 hpi, tissue samples were collected, and luciferase activity present in clarified tissue homogenates was measured. (A) Luciferase (Luc) expression in spleen (P < 0.05 [*]), liver, and ovaries (P = 0.4). The data from four different animals in one experiment are shown as means plus standard deviations (error bars). Comparable results were obtained in three independent experiments. RLU/mg, relative light units per milligram of protein. (B) Two groups of 12-week-old female C57BL/6 and TgE3L mice (n = 4) were inoculated i.p. with 2 x 106 PFU of WRluc per mouse. At 38, 48, and 73 hpi (P < 0.005 [**]), cells from the peritoneal cavity were extracted and luciferase activity was measured from pools. (C) Two groups of 16-week-old female C57BL/6 and TgE3L mice (n = 3) were inoculated i.p. with 6 x 107 PFU of vv{Delta}E3L per mouse. At 24 hpi, tissue samples were collected, and viral titer from pools was determined by immunostaining and expressed as PFU/g of tissue.

TgE3L mice are more susceptible to infection with the parasite Leishmania major. We next asked whether the enhanced sensitivity to virus infection of TgE3L mice could also be demonstrated for a parasitic infection. To do this, we used the murine Leishmania model as infection can be readily monitored. C57BL/6, TgE3L, and susceptible BALB/c mice were inoculated with 5 x 104 L. major by the s.c. route in the right hind footpad. The increase of the lesion size was monitored weekly for 8 weeks. Differences in lesion development at the site of inoculation between transgenic and control mice were observed at week 5 after infection. TgE3L mice developed lesions 3.3-fold larger than those of the C57BL/6 mice (P < 0.005) (Fig. 8A), although these lesions did not reach the size obtained in the susceptible BALB/c mice. In order to confirm that the lesion size was related to the number of parasites in the lymph nodes, the parasite loads were determined for each group 8 weeks postinoculation. As expected, the highest parasite load was detected in susceptible BALB/c mice (not shown). We found that the number of parasites/mg of tissue in the TgE3L group was 2.23-fold higher than that in the C57BL/6 group (Fig. 8B), and the weight of the lymph nodes of TgE3L mice were 2-fold higher (not shown), which correlates with higher replication of the parasite (4). Therefore, TgE3L mice are more susceptible to L. major infection than C57BL/6 mice.


Figure 8
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  		FIG. 8. Susceptibility of TgE3L mice to the parasite Leishmania major. Three groups of 10- to 12-week-old BALB/c mice (n = 2), C57BL/6 mice (n = 6), and TgE3L mice (n = 6) were infected s.c. with 5 x 104 L. major. (A) Increase of lesion size 5 weeks after challenge. The average lesion size was measured as the increase in thickness of an infected foot compared to an uninfected foot of the mice in each immunization group. TgE3L mice developed larger lesions than the C57BL/6 mice did (P < 0.005 **). Each symbol shows the results for one mouse. The short horizontal black bars are the mean values for the groups of mice. (B) Parasite loads detected in each immunization group 8 weeks postchallenge. Draining lymph nodes were excised, weighed, and homogenized. The single-cell suspension from each pool was serially diluted in 96-well flat-bottom microtiter plates. The number of viable parasites per mg of tissue was determined from the highest dilution at which promastigotes could be detected after incubation for 14 days at 27°C.

E3L transgenic mice have reduced cellular immune responses to VACV antigens and have alterations in the levels of cells of the immune system. The fact that TgE3L mice were more susceptible to VACV and L. major infections and developed delayed healing after virus inoculation by scarification suggest that these animals might have alterations in their immune responses to the pathogens. Thus, we next compared the cellular immune responses elicited in mice inoculated by t.s. with vv{Delta}E3L or VACV-WR at day 16 postinoculation by ELISPOT assay. The number of IFN-{gamma}-secreting cells specific for VACV antigens found in the spleen was similar for transgenic and C57BL/6 mice when the mice were inoculated with the mutant vv{Delta}E3L virus. However, after VACV-WR inoculation, the number of IFN-{gamma}-secreting cells in the spleen was lower in transgenic mice than in C57BL/6 mice (P < 0.05) (Fig. 9A). The same result was observed in lymphocytes from lymph nodes (P < 0.05) (Fig. 9B). A comparison of the antigen-specific IL-2-secreting cells from spleen and lymph nodes was also determined. The levels of IL-2-secreting cells from the spleen were similar for both transgenic and C57BL/6 mice infected with either VACV WR or vv{Delta}E3L (Fig. 9C). It is noteworthy that the number of IL-2-secreting cells from the lymph nodes of vv{Delta}E3L-inoculated mice was about twofold lower in TgE3L mice than in C57BL/6 mice (P < 0.05) (Fig. 9D). Similarly, in mice inoculated with VACV-WR, the number of IL-2-secreting cells from lymph nodes was 1.85-fold lower in TgE3L mice than in C57BL/6 mice (P < 0.05) (Fig. 9D).


Figure 9
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  		FIG. 9. Cellular immune responses elicited in TgE3L mice against VACV antigens. (A) Two groups of 10-week-old C57BL/6 and TgE3L mice (n = 4) were inoculated by t.s. with vv{Delta}E3L (8 x 106 PFU/mouse) or VACV-WR (1 x 106 PFU/mouse), and 16 days after infection, T-cell immune responses specific to viral antigens were determined in splenocytes by ELISPOT assays. (B to D) Two groups of 10-week-old C57BL/6 and TgE3L mice (n = 4) were inoculated by t.s. with vv{Delta}E3L or VACV-WR (1 x 106 PFU/mouse), and 16 days after infection, the T-cell immune responses specific to viral antigens were determined by ELISPOT assays. The number of IFN-{gamma}-secreting cells in lymph nodes (LN) (B) and the number of IL-2-secreting cells in the spleen (C) and in lymph nodes (D) were determined. The values are shown as means plus standard deviations (error bars) of pooled samples from triplicate cultures. Values that were statistically significantly different (P < 0.05) are indicated by an asterisk and arrow. Comparable results were obtained in three independent experiments.

The findings shown in Fig. 9 reveal that E3 transgenic mice trigger a reduced anti-VACV T-cell immune response compared to that in C57BL/6 mice, particularly in lymph nodes.

To define whether the effects of E3 in transgenic mice are due to impairment of specific innate cell functions, we analyzed the percentage of NK cells, macrophages, and T cells present in uninfected and VACV-infected control and TgE3L mice. C57BL/6 and TgE3L mice were infected with WRluc (2 x 106 PFU/mice) by the i.p. route for 16 h. Cells from the peritoneal cavity, spleen, and PBMCs were extracted and NK cells, macrophages, and T lymphocytes (CD4, CD8, and CD3) were determined by specific receptor staining and flow cytometry. As shown in Table 1, in uninfected cells (mock-infected cells), the endogenous population of NK cells in the spleen and PC was higher in TgE3L mice than in C57BL/6 mice. The basal population of macrophages in TgE3L mice was also higher than in control mice, particularly in the PC. The CD4, CD8, and CD3 populations were similar for the control and transgenic mice. After VACV infection of transgenic and control mice, the percentage of NK cells was higher in PBMCs from TgE3L mice than in PBMCs from control mice, the macrophage population of spleen cells was lower in transgenic mice than in control mice, and the T-cell populations of CD4, CD8, and CD3 were all higher in transgenic mice than in control mice. Comparing uninfected cells with VACV-infected cells, in the PC, the population of macrophages in uninfected cells from control mice was considerably increased after VACV infection, while in TgE3L mice there was only a slight increase after VACV infection. In PBMCs, the only difference observed between infected cells versus uninfected cells was that the percentage of CD8 in control mice diminished after infection, whereas in transgenic mice, CD8 levels remained steady. We also quantified as an index of innate response, the levels of IFN-β in serum from 12- and 16-week-old control and transgenic mice. The levels of IFN-β from uninfected TgE3L mice were about two times lower than in C57BL/6 mice (258 versus 461.7 pg/ml at 12 weeks; 322 versus 741 pg/ml at 16 weeks, mean values from two or three animals per group). In both transgenic and control mice, by 6 hpi, the levels of IFN-β in serum were reduced more than threefold when mice were infected with VACV WR by the i.p. route with 2 x 106 PFU/mouse of WRluc (not shown).


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  		TABLE 1. Different innate immune cell populations in transgenic and normal micea

The above findings show that compared to control uninfected mice, TgE3L mice exhibit differences in cells of the innate immune system, particularly an enhancement of NK cells and macrophages in the PC and spleen. After VACV infection, some differences were also observed in the levels of immune cells of tissues. These observations support that E3 effects in transgenic mice might be due to alterations in the levels of specific innate cell populations.


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DISCUSSION
 
IFNs are a large family of multifunctional cytokines that inhibit virus replication and have immunoregulatory activities. Fibroblasts, hepatocytes, and conventional dendritic cells use the so-called classical pathway to sense viruses and activate their type I IFN genes (30). The idea that IFNs play a central role in antiviral defense has been reinforced by the discovery of numerous viral mechanisms of IFN inhibition (19). Many viruses have developed different strategies to inhibit IFN action; some of these inhibitors directly sequester IFNs, interfere with IFN-induced signaling, or act against IFN-induced proteins (1, 20, 43, 44). Several viruses, like influenza virus, reovirus, and VACV, encode dsRNA-binding proteins which inhibit the IFN antiviral response. Members of the Poxviridae family, including VACV, are paradigmatic in the elucidation of the mechanisms by which the IFN-induced antiviral response is impaired by virus-encoded products. The VACV E3L and K3L gene products stimulate viral translation through inhibition of PKR (27). E3 is a potent inhibitor not only of PKR (12) but also of the 2'-5'OAS (27, 37) and ADAR-1 that acts by sequestering dsRNA (28).

In this investigation, we have developed two strategies to study E3 effects in the inhibition of host responses after virus infection. We have generated an inducible cell line expressing E3 protein which inhibits the antiviral response elicited by IFN against different viruses such as vv{Delta}E3L and Sindbis viruses. Hundreds of genes are induced by IFN (14), and many of them are involved in generating an antiviral state. We showed that in the absence of E3, replication of both vv{Delta}E3L and Sindbis viruses was impaired when the cells had been previously treated with IFN-{alpha}/β. In contrast, when cells expressed E3, the inhibition of the replication of both vv{Delta}E3L and Sindbis viruses by IFN was partially reversed. These antiviral effects were partly mediated by E3 inhibition of eIF-2{alpha} phosphorylation and hence of PKR activation, since this factor is the main substrate of PKR (17). This was not due to inhibition of E3 expression on induction of PKR, since IFN treatment of E3-expressing cells increased production of PKR and another IFN-induced gene, ISG15 (Fig. 1B). Nevertheless, E3 expression of cells treated with IFN had reduced levels of PKR in mock-infected and infected cells compared with TetON cells, probably due to E3-mediated inhibition of transcription factors IRF3 and IRF7 leading to lower IFN secretion and amplification of the pathway (42, 50).

These results confirm and extend E3 functions that had been described before with different virus-cell systems with the advantage that here we used an inducible cell system that expresses high levels of E3 protein. This cell system would also help us to define new interactions of E3 with cellular proteins without the viral environment.

The second strategy used in this work was the generation of transgenic E3L mice with the aim of studying the biological effects of E3 in an organism. Transgenic mice expressing low levels of both E3L mRNA and E3 protein were generated, but significantly, these animals showed different responses to infections than the control animals did. We do not know the locus of integration and the surrounding genomic promoter region, which could play an important role in the transcription and translation of the transgene (48). Although transgenic MEFs showed low levels of E3 expression, E3 is able to reduce the antiviral response of the host after Sindbis virus infection and to prevent the phosphorylation of eIF-2{alpha} (Fig. 5). E3 might be inhibiting the IFN pathway at multiple and different levels, if the low levels of E3 expression were sufficient to partly overcome the host antiviral response in transgenic mice.

The functional significance of E3 expression in transgenic mice was established by inoculating both TgE3L and parental C57BL/6 mice with different viruses and parasites, revealing that transgenic mice are more susceptible to infections. C57BL/6 mice inoculated by t.s. with vv{Delta}E3L did not lose weight or exhibit signs of illness (Fig. 6A). It is interesting that the TgE3L group inoculated with the non-replication-competent vv{Delta}E3L by the t.s. route showed more signs of illness than the control mice did and that they lost more weight during infection (Fig. 6A). Moreover, the TgE3L mice inoculated i.p. with vv{Delta}E3L showed higher viral titers than the control mice did (Fig. 7C). These differences were more evident when replication-competent strain VACV-WR was used (Fig. 7A and B). The difference in virus susceptibility of transgenic and control mice to VACV infection was also confirmed with a different pathogen, L. major. Immunity to infection in mice requires a L. major-specific Th1 response as well as the participation of dendritic cells and macrophages for resistance (6). Inbred BALB/c mice are susceptible, primarily because early after infection, they mount a deleterious Th2 response against the immunodominant LACK antigen of Leishmania that leads to the dissemination of the parasite (40). Since we generated the TgE3L mice with the C57BL/6 background and these animals are naturally resistant to leishmaniasis, we monitored parasite infection until the resistance state was developed in the infected animals. We hypothesized that if the development of immune responses was affected in transgenic mice, we should detect differences in the pathogenesis of the parasite. We found that TgE3L mice are more susceptible to the parasite infection since these animals developed larger lesions in the footpad at week 5 postinfection and the number of parasites in infected draining lymph nodes was higher than in C57BL/6 mice. IL-4 and IL-10 were measured to analyze the Th2 response, but the levels of both cytokines were low and only the IL-10 levels were slightly higher in TgE3L mice than in C57BL/6 mice (not shown). Transgenic mice were susceptible to Leishmania major infection, suggesting that, at least in part, a Th2 immune response could be elicited.

Since TgE3L mice were found to be more susceptible to VACV and L. major infections than control mice, we asked whether these differences were due to an impairment of immune responses. This was addressed by an evaluation of virus-specific cellular immune responses in animals inoculated with wild-type and mutant VACVs. We observed that TgE3L mice inoculated with VACV-WR had lower numbers of viral antigen-specific IFN-{gamma}-secreting cells in the spleen than C57BL/6 mice did. The differences were more noticeable in the lymph nodes, as transgenic mice inoculated with vv{Delta}E3L or VACV-WR virus had lower numbers of virus-specific IFN-{gamma}- and IL-2-secreting cells (Fig. 9B and D). Together, these results indicate that in transgenic mice there is reduced activation of a specific Th1 immune response after inoculation with both viruses, an observation that correlated with the higher susceptibility to viral infection of transgenic mice. The enhanced infection of TgE3L mice would be expected, as E3 blocks the antiviral action of IFN in cultured cells (Fig. 1 and 2). In fact, the levels of IFN-β in serum from uninfected 12- and 16-week-old transgenic mice were lower than in serum from C57BL/6 mice of the same ages, an effect which could be due to E3 inhibition of IFN-β synthesis through a blockade of IRF3 and IRF7 functions (42, 50). E3 expression could block the innate response of the TgE3L mice through inhibition of type I IFN pathway, and this blockade could, in turn, affect the differentiation and activation of the Th1 immune responses (5, 42).

When we analyzed by flow cytometry different innate cell populations in the tissues of transgenic versus control mice in the absence or presence of VACV infection, we found that uninfected transgenic mice had enhanced basal populations of NK cells and macrophages in the peritoneal cavity and in the spleen (Table 1), an effect which could be due to regulation of type I IFN secretion by E3 blockade. After VACV infection, the levels of NK cells and macrophages in the spleens of transgenic mice were reduced compared with control infected mice (Table 1). It has been previously described that NK cells migrate to the site of infection after VACV inoculation (33) and that NK cell trafficking after viral infection is driven by type I IFN (41). Since the differences in innate immune cell populations did not enhance protection of the transgenic mice upon VACV infection, E3 expression could be altering recognition of the pathogen and innate immune activation, thus favoring virus evasion. Clearly, the E3 effects observed in transgenic mice correlate with alterations of specific innate cell functions.

In conclusion, our results demonstrate that TgE3L mice are more susceptible to viral and parasitic infections, having reduced adaptive T-cell immune responses to viral antigens and altered levels of innate immune cell populations in some tissues after VACV infection compared to control mice. As a consequence, the pathogens spread through tissues developing larger lesions and more signs of illness than control mice. Our findings that E3 expression partly reverses the antiviral action of the host (described for cultured NIH 3T3 cells expressing E3L and for MEFs from TgE3L mice) and the observed alterations in specific immune responses and T-cell populations all suggest that E3 effects in transgenic mice are probably due to the contribution of inhibition by E3 of the endogenous host antiviral responses (like PKR) and to modulation of levels of innate immune cells, like NK cells and macrophages. The TgE3L system may provide a way to study pathogens that are resistant to IFN pathways and to isolate infectious agents that normally cannot escape immune surveillance.


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ACKNOWLEDGMENTS
 
This investigation was supported partly by Spanish grant BIO-2005-06264 and by the Fundación Botín.

We are grateful to Bertram L. Jacobs and Karen Kibler (Arizona State University) for providing vv{Delta}E3L and E3 polyclonal antibody. We thank Mónica Górdon Alonso and Manuel Gómez Gutiérrez for expert help with cell staining and FACS analysis, Patricia Molina Ortiz for expert help with MEF extraction, Javier Martin for expert help with the animals, Victoria Jiménez for expert help with tissue culture, and Silvia Carrasco for comments on the genotyping and characterization of transgenic mice.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Cellular and Molecular Biology, Centro Nacional de Biotecnología, CSIC, Ciudad Universitaria Cantoblanco, 28049 Madrid, Spain. Phone: 34-91-585-4553. Fax: 34-91-585-4506. E-mail: mesteban{at}cnb.uam.es Back

{triangledown} Published ahead of print on 24 October 2007. Back

{dagger} Present address: Immunobiology Center, Mount Sinai School of Medicine, Box 1630, One Gustave L. Levy Place, New York, NY 10029. Back

{ddagger} Present address: Centro de Biología Molecular "Severo Ochoa," Consejo Superior de Investigaciones Científicas-Universidad Autónoma, Facultad de Ciencias, Cantoblanco, Universidad Autónoma de Madrid, 28049 Madrid, Spain. Back


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Journal of Virology, January 2008, p. 254-267, Vol. 82, No. 1
0022-538X/08/$08.00+0     doi:10.1128/JVI.01384-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Myskiw, C., Arsenio, J., van Bruggen, R., Deschambault, Y., Cao, J. (2009). Vaccinia Virus E3 Suppresses Expression of Diverse Cytokines through Inhibition of the PKR, NF-{kappa}B, and IRF3 Pathways. J. Virol. 83: 6757-6768 [Abstract] [Full Text]  

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