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1 Department of Molecular Microbiology and Immunology, Saint Louis University Health Sciences Center, 1402 South Grand Blvd, St Louis, MO 63104, USA
2 Department of Biochemistry and Molecular Biology, Saint Louis University Health Sciences Center, 1402 South Grand Blvd, St Louis, MO 63104, USA
3 Howard Hughes Medical Institute, Rheumatology Division, Department of Medicine, Washington University School of Medicine, St Louis, MO 63110, USA
4 Genelux Corporation, San Diego Science Center, 3030 Bunker Hill Street, Suite 310, San Diego, CA 92109, USA
Correspondence
R. Mark L. Buller
bullerrm{at}slu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) production was diminished and infected cells ceased proliferating and lost viability. ECTV infection of NK cells led to early and late virus gene expression and visualization of immature and mature virus particles, but no detectable increase in viable progeny virus. It was not unexpected that early gene expression occurred in infected NK cells, as the complete early transcription system is packaged within the virions. The detection of the secreted early virus-encoded immunomodulatory proteins IFN--binding protein and ectromelia inhibitor of complement enzymes (EMICE) in NK cell culture supernatants suggests that even semi-permissive infection may permit immunomodulation of the local environment. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Yokoyama et al., 2004). Studies have shown NK cells to play a pivotal role in the innate immune response to viral infections, particularly murine cytomegalovirus (MCMV) and human herpesviruses (Biron et al., 1989). In the case of MCMV, the autosomal dominant Cmv1 locus encoding the NK cell activation receptor Ly49H has been shown to be responsible for enhanced resistance of C57BL/6 mice to a lethal MCMV challenge (Brown et al., 2001). Further studies identified the MCMV viral protein m157 as the ligand responsible for triggering the Ly49H receptor and the subsequent specific proliferation and activation of the Ly49H+ subset of NK cells (Smith et al., 2002). It is not known whether MCMV productively infects NK cells.
Previous studies have demonstrated an important role for NK cells in recovery from mousepox, a fulminant disease of selected mouse strains that is used as a small-animal model of smallpox. Depletion of NK cells through the use of anti-asialo GM1 gammaglobulin or anti-NK1.1 antibody treatment increased the mortality and tissue infectivity titres following infection of C57BL/6 mice with ectromelia virus (ECTV); however, interpretations of these studies were limited by the observation that treatment with anti-asialo GM1 also depletes CD8+ T cells, thus diminishing a virus-specific cytotoxic lymphocyte response, and anti-NK1.1 treatment depleted both NK and NKT cells (a subset of T cells that co-express an 
β T-cell receptor and NK cell receptors) (Delano & Brownstein, 1995; Jacoby et al., 1989; Karupiah et al., 1996). Recent studies of NK1.1-depleted mice and mice genetically deficient in NK cells (NKD mice) have confirmed and extended these studies (Parker et al., 2007).
These past studies have raised a number of additional questions. Are NK cells susceptible to infection? Does infection affect NK cell antiviral function? Does infection result in the production of host response modifiers, such as gamma interferon (IFN-)-binding protein (IFN-BP) and ectromelia inhibitor of complement enzymes (EMICE), that target innate responses to viral infection? Are infections productive or abortive?
In this study, we assessed the effect of viral infection on lymphokine-activated killers (LAKs) and naïve NK cells and demonstrated that ECTV is capable of infecting NK cells and suppressing NK cell function. Although the production of viable progeny was not efficient, ECTV-infected NK cells produced viral proteins, including immunomodulatory proteins, which have the potential to contribute to immune dysfunction in the local microenvironment.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Media.
LAKs and freshly isolated NK cell cultures were prepared in complete medium consisting of RPMI 1640 supplemented with L-glutamine (300 µg ml–1) and 10 % heat-inactivated fetal calf serum (FCS; HyClone). BS-C-1, a green monkey kidney epithelial cell line, was cultured in Dulbecco's modified Eagle's medium supplemented with L-glutamine (2 mM), penicillin (100 U ml–1), streptomycin (100 µg ml–1) and 10 % FCS.
Flow cytometry.
Leukocytes were blocked in anti-mouse CD16/CD32 (BD Biosciences) for 10 min at 4 °C to prevent non-specific antibody binding. Samples were then stained for 30 min, washed and fixed in 1 % formaldehyde or stained for 30 min, washed and subjected to cell sorting. Phycoerythrin (PE)-labelled PK136 (anti-NK1.1), PE-labelled DX5 (anti-CD49b), peridinin–chlorophyll protein complex-labelled Cy5.5-145-2C11 (anti-CD3), allophycocyanin (APC)-labelled 30-F11 (anti-CD45) and APC-labelled XMG1.2 (anti-IFN-) were obtained from BD Biosciences. Intracellular cytokine staining of IFN- was performed as described by the manufacturer (BD Biosciences). Samples were run on a BD FACSCalibur. Cell sorting was performed on a BD FACSAria.
Production of LAKs.
Spleens from C57BL/6 mice were harvested and single-cell suspensions (1x108 cells ml–1) were prepared in PBS containing 10 % FCS. NK cells were positively selected using DX5 magnetic beads and magnetic cell sorting (Miltenyi Biotech) according to manufacturer's instructions. NK cells were grown in interleukin (IL)-2 (800 U ml–1) containing RPMI 1640 for 6–8 days in T75 flasks at a density of 1x106 cells ml–1. The purity of NK1.1+ CD3– cells was determined by flow cytometry to be >90 % after 6 days culture.
Freshly isolated NK cell preparation.
C57BL/6 spleens were harvested and single-cell suspensions were prepared in PBS containing 10 % FCS. NK cells were negatively selected from splenocyte suspensions using magnetic cell sorting (Miltenyi Biotech). The negatively selected NK cell population was subjected to cell sorting of CD49b+ CD3– to increase the purity of the NK cell population. Purity after cell sorting was determined to be >90 %.
Construction of the targeting plasmid pNCEVdrvT-NP-S-eGFP.
The left and right flanking sequences of the disrupted region V in the ECTV Moscow strain (ECTV-MOS) genome were PCR amplified using ECTV-MOS genomic DNA as the template and primers 5'-GCGGGCGCCGACTTGTTAGATAGAGATGAGATG-3' (NarI site underlined) and 5'-GCGAGATCTGCTAGCGGTACCGTCGACCACTATCTTACGCATCTCTAG-3' (BglII site underlined) for the left flank, and 5'-GCGGGATCCGCGGCCGCAAGCTTGGTAAACGGGTCATGTGACG-3' (BamHI site underlined) and 5'-GCGGAATTCGTAGCTATATAATGTATGGTAACATG-3' (EcoRI site underlined) for the right flank. The left and right flanks were digested with BglII and BamHI, respectively, and ligated together using T4 DNA ligase in the presence of BglII and BamHI to prevent self-ligation. The gel-purified fragment containing both left and right flanks was then digested with NarI and EcoRI, and cloned into pUCP7.5gpt-1 cut with the same enzymes (kindly provided by Dr Kangla Tsung, Washington University, MO, USA) to yield pNCEVdrvT. The plasmid pSC11-NP-SIINFEKL-eGFP (a gift from John Yewdell, NIH, MD, USA) was cut with XbaI and NotI. The fragment containing the NP–SIINFEKL–eGFP fusion gene (NP–S–eGFP, comprising the influenza nucleoprotein, SIINFEKL and enhanced green fluorescent protein sequences) under the control of the vaccinia virus (VACV) p7.5 promoter was gel purified and cloned into pNCEVdrvT digested with NheI and NotI to obtain the final targeting plasmid pNCEVdrvT-NP-S-eGFP. The sequences of the left and right flank and of the NP–S–eGFP expression cassette were verified by sequencing.
Generation of recombinant ECTV expressing NP–S–eGFP.
The NP–S–eGFP cassette was recombined into ECTV-MOS as described previously (Falkner & Moss, 1990). Briefly, CV-1 cells in a six-well plate (2.5x106 cells ml–1) were infected with ECTV-MOS and then transfected with 2 µg pNCEVdrvT-NP-S-eGFP using Lipofectamine 2000 (Invitrogen). After 48 h, cell lysates were prepared and recombinant isolates were subject to two rounds of plaque purification in the presence of mycophenolic acid followed by three rounds of plaque purification without mycophenolic acid. The genotypes of the final isolates were determined by PCR and expression of GFP was detected by fluorescence microscopy. One recombinant ECTV–eGFP was used in subsequent experiments.
Virus.
Plaque purified ECTV-MOS was propagated in murine L929 cells as described previously (Chen et al., 1992). Virus was purified through a sucrose cushion as described previously (Earl et al., 1998). In strain A mice, wild-type ECTV and the recombinant ECTV–eGFG had similar LD50 values of 0.02 and 0.04 p.f.u., respectively.
Infection of NK cells.
LAKs and naïve NK cells were cultured in 24-well plates at a concentration of 2.5x105 cells ml–1. Medium was removed by aspiration and cells were infected with wild-type ECTV or recombinant ECTV–GFP under the p7.5 early/late promoter at an m.o.i. of 5 in 0.1 ml. Virus was allowed to adsorb for 1 h at 37 °C, followed by the addition of 1–2 ml complete RPMI 1640.
Trypan blue exclusion.
Infected and uninfected cultures (1.5 ml containing 3.3x105 cells ml–1) were centrifuged and resuspended in 10 µl PBS, combined with 10 µl 0.4 % trypan blue and viewed in a haemocytometer. Cells were assessed in 16 squares and the number of cells excluding trypan blue was used to determine viability.
Single-cycle growth curves.
LAKs, naïve NK cells and BS-C-1 cells were cultured in 24-well plates at a concentration of 2.5x105 cells ml–1 and infected as described above with ECTV at an m.o.i. of 5. Virus growth was assessed at 1, 12, 24, 48 and 72 h post-infection (p.i.) by plaque assay on BS-C-1 cells. Virus titre was expressed as p.f.u. ml–1.
SDS-PAGE and Western blotting.
Proteins from supernatants and cell pellets (2.5x105 cells) were separated by SDS-PAGE in a 4–12 % NuPAGE Bis/Tris gel prior to electrophoretic transfer onto nitrocellulose filters as described previously (Bai et al., 2005). Briefly, filters were blocked for at least 1 h at room temperature in 0.1 % Tween 20/5 % non-fat dried milk in PBS followed sequentially by a 1 h incubation at room temperature with anti-E3 (diluted 1 : 1000), anti-ATI (1 : 5000), anti-IFNBP (1 : 10 000) or anti-EMICE (1 : 5000). Reactive proteins were detected with horseradish peroxidase-conjugated goat anti-rabbit IgG (H+L) (Kirkegaard & Perry Laboratories) using an ECL Plus protein identification system according to the manufacturer's instructions (Amersham Pharmacia Biotech). Total protein loaded onto gels varied less than twofold for all samples. Films were scanned into Adobe Photoshop using an Epson Perfection 4990 Photo Scanner.
Electron microscopy.
Virus-infected cells (1x106) were washed with PBS and fixed with 2 % glutaraldehyde in phosphate buffer (pH 7.2) overnight and then post-fixed in a 1 % OsO4 solution. Cells were dehydrated in a series of graded ethanol solutions and embedded in plastic. Sections were viewed with a JOEL 100CX transmission electron microscope.
NK1.1 cross-linking assay.
Twelve-well polystyrene plates were coated with mouse monoclonal antibody (mAb) PK136 as described previously (Kim et al., 2005). LAKs (1x106 cells ml–1) were infected with ECTV–eGFP at an m.o.i. of 1 for 24 h and incubated with PK136-coated plates for 1 h at 37 °C. Brefeldin A (0.12 % final concentration; BD Biosciences) was added and the plates were allowed to incubate for an additional 6–8 h. Cells were stained for NK1.1, CD3 and IFN- to determine the activation state of the infected NK cells.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Not only were more LAKs infected than naïve NK cells, but the mean fluorescence intensity was also nearly 1 log higher. Together, these results indicated that NK cells could be infected as a consequence of an experimental in vivo or in vitro infection, and that activated NK cells may be more permissive to ECTV infection than naïve NK cells.
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). The population of uninfected LAKs maintained 90 % viability and increased in concentration fourfold over the course of the experiment. In contrast, ECTV-infected LAKs did not increase in number and exhibited 
40 % viability at the termination of the experiment at 72 h p.i. These results revealed that infection with ECTV can decrease the viability of LAK cells.
ECTV affects IFN- synthesis by LAKs
To determine further the effect of ECTV infection on NK cell effector function, we assessed the ability of NK cells to produce IFN- after a 24 h ECTV infection. Uninfected and ECTV-infected LAK cells were stimulated to produce IFN- through binding of mAb PK136 to the NK1.1 activation receptor (Kim et al., 2002, 2005). For uninfected LAKs incubated with anti-NK1.1-coated plates for 8 h, 14.7±1.2 % produced IFN-, whilst for similarly treated LAKs from cultures pre-infected with ECTV for 24 h, 2.2±0.3 % of the GFP+ and 3.9±0.4 % of the GFP– LAKs produced IFN- (Fig. 3, P<0.001). Uninfected and ECTV-infected LAKs incubated on plates coated with no mAb or with an isotype control (anti-MAR18.5) demonstrated only 1.0–2.3 % NK1.1+ IFN-+ cells. These results revealed that infection of LAKs with ECTV significantly eliminated the ability of LAKs to be stimulated to produce IFN- (P<0.02), possibly by decreasing viability. The decrease in the percentage of infected NK cells producing IFN- was not due to the virus-encoded IFN-BP interfering with the IFN- assay, as similar results were obtained with an ECTV IFN-BP– mutant (data not shown). In addition, uninfected LAKs (GFP–) in the presence of ECTV-infected LAKs also displayed significantly reduced IFN- production (P<0.001).
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), IFN-BP and EMICE (Esteban & Buller, 2005). Late gene expression was followed through synthesis of a major late non-structural protein, ATI (A-type inclusion protein; Patel et al., 1986). BS-C-1 cells showed E3 expression as early as 4 h p.i. (data not shown) and this increased in intensity up to the 48 h p.i. time point. LAKs exhibited delayed expression of E3, but nowhere near the level of that seen with BS-C-1 cells (Fig. 5a). Naïve NK cells showed results similar to those seen with LAKs (data not shown). Expression of the immunomodulators EMICE and IFN-BP was also seen in LAKs. EMICE could be detected by 12 h p.i. in the supernatant of BS-C-1 cells and the cell pellet of LAKs (Fig. 5b). Although EMICE is a secreted protein, previous studies have shown its presence in cell pellets, which may indicate binding to the cell surface via a heparin motif (Liszewski et al., 2006). The IFN-BP was also expressed by both BS-C-1 cells and LAKs; however, the secreted immunomodulator was found in BS-C-1 cell supernatants earlier than in LAK supernatants (Fig. 5c). The apparent delay in secretion of IFN-BP in LAKs in not understood. The late viral protein ATI appeared at approximately 12 h p.i. in BS-C-1 cells. In both LAKs and naïve NK cells, ATI gene expression appeared to be decreased and delayed compared with that seen in BS-C-1 cells (Fig. 5d). Intracellular viral proteins E3 and ATI were detected in the culture supernatants from both cell types and may result from membrane permeability changes following infection, which may be cell-type specific. As both early and late viral proteins were expressed, but viable progeny were not produced, we next examined virion assembly.
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). In infected LAKs, we observed that 36.1±8.2 % of cells contained immature and/or mature virions by 24 h p.i. (Fig. 6). LAKs containing virions demonstrated significantly lower numbers of particles, with 6.1±0.9 immature particles and 8.1±1.8 mature particles per infected cell compared with BS-C-1 cells, which contained 28.9±4.5 and 35.1±7.1 immature and mature particles per infected cell, respectively (Fig. 6). The lower levels of viral proteins, the lack of infectious progeny following single-cycle replication conditions in naïve NK cells and LAKs, and the presence of fewer immature and intracellular mature virions following infection indicated that NK cells are not as fully permissive to ECTV replication as BS-C-1 cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, and kill virus-infected cells via the perforin/granzyme pathway. The importance of these cells during virus infections has become apparent in several systems. In the MCMV system, the genetic resistance of the C57BL/6 mouse is mediated through the NKC (NK cell gene complex) locus (Brown et al., 2001; Daniels et al., 2001; Scalzo et al., 1990). The importance of NK cells during viral infections is also demonstrated in the poxvirus system, where NK cells are crucial for early control and recovery from ECTV infection in C57BL/6 mice (Parker et al., 2007).
Another orthopoxvirus, VACV, readily infects human peripheral blood lymphocytes including NK cells, monocytes and B cells (Kirwan et al., 2006; Sanchez-Puig et al., 2004). Kirwan et al. (2006) demonstrated VACV infection of human NK cell lines, naïve peripheral blood NK cells and blood-derived LAKs by both direct infection and co-culture with infected cells. Here, we have shown that, like VACV, ECTV infects both resting and activated NK cells (Fig. 2). Assessment of late viral gene expression and progeny virus infectivity following infection revealed comparable results for VACV and ECTV. Both VACV and ECTV were capable of infecting and expressing late genes weakly, which did not lead to efficient production of infectious virus particles (Figs 4 and 5) (Kirwan et al., 2006). Consistent with a minimal increase in progeny, fewer immature and mature virion particles were detected in LAKs compared with BS-C-1 cells (Fig. 6). Studies of NK cell function following poxvirus infection also yielded analogous results. VACV infection of NK cells leads to decreased NK cell lytic activity, whilst ECTV infection resulted in decreased IFN- production following stimulation through the NK1.1 receptor (Fig. 3) (Kirwan et al., 2006). Our studies also demonstrated decreased viability of LAKs following ECTV infection; the effect of VACV on NK cell viability has not been reported in the literature.
Although NK cells have been tested for infectability with a number of non-poxviruses, most studies did not monitor the entire replication cycle. A recombinant Epstein–Barr virus expressing GFP under the control of the HCMV immediate-early promoter demonstrated reporter gene expression in human NK cell clones and peripheral blood NK cells, but infected cultures were not monitored for virus DNA synthesis, virion assembly and progeny virus infectivity. These infected NK cells underwent apoptosis as early as 72 h.p.i. (Isobe et al., 2004). In the case of human herpesvirus 6 (HHV-6), NK cell clones that do not kill HHV-6-infected peripheral blood mononuclear cells (PBMCs) are infectable and express an early/late viral phosphoprotein, whilst NK cells capable of killing HHV-6-infected PBMCs appear refractile to virus replication (Lusso et al., 1993). Clonal and polyclonal populations of NK cells from peripheral blood can be infected with human immunodeficiency virus type 1, as indicated by p24 antigen production, and show decreased viability similar to that seen with ECTV (Chehimi et al., 1991).
Other cell types have been shown to be semi-permissive for poxvirus infections. VACV infection of dendritic cells (DCs) and monocytes results in abortive infection, with only early virus gene expression (Drillien et al., 2000; Engelmayer et al., 1999; Jenne et al., 2000). Further studies have revealed that VACV infection of immature DCs blocks maturation, whilst infection of mature DCs impairs antigen-presenting abilities, suggesting yet another means of dampening the immune response (Engelmayer et al., 1999; Jenne et al., 2000). Semi-permissive poxvirus infections of DCs, monocytes and NK cells all appear to express early viral genes. In agreement with these studies, we detected synthesis of early proteins, including the immunomodulatory proteins EMICE and IFN-BP, which may skew the cytokine milieu in the surrounding microenvironment. A possible demonstration of this principle may be observed in the experiment in which infected LAKs failed to produce IFN- upon stimulation and a significant proportion of the uninfected LAKs within the same culture failed to synthesize IFN-, possibly through virus immunomodulation (Fig. 3).
Studies have shown the ability of NK cells to kill VACV-infected cells (Baraz et al., 1999; Chisholm & Reyburn, 2006), as well as the importance of the cytokine IFN- in recovery from ECTV infection (Chaudhri et al., 2004). Diminution of NK cell antiviral activities and disruption of the innate immune response by infection may be advantageous to the virus, particularly for a virus that, unlike poxviruses, does not produce a large range of immunomodulators. For poxviruses, the ability to rapidly transcribe early genes, a number of which have immunomodulatory properties, may provide an additional mechanism to modify the local environment through semi-permissive infection of NK cells.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Chaudhri, G., Panchanathan, V., Buller, R. M., van den Eertwegh, A. J., Claassen, E., Zhou, J., de Chazal, R., Laman, J. D. & Karupiah, G. (2004). Polarized type 1 cytokine response and cell-mediated immunity determine genetic resistance to mousepox. Proc Natl Acad Sci U S A 101, 9057–9062.
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Received 1 June 2007;
accepted 6 November 2007.
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) or infected with ECTV (
) for 24, 48 or 72 h. Viability was determined by trypan blue exclusion. The total number of viable cells (c) and the percentage of viable cells (d) were measured over time. Experiments were repeated four times and the results are shown as means±SEM.
, naïve NK; 
