Murine Gammaherpesvirus 68 Pathogenesis Is Independent of Caspase-1 and Caspase-11 in Mice and Impairs Interleukin-1β Production upon Extrinsic Stimulation in Culture

ABSTRACT

Gammaherpesviruses establish lifelong infections that are associated with the development of cancer. These viruses subvert many aspects of the innate and adaptive immune response of the host. The inflammasome, a macromolecular protein complex that controls inflammatory responses to intracellular danger signals generated by pathogens, is both activated and subverted during human gammaherpesvirus infection in culture. The impact of the inflammasome response on gammaherpesvirus replication and latency in vivo is not known. Caspase-1 is the inflammasome effector protease that cleaves the proinflammatory cytokines interleukin-1β (IL-1β) and IL-18. We infected caspase-1-deficient mice with murine gammaherpesvirus 68 (MHV68) and observed no impact on acute replication in the lung or latency and reactivation from latency in the spleen. This led us to examine the effect of viral infection on inflammasome responses in bone marrow-derived macrophages. We determined that infection of macrophages with MHV68 led to a robust interferon response but failed to activate caspase-1 or induce the secretion of IL-1β. In addition, MHV68 infection led to a reduction in IL-1β production after extrinsic lipopolysaccharide stimulation or upon coinfection with Salmonella enterica serovar Typhimurium. Interestingly, this impairment occurred at the proIL-1β transcript level and was independent of the RTA, the viral lytic replication and transcription activator. Taken together, MHV68 impairs the inflammasome response by inhibiting IL-1β production during the initial stages of infection.

IMPORTANCE Gammaherpesviruses persist for the lifetime of the host. To accomplish this, they must evade recognition and clearance by the immune system. The inflammasome consists of proteins that detect foreign molecules in the cell and respond by secreting proinflammatory signaling proteins that recruit immune cells to clear the infection. Unexpectedly, we found that murine gammaherpesvirus pathogenesis was not enhanced in mice lacking caspase-1, a critical inflammasome component. This led us to investigate whether the virus actively impairs the inflammasome response. We found that the inflammasome was not activated upon macrophage cell infection with murine gammaherpesvirus 68. Infection also prevented the host cell inflammasome response to other pathogen-associated molecular patterns, indicated by reduced production of the proinflammatory cytokine IL-1β upon bacterial coinfection. Taken together, murine gammaherpesvirus impairment of the inflammatory cytokine IL-1β in macrophages identifies one mechanism by which the virus may inhibit caspase-1-dependent immune responses in the infected animal.

INTRODUCTION

Gammaherpesviruses establish lifelong latent infections that are associated with significant morbidity and the development of malignancies (1–3). Gammaherpesviruses initially infect the mucosal tissues of naive hosts and undergo productive replication prior to colonization of latency reservoirs, including B lymphocytes and macrophages (4–6). Murine gammaherpesvirus 68 (MHV68) is a natural pathogen of murid rodents that shares genetic and pathological similarities to the human gammaherpesviruses (7, 8). The analysis of MHV68 in the context of a mouse infection enables the investigation of the dynamic interplay between the virus and the innate and adaptive immune responses of the host (9, 10).

The inflammasome is a critical mediator of inflammatory responses that restricts the pathogenesis of numerous viruses and bacteria (11). Pathogen-associated molecular patterns (PAMPs) are detected by multiple cellular sensors, including Toll-like receptors (TLRs), NOD-like receptors (NLRs), and other RNA and DNA sensors. Triggering of this comprehensive surveillance system leads to upregulation of inflammasome components and induction of the rapid formation of a multiprotein complex containing procaspase-1. Upon procaspase-1 cleavage, the inflammatory cytokines inteleukin-1β (IL-1β) and IL-18 are processed and secreted (11–13). IL-1β is a critical proinflammatory cytokine that mediates macrophage and neutrophil recruitment to the site of infection (14, 15). IL-18 induces robust gamma interferon (IFN-γ) expression from natural killer (NK) and T cells, promoting a Th1 proinflammatory response (15).

Inflammasome signaling serves as an important restriction factor against herpesviruses in vivo. In the absence of IL-1β, herpes simplex virus 1 (HSV-1) infection of mice rapidly advances to lethal encephalitis (16). Similarly, the removal of AIM2, a cytosolic DNA sensor that activates the inflammasome in response to murine cytomegalovirus (MCMV), leads to an impaired NK cell response and an increase in virus titers during infection (17). The loss of the inflammasome components NLRP3 or ASC, but not the AIM2 sensor, results in an increase of MHV68 genomes in the spleen (18). Thus, the inflammasome response is a critical innate defense with the potential to activate immune cells to control herpesvirus replication.

Recently, herpesviruses, including the oncogenic gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8), were found to be sensed by the host DNA sensor IFI16, leading to inflammasome complex formation (19–21). However, like many other viral and bacterial pathogens, herpesviruses use multiple strategies to inhibit the inflammasome upon infection. HSV-1 rapidly induces IFI16 degradation upon expression of the immediate-early gene ICP0 (22). EBV encodes a miRNA that reduces transcription of the NLRP3 sensor protein (23). The KSHV open reading frame 63 (ORF63) tegument protein impairs both the NLRP1- and the NLRP3-mediated inflammasomes (24), while the MHV68 ORF64 deubiquinase impairs sensing that reduces type I IFN and inflammasome responses upon infection of dendritic cells (18). Gammaherpesviruses activate and inhibit inflammasome responses in culture (19–21, 23, 24), but the influence of caspase-1-dependent inflammatory responses on pathogenesis in vivo is unclear.

We assessed the impact of caspase-1-dependent inflammasome activation on murine gammaherpesvirus pathogenesis. The absence of caspase-1 had no effect on viral replication in cell culture or in the lungs of mice after intranasal infection. Establishment of latency and reactivation from latency in the spleen were not altered. The lack of a phenotype in vivo led us to examine whether MHV68 might counteract inflammasome responses. MHV68 did not induce inflammasome activation upon infection of primary bone marrow-derived macrophages (BMDMs). Furthermore, MHV68 inhibited IL-1β production in response to extrinsic lipopolysaccharide (LPS) stimulation and upon coinfection with Salmonella enterica serovar Typhimurium. We determined that the repression of IL-1β occurred at the transcript level and was mediated by the tegument or a replication and transcription activator (RTA)-independent gene product during the early stages of productive infection. MHV68 subversion of the inflammatory cytokine IL-1β is consistent with the lack of any change in the course of infection in mice lacking caspase-1 and caspase-11.

MATERIALS AND METHODS

Mice.

Mice bearing germ line caspase-1 and caspase-11 deletions on a C57BL/6 background (12, 25) were bred in our facility. Age- and sex-matched C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and maintained in our facility. We used 8- to 12-week-old animals of mixed genders in groups of three to seven for most experiments. Isoflurane was used for anesthesia to conduct infections and terminal harvests. The animal experiments described here were performed according to a protocol approved by the Stony Brook University Institutional Animal Care and Use Committee. IFNAR1^−/− mice (C57BL/6 background) were kindly provided by Mitchell Grayson and maintained at the Medical College of Wisconsin (26).

Cell culture.

Low-passage murine embryonic fibroblasts (MEFs) were cultured at 37°C with 5% CO₂ in Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 2 μM l-glutamine, 50 U of penicillin/ml, and 50 μg of streptomycin/ml (10% cMEM). National Institutes of Health (NIH) murine 3T12 fibroblasts were cultured at 37°C with 5% CO₂ in DMEM containing 8% FBS, 2 μM l-glutamine, 50 U of penicillin/ml, and 50 μg of streptomycin/ml (8% cMEM). To generate bone marrow-derived macrophages (BMDMs), bone marrow was flushed from the mouse femur and differentiated for 5 days in DMEM with GlutaMAX (Life Technologies, Grand Island, NY) containing 20% FBS and 30% L-929 conditioned medium (BMM-Hi) in low-binding dishes. Cells were then seeded in DMEM with GlutaMAX containing 10% FBS and 15% L-929 conditioned medium (BMM-Low) in tissue culture-treated plates for experiments.

Virus and infections.

For in vivo infection experiments with murine MHV68, we used the murid herpesvirus 4 WUMS strain (ATCC VR1465) that was propagated as previously described (27). Unless otherwise indicated, MHV68-eYFP (28) or MHV68-H2bYFP (29) was used for cell culture infections. Virus stocks were concentrated to >10⁸ PFU/ml by centrifugation at 4°C for 2 h at 13,000 × g in a Sorvall (Thermo Scientific) GSA rotor. For intranasal infection, mice were lightly anesthetized using isoflurane and infected with 1,000 PFU of virus in a 20-μl bolus of 10% cMEM applied to the nose. For intraperitoneal infections, mice were lightly anesthetized using isoflurane and injected with 1,000 PFU of virus in 500 μl of 10% cMEM. Back-titers of inoculate were performed to confirm infectious dose.

MHV68-ORF50stop was produced as previously described (30) with the following modifications. ORF50 cDNA was cloned into pMSCV-puro (Clontech, Mountain View, CA). RTA-encoding retroviruses were packaged by transfecting BOSC23 cells with pMSCV-puro-RTA, and NIH 3T12 fibroblasts were transduced with the resulting virus. Stable cell lines were selected using puromycin in the culture media at a final concentration of 5 μg/ml. RTA-expressing cell lines or empty vector control cell lines were transfected with ORF50stop bacterial artificial chromosome (BAC) or wild-type (WT) BAC. Transfected cells were observed for cytopathic effect to ensure that WT contaminant viruses were not present in empty vector control cultures, and viral stocks were generated according to standard MHV68 protocols (27). WT MHV68 and ORF50stop P2 stocks were concentrated to >10⁸ PFU/ml by centrifugation at 4°C for 90 min at 35,000 × g in a Beckman-Coulter (Brea, CA) JA-20 rotor. Concentrated ORF50stop MHV68 viral stocks were re-evaluated by plaque assay on empty vector control cell lines to confirm that WT contaminants were not present in stocks used for experiments.

Flow cytometry.

For analysis of immune cell responses in mouse tissues, the spleens and lungs from infected mice were homogenized into single cell suspensions. Lungs were digested in 3 ml of 10% DMEM with 150 U of collagenase/ml and 10 U of DNase I/ml at 37°C. Then, 2 × 10⁶ cells per sample were resuspended in 200 μl of phosphate-buffered saline (PBS) plus 2% FBS (for fluorescence-activated cell sorting [FACS]). Cells were blocked with TruStain fcX (clone 93; BioLegend, San Diego, CA), washed, and stained to detect the following markers for T cells (CD3, clone 145-2C11; CD4, clone GK1.5; CD8, clone 53-6.7; CD62L, clone MEL-14; CD44, clone IM7; Vβ4, clone KT4) and B cells (CD19, clone 6D5; CD69, clone H1.2F3; CD95, clone Jo2; GL7, clone GL7). All conjugated antibodies were purchased from BioLegend except antibodies against CD95 and Vβ4 (BD Pharmingen, San Jose, CA) and anti-GL7 (eBioscience, San Diego, CA). The p79 tetramer (TSINFVKI/H-2K^b) was kindly provided by the NIH tetramer facility. Cells were analyzed using a FACSCalibur (BD Biosciences) or Dxp8 FACScan (BD Biosciences/Cytek Development), and data were analyzed using FlowJo vX (Tree Star, Ashland, OR).

Viral pathogenesis assays.

For acute titers, mice were sacrificed with isoflurane at the indicated days postinfection, and the left lung was removed and frozen at −80°C. Lungs were disrupted in 1 ml of 8% cMEM using 1-mm zirconia beads in a bead beater (Biospec, Bartlesville, OK). Serial dilutions in 10% cMEM were plated on subconfluent monolayers of NIH 3T12 murine fibroblasts in six-well plates. Plates were rocked intermittently for 1 h and then overlaid with 3 ml of 5% cMEM plus 1.5% methylcellulose. The cells were fixed after 1 week and stained with crystal violet, and the plaques were counted.

To analyze latently infected cells, mice were sacrificed with isoflurane at 16 or 42 days postinfection (dpi). Spleens were excised, homogenized, and resuspended in 10% cMEM. Peritoneal exudate cells were isolated by peritoneal injection of 10 ml of 10% cMEM, followed by agitation of the abdomen and withdrawal of the peritoneal wash by syringe.

For quantitation of latency, limiting-dilution nested PCR with primers for the MHV68 orf50 genomic region was used to determine the frequency of virally infected cells as previously described (31). Briefly, frozen samples were thawed, resuspended in isotonic buffer, counted, and plated in serial 3-fold dilutions in a background of 10⁴ NIH 3T12 murine fibroblasts into a 96-well plate. The resultant PCR products were resolved on 2% agarose gels, and each dilution was scored for amplimers of the expected sizes. Control wells containing uninfected cells or 10, 1, and 0.1 plasmid copies of ORF50 target sequence were run with each plate to ensure single-copy sensitivity and no false positives.

For quantitation of reactivation, a limiting-dilution reactivation assay was performed as previously described (31). Briefly, bulk splenocytes in 10% cMEM were plated in serial 2-fold dilutions (starting with 10⁵ cells) onto MEF monolayers in each well of a 96-well tissue culture plate. Twelve dilutions were plated per sample, and 24 wells were plated per dilution. Wells were scored for cytopathic effect at 14 and 21 days after plating. To detect preformed infectious virus, parallel samples of mechanically disrupted cells were plated onto MEF monolayers.

BMDM cell infection and stimulation.

Differentiated BMDMs were seeded onto cell culture-treated dishes. Subconfluent cells were infected with MHV68-H2bYFP, MHV68-eYFP, or MHV68-ORF50stop at a multiplicity of infection (MOI) of 10 for 90 min with intermittent rocking. At 6 or 22 h postinfection (hpi), the medium was replaced with fresh BMM-Low plus 100 ng of LPS/ml. After 2 h, the medium was replaced with fresh BMM-Low plus 100 ng of LPS/ml plus 5 mM ATP and then incubated for 1 h. The supernatant was removed, and then cells were lysed in 500 μl of fresh BMM-Low by freezing. Samples were stored at −80°C. Triplicate wells were examined per experiment.

DNA stimulation.

Subconfluent BMDMs were incubated in BMM-Low media containing 0.5 μM CpG DNA (ODN 1826; Invivogen, San Diego, CA) for 16 h. After incubation, tumor necrosis factor alpha (TNF-α) secretion was measured by enzyme-linked immunosorbent assay (ELISA). For detection of DNA-induced inflammasome activation, subconfluent WT BMDMs were prestimulated for 4 h with 100 ng of LPS/ml. After prestimulation, 1 μg of poly(dA-dT) (Invivogen) was transfected into cells using TransIT LT-1 (Mirus, Madison, WI). After 6 h, IL-1β secretion was measured by ELISA.

Bacterial infection.

Salmonella enterica serovar Typhimurium strain ATCC 14028 was used as the wild-type strain. Using standard microbiological techniques, bacteria were grown aerobically overnight at 37°C in Luria-Bertani (LB) broth. Bacteria were then subcultured at 1:20 in LB broth for 3 h to reach late-log phase growth. To infect BMDM, WT S. Typhimurium was inoculated at an MOI of 1 or 4 CFU/cell. Infected cells were spun at 1,000 rpm for 5 min, followed by incubation at 37°C for 2 h. To measure invasion, BMDMs were washed using PBS three times in order to remove unattached bacteria, followed by lysing with Triton X-100 (Sigma-Aldrich). Cell lysates were diluted in PBS, plated on LB agar plates, and colonies (i.e., CFU) were counted after overnight incubation at 37°C.

ELISA and cell death assay.

IL-1β and TNF-α were quantified by using a mouse Duoset IL-1β or a TNF-α ELISA kit (R&D Systems, Minneapolis, MN). IL-18 was quantified using a mouse IL-18 ELISA kit (MBL International, Japan). Plates were prepared and assayed according to the manufacturer's protocol, and signals were read at 450 nm with the background subtracted. Lactate dehydrogenase (LDH) release into the conditioned medium was measured using a CytoTox 96 nonradioactive cytotoxicity assay according to the manufacturer's instructions (Promega, Madison, WI).

Immunoblotting.

Infected BMDMs were lysed in radioimmunoprecipitation assay buffer for 20 min. Samples were quantified by Bradford assay (Bio-Rad, Berkeley, CA), and the protein was diluted in NuPAGE LDS sample buffer (Invitrogen) with reducing agent (Invitrogen) before boiling at 95°C for 5 min. Samples were separated by using NuPAGE Bis-TRis minigels (Invitrogen), and transferred to Immobilon-P membranes (Millipore, Darmstadt, Germany). Primary antibodies against caspase-1 p10 (Santa Cruz Biotechnology, Dallas, TX), IL-1β (R&D Systems), IL-18 (BioVision, Milpitas, CA), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Sigma, St. Louis, MO) were used. Rabbit anti-LANA antibody was kindly provided by Scott Tibbetts. Affinity-purified chicken anti-ORF75c antibody (Gallus Immunotech, Cary, NC) was previously described (32). Detection was performed with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG or anti-mouse IgG (GE Healthcare, Buckinghamshire, United Kingdom), HRP-conjugated streptavidin IgG (Rockland, Gilbertsville, PA), or HRP-conjugated goat anti-chicken (Gallus Immunotech). The data were captured by using a GE charge-coupled device camera and analyzed by ImageQuant software (v7.0; GE Healthcare).

Quantitative reverse transcription-PCR.

BMDMs were lysed in RLT buffer (Qiagen, Hilden, Germany) supplemented with 1% β-mercaptoethanol (BME) and stored at −80°C before RNA extraction. Total RNA was extracted using a Qiagen RNeasy minikit according to the manufacturer's instruction. RNA concentration and quality was determined by measuring the absorbance at 260 and 280 nm. RNA was DNase treated (Ambion Turbo DNA-Free kit), RNA was reverse transcribed into cDNA using SuperScript III (Invitrogen), and the cDNA was subjected to quantitative PCR using ABsolute Blue QPCR SYBR with Low ROX (Thermo Scientific) in an ABI 7500 real-time PCR system (Applied Biosystems) according to the manufacturer's instructions. PCR conditions were 95°C for 15 min, followed by 40 cycles of 95°C for 15 s and 60°C for 30 s. Primer sets were purchased from Eurofins Genomics (Huntsville, AL) as follows: IL-1β, For (5′-TCTTTGAAGTTGACGGACCC-3′) and Rev (5′-TGAGTGATACTGCCTGCCTG-3′) (33); IL-18, For (5′-CAGGCCTGACATCTTCTGCAA-3′) and Rev (5′-CTGACATGGCAGCCATTGT-3′) (34); TNF-α, For (5′-GCCTCTTCTCATTCCTGCTTGT-3′) and Rev (5′-GATGATCTGAGTGTGAGGGTCTG-3′) (35), caspase-1, For (5′-AGATGGCACATTTCCAGGAC-3′) and Rev (5′-GATCCTCCAGCAGCAACTTC-3′) (33); and HPRT (hypoxanthine phosphoribosyltransferase), For (5′-GTAATGATCAGTCAACGGGGGAC-3′) and Rev (5′-CAGCAAGCTTGCAACCTTAACCA-3′) (36). Relative transcript levels were calculated as E = 2^−ΔΔCT, where E is the gene expression value and ΔΔC_T is the difference between HPRT and target genes of the experimental group over the mock treatment group.

Statistical analyses.

All data were analyzed using Prism software (GraphPad, La Jolla, CA). Titer data were statistically analyzed with a two-tailed Student t test. Based on the Poisson distribution, frequencies of reactivation and viral genome-positive cells were obtained from the nonlinear regression fit of the data where the regression line intersected 63.2%. The frequencies of reactivation and genome-positive cells were statistically analyzed using an unpaired two-tailed Student t test.

RESULTS

Lytic replication and early pathogenesis of MHV68 in vivo is not affected by the loss of caspase-1 and caspase-11.

To examine the role of inflammatory processes dependent on caspase-1 during gammaherpesvirus pathogenesis, we examined MVH68 replication, latency, and reactivation from latency in the cells and tissues of mice lacking caspase-1 (12). These caspase-1-deficient mice also lack caspase-11 and are referred to here as Caspase-1/11^−/− mice (25). Caspase-1 is the critical effector protease for IL-1β and IL-18 (37). Caspase-11 controls a noncanonical inflammasome pathway that is activated by LPS from Gram-negative bacteria (25, 38). BMDMs were prepared from Caspase-1/11^−/− and WT mice. Cells were pretreated with LPS for 2 h and then stimulated with LPS and ATP for 1 h to activate the inflammasome. As expected, Caspase-1/11^−/− BMDMs failed to secrete IL-1β in response to inflammasome activation (Fig. 1A). TNF-α, an inflammasome-independent cytokine, was released from both Caspase-1/11^−/− and WT BMDMs (Fig. 1A). Caspase-1 has been reported to promote EBV replication (39). We assessed the growth of MHV68 in cells that lacked caspase-1. WT and Caspase-1/11^−/− primary murine BMDMs were infected at a low multiplicity of infection (MOI of 0.1), and replication was examined over the course of a multistep growth curve. There was no difference in the kinetics of replication in Caspase-1/11^−/− and WT BMDMs (Fig. 1B). Virus replication was also comparable between Caspase-1/11^−/− and WT MEFs upon infection at an MOI of 0.01 (Fig. 1C), indicating that caspase-1 and caspase-11 are not required to support viral replication. Next, Caspase-1/11^−/− and WT mice were infected with 1,000 PFU of MHV68 via the intranasal route, and viral replication in the lungs was measured over a 12-day time course. No differences were observed in replication kinetics or peak virus titers in the lungs of Caspase-1/11^−/− mice compared to WT mice (Fig. 1D). In addition, the acute phase of infection was cleared with normal kinetics in Caspase-1/11^−/− mice, with virus titers dropping below the limit of detection by 12 dpi.

FIG 1.

Loss of caspase-1 and caspase-11 does not impact MHV68 replication. (A) WT and Caspase-1/11^−/− BMDMs were treated for 2 h with LPS, followed by 1 h with both LPS and ATP. Cytokine release was measured by ELISA. Dark bars represent WT; light bars represent Caspase-1/11^−/−. (B) Multistep growth curve in WT and Caspase-1/11^−/− BMDMs upon infection with MHV68-eYFP at a low MOI (0.1 PFU/cell). (C) Multistep growth curve in WT and Caspase-1/11^−/− MEFs upon infection with MHV68-eYFP at a low MOI (0.01 PFU/cell). At the indicated times postinfection, the virus titer was assayed by plaque assay. Symbols represent means ± the standard deviations (SD) of triplicate samples. (D) C57BL/6 WT or Caspase-1/11^−/− mice were infected with 1,000 PFU of WT MHV68 via the intranasal route. At the indicated days postinfection, lungs were removed and homogenized, and virus titers were determined by plaque assay. Symbols represent individual mice, and lines represent the mean titer; the dashed line indicates the limit of detection (50 PFU/ml).

Long-term latency of MHV68 is unaffected by the absence of caspase-1 and caspase-11.

Following acute lytic replication in the respiratory tract after an intranasal infection, MHV68 establishes latency in B cells and macrophages of secondary lymphoid tissues (4, 27, 40). Spleens from infected mice were harvested 16 dpi, and levels of viral latency were determined using a limiting-dilution, nested PCR assay to enumerate the frequency of splenocytes that harbor viral DNA. We found that the absence of caspase-1 and caspase-11 had no effect on the establishment of latency in the spleen (Fig. 2A). Further, a caspase-1 and caspase-11 deficiency did not impact reactivation from latency in intact cells (Fig. 2B, solid lines) or the low levels of productive replication detected in disrupted cells (Fig. 2B, dotted lines) as assessed using a limiting-dilution explant reactivation assay. We evaluated long-term latency at 42 dpi and found no significant change in the maintenance of latency in Caspase-1/11^−/− splenocytes (Fig. 2C). After intraperitoneal infection, the absence of caspase-1 and caspase-11 did not alter latency (Fig. 2D) or reactivation (Fig. 2E) from peritoneal exudate cells, a compartment rich in the macrophage reservoir of latency (4, 27). These data indicate that MHV68 pathogenesis was not enhanced in the absence of caspase-1 and caspase-11 during the course of lytic and latent infection in vivo.

FIG 2.

Loss of caspase-1 and caspase-11 does not impact latency and reactivation of MHV68 in mice. C57BL/6 WT or Caspase-1/11^−/− mice were infected with 1,000 PFU of WT MHV68 via the intranasal (A to C) or intraperitoneal (D and E) route. (A) Latency was measured at 16 dpi, as indicated by the frequency of intact splenocytes that harbor the virus genome using a limiting-dilution nested-PCR assay. (B) Reactivation from latency was measured using a limiting-dilution explant reactivation coculture assay. Dotted lines represent disrupted splenocytes used to measure preformed infectious virus. (C) Latency analysis of splenocytes at 42 dpi. (D) Latency analysis of peritoneal exudate cells at 16 dpi. (E) Reactivation from latency in the peritoneal exudate cells at 16 dpi. Dotted lines represent disrupted peritoneal exudate cells used to measure preformed infectious virus. The intersection of the nonlinear regression curves with the dashed line at 63.2% was used to determine the frequency of cells that were either positive for the viral genome or reactivating virus. Graphs represent two to three independent experiments using three to five mice.

The adaptive immune response to MHV68 infection is not altered by the absence of caspase-1 and caspase-11.

Detection of invading pathogens by cells and the subsequent release of chemokines and cytokines recruits numerous immune cells to the site of infection. IL-1β and IL-18 are critical activators of inflammatory responses by macrophages and NK cells, respectively, and promote the clearance of many bacterial and viral pathogens (41–43). Given that the absence of caspase-1 and caspase-11 prevents the secretion of IL-1β and IL-18, we next examined the immune response in the lungs and spleen at various times postinfection. We measured the level of CD8⁺ T cells specific to the p79 epitope of ORF61, a dominant antigen during MHV68 infection (44), in the lungs at 10 dpi and found that WT and Caspase-1/11^−/− mice had comparable frequencies (Table 1) and numbers (data not shown) of virus-specific CD8⁺ T cells.

TABLE 1.

Immune response to MHV68 infection of Caspase-1/11^−/− and WT mice

Cell typea	% mean frequency (SD)b
	Caspase-1/11−/− mice		WT mice
	Naive	Infected	Naive	Infected
Lung, 10 dpi
p79+ CD8+ T cells*	1.1 (0.2)	4.1 (2.0)	1.2 (0.9)	3.5 (1.1)
Spleen, 16 dpi
CD19+ B cells	40.5 (10.5)	43.8 (7.2)	46.7 (8.6)	46.3 (9.8)
Activated B cells†	1.7 (0.3)	4.5 (1.8)	1.6 (0.2)	6.1 (1.9)
CD4+ T cell	18.6 (1.1)	11.9 (1.6)	15.9 (3.5)	11.0 (1.6)
Effector CD4+ T cells‡	14.5 (0.6)	23.0 (3.3)	16.5 (1.7)	26.3 (5.8)
CD8+ T cell	13.6 (0.3)	14.7 (2.0)	12.5 (1.2)	13.4 (3.1)
Effector CD8+ T cells‡	8.0 (1.0)	33.3 (6.7)	7.5 (1.3)	30.9 (8.2)
p79+ CD8+ T cells	0.38 (0.1)	8.4 (3.4)	0.74 (0.3)	3.9 (1.9)
Spleen, 42 dpi
CD19+ B cells	41.5 (0.6)	38.7 (5.2)	42.6 (3.3)	38.2 (1.7)
Activated B cells	1.8 (0.5)	1.0 (0.2)	1.3 (0.1)	0.91 (0.1)
CD4+ T cells	17.5 (1.1)	17.8 (2.4)	18.5 (1.4)	16.4 (2.3)
Effector CD4+ T cells	17.8 (2.4)	17.2 (2.4)	15.4 (1.7)	16.4 (6.4)
CD8+ T cells	13.9 (1.8)	22.4 (2.8)	15.2 (1.9)	19.3 (5.6)
Effector CD8+ T cells	7.1 (0.5)	43.2 (6.3)	9.0 (1.3)	41 (5.8)
p79+ CD8+ T cells	0.28 (0.1)	2.7 (0.7)	0.36 (0.1)	2.3 (1.1)
Vβ4+ CD8+ T cells§	7.7 (2.5)	33.8 (9.4)	6.0 (0.4)	30.5 (10.2)

^a*, p79 tetramer CD8⁺ T cell surface markers: CD4, CD8, CD62L, and p79 tetramer; †, activated B cell surface markers: CD19 and CD69; ‡, effector T cell surface markers: CD4, CD8, CD62L, and CD44; §, Vβ4 T cell surface markers: CD3, CD8, CD62L, and Vβ4.

^bThe data shown are the mean percentages and standard deviations of values obtained by FACS analysis of individual infected mice. Three to five mice in one or two experiments were analyzed per time point. No significant differences were observed for any population between Caspase-1/11^−/− mice and WT mice infected with MHV68.

Examination of B and T cell populations in the spleen at 16 dpi revealed no changes in the total number (data not shown) or the proportion of B and T cells and their activation status (Table 1). CD4⁺ and CD8⁺ T cells were recruited and activated to the same extent, and virus-specific p79 CD8⁺ T cells were efficiently expanded in Caspase-1/11^−/− mice. T cells expressing Vβ4 T-cell receptors, a hallmark of MHV68 infection of C57BL/6 mice (45, 46), were enriched in Caspase-1/11^−/− mice to comparable levels in WT mice. These data demonstrate that the absence of caspase-1 and caspase-11 does not influence the adaptive immune response against MHV68, a finding consistent with our data indicating that the deletion of caspase-1 and caspase-11 does not enhance MHV68 pathogenesis (Fig. 2). Given the importance of caspase-1 and caspase-11 in mediating innate immune responses to other intracellular pathogens (17, 41–43), we hypothesized that WT MHV68 is refractory to caspase-1- and caspase-11-dependendent inflammasome functions in vivo due to a subversion of either the detection or the effector responses of the host.

MHV68 infection impairs inflammatory cytokine responses to extrinsic stimulation.

Given our findings that caspase-1 and caspase-11 do not contribute to the immune response to MHV68 in vivo, we next investigated the inflammasome response to MHV68 infection in primary WT BMDMs. Macrophages express a wide range of innate immune sensors, making them ideal for dissecting viral interactions with intrinsic and innate host defenses. Inflammasome activation upon infection with MHV68-H2b-YFP was assessed by procaspase-1 cleavage and IL-1β release over a 24-h time course of infection (MOI of 10). Despite infection levels reaching ca. 40% of cells (data not shown), procaspase-1 cleavage was not observed by Western blotting at any time point (Fig. 3A). The secretion of IL-1β was also not detected in the conditioned media of infected BMDMs, as determined by ELISA (Fig. 3A). The lack of response to infection contrasted sharply with robust caspase-1 cleavage and IL-1β release upon stimulation with LPS and ATP, which are classic stimuli of the NLRP3 inflammasome in BMDMs (Fig. 3A). We confirmed that BMDMs responded to the foreign DNA ligands CpG and poly(dA-dT) (Fig. 3B) that bind the TLR9 and AIM2 sensors, respectively, that have been previously shown to detect herpesviruses (17, 47, 48).

FIG 3.

MHV68 impairs inflammatory response in primary macrophage cells. (A) WT C57BL/6 BMDMs were infected with WT MHV68 at an MOI of 10. For each panel, the procaspase-1 and caspase-1 p10 levels were detected by immunoblotting, and secreted IL-1β was measured by ELISA. BMDMs treated for 2 h with LPS, followed by 1 h with both LPS and ATP, served as a positive control for caspase-1 cleavage and IL-1β secretion (P < 0.0001 for LPS and ATP compared to an untreated mock control). GAPDH was used as a loading control. (B) WT BMDMs were treated with CpG DNA for 16 h, and TNF-α secretion was measured by ELISA. For the detection of DNA-induced inflammasome activation, WT BMDMs were treated for 4 h with LPS, followed by transfection with poly(dA-dT) for 6 h, and then IL-1β secretion was measured by ELISA. (C) An infection and stimulation schematic for panels D to F describes infection of WT C57BL/6 BMDMs at an MOI of 10 for 22 h, followed by 2 h of LPS and an additional 1 h with LPS alone or LPS plus ATP. (D) Proform and mature cleaved forms of the indicated proteins at 25 hpi were detected by immunoblotting and normalized to the GAPDH loading control. Values are the protein levels relative to the uninfected cultures treated with LPS and ATP. (E) The levels of the secreted cytokines were determined by ELISA. (F) Cell death at 25 hpi was monitored by LDH release into conditioned medium relative to the freeze-thawed samples. Bars represent means ± the SD of triplicate samples. The dashed line represents the limit of detection. BLD, below limit of detection. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Having found that de novo infection of BMDMs with MHV68 did not trigger an inflammasome response, we sought to determine whether MHV68 infection hindered inflammasome activation by extrinsic stimuli. Inflammasome function is dependent on the activation of two distinct pathways (49, 50). A first signal is typically TLR activation by PAMPs such as LPS to induce the production of proforms of IL-1β and IL-18. The second signal is the detection of conserved pathogen- or damage-associated molecular patterns, such as ATP, by sensors such as NALP1, NLRP3, AIM2, or IFI16. This drives the assembly of the multiprotein inflammasome complex to mediate the cleavage of procaspase-1 and subsequent processing of proinflammatory cytokines IL-1β and IL-18 (51, 52).

To determine whether MHV68 infection impaired procaspase-1 or pro-IL-1β cleavage, intracellular levels of IL-1β and caspase-1 were examined in infected BMDMs treated with LPS alone (signal 1 via TLR4) or primed with LPS (signal 1), followed by ATP (signal 2 via NLRP3) (Fig. 3C). Although the levels of both proforms and mature forms of IL-1β were reduced in infected macrophages, the levels of cleaved caspase-1 were similar between infected and uninfected cells stimulated with LPS and ATP at 25 hpi (Fig. 3D). In agreement with the reduction in intracellular IL-1β protein levels, infection with MHV68 significantly reduced IL-1β secretion upon extrinsic stimulation with LPS and ATP treatment (Fig. 3E, left). We next examined IL-18, a cytokine that is also dependent on caspase-1 cleavage for secretion. IL-18 secretion in response to LPS and ATP was also reduced by MHV68 infection (Fig. 3E, middle). TNF-α is an NF-κB-responsive cytokine that is not dependent on inflammasome activation for secretion. The levels of TNF-α produced in response to either LPS or LPS and ATP treatment were greatly reduced in the infected BMDMs compared to uninfected cells (Fig. 3E, right). These data indicate that infection of BMDMs reduces cytokine secretion in response to LPS and ATP stimulation, but this inhibition is not due to interference with caspase-1 activation.

Viral particle release initiates at around 24 hpi (Fig. 1C). We found that infection did not induce cell death, as indicated by no increase in LDH release compared to uninfected cells (Fig. 3F, left). However, upon treatment with either LPS alone or LPS and ATP, there was significantly more cell death in the infected BMDMs than in the uninfected cells (Fig. 3E, middle, right). Due to the increase in cell death observed at 25 hpi, we next examined whether MHV68 exerted a repression on cytokine secretion at an earlier stage of infection.

Next, BMDMs were stimulated at 6 hpi, and both secreted and intracellular levels of IL-1β, IL-18, and TNF-α were measured by ELISA at 9 hpi (Fig. 4A). When MHV68-infected macrophages were examined, we observed a dramatic reduction in intracellular IL-1β levels in LPS- or LPS+ATP-treated cells (Fig. 4B, top left), and a reduction in secreted IL-1β in response to LPS and ATP (Fig. 4B, bottom left). We observed a slight but significant reduction in intracellular IL-18 levels from infected cells that were treated with LPS and ATP (Fig. 4B, top middle), an observation concomitant with the increase in the secretion of IL-18 (Fig. 4B, bottom middle). At this earlier time point, infection did not alter intracellular or secreted TNF-α in response to either LPS or LPS+ATP (Fig. 4B, right). Although LPS and ATP treatment resulted in a significant increase in cell death, infection did not significantly affect cell death upon LPS treatment alone (Fig. 4C). Taken together, MHV68 infection alone does not trigger inflammatory cytokine production. In addition, infection potently impairs IL-1β production by extrinsic LPS stimulation at 9 hpi. This inhibition is not attributed to an increase in cell death.

FIG 4.

MHV68 impairment of IL-1β production in response to LPS and ATP stimulation is independent of the type I IFN response. (A) An infection and stimulation schematic describes the infection of WT C57BL/6 BMDMs at an MOI of 10 for 6 h, followed by treatment with LPS for 2 h and LPS alone or LPS plus ATP for an additional 1 h. (B) The levels of intracellular (top panels) or secreted (bottom panels) cytokines were measured by ELISA. For IL-1β, both the proform and the mature form are detected. For IL-18, only the mature form is detected. (C) Cell death at 9 hpi was monitored by LDH release into conditioned medium relative to freeze-thawed samples. (D) IFN-α/βR^−/− BMDMs were infected with MHV68 at an MOI of 10, followed by the treatment described above. The intracellular levels of the indicated cytokines were measured by ELISA. Bars represent means ± the SD of triplicate samples. The dashed line represents the limit of detection. BLD, below limit of detection. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Inflammasome components and inflammasome activation can be reduced by type I IFN signaling (53). We observed a potent type I IFN response based on upregulation of IFN-stimulated genes (data not shown), which is consistent with a previous report of macrophage infection with MHV68 (54). To assess the role of paracrine IFN-β signaling in the repression of IL-1β, we infected IFN-α/β receptor^−/− BMDMs with MHV68 and evaluated IL-1β production after LPS stimulation. MHV68 infection reduced IL-1β levels upon extrinsic stimulation even in the absence of the IFN-α/β receptor (Fig. 4D). This indicates that MHV68 infection inhibits IL-1β production independent of type I IFN signaling.

MHV68 impairs IL-1β production induced by Salmonella Typhimurium.

Having found that MHV68 inhibits IL-1β production induced by LPS and ATP treatment (Fig. 4), we next examined whether MHV68 infection could impair IL-1β induction in response to a bacterial pathogen that activates multiple sensors. Salmonella enterica serovar Typhimurium triggers inflammasome responses via sensing by NLRC4 and NLRP3 (11, 55). To determine the effect of MHV68 infection on the inflammasome response to S. Typhimurium, BMDMs were infected with MHV68 at an MOI of 10 for 7 h prior to infection with S. Typhimurium at an MOI of 1 or 4. Cytokine secretion was measured after 2 h of coinfection (Fig. 5A). In the absence of MHV68, we observed a dose-dependent increase in the inflammasome response upon infection with S. Typhimurium, as indicated by elevated levels of both intracellular and secreted IL-1β (Fig. 5B). MHV68 infection significantly reduced both intracellular production and secretion of IL-1β induced by secondary S. Typhimurium infection (Fig. 5B). TNF-α secretion was induced in response to S. Typhimurium but was not changed by MHV68 infection (data not shown), suggesting that this inhibition is not common to all NF-κB-driven genes. Under the growth conditions used to prepare the bacteria, S. Typhimurium infection of BMDMs is known to trigger inflammasome activation and a type of caspase-1-dependent cell death termed pyroptosis (56, 57). Indeed, cell viability was reduced upon S. Typhimurium alone, and a slight further reduction upon MHV68 coinfection was observed at an MOI of 1 but not at the higher MOI of 4.0 CFU/cell (Fig. 5C). We examined whether MHV68 reduced the infectivity and survival of S. Typhimurium that might, in turn, reduce inflammasome activation. MHV68 infection did not impair infectivity; S. Typhimurium invasion was slightly increased at the lower MOI (Fig. 5D). Taken together, the block in IL-1β production by MHV68 extends beyond extrinsic stimulation with LPS to challenge with a secondary bacterial infection.

FIG 5.

MHV68 impairs IL-1β production induced by S. Typhimurium coinfection. (A) An infection and stimulation schematic describes the infection of WT C57BL/6 BMDMs at an MOI of 10 for 7 h, followed by S. Typhimurium infection at an MOI of 1 or 4 CFU/cell for 2 h. (B) Intracellular levels (left panel) or secreted levels (right panel) of IL-1β were analyzed by ELISA at 9 hpi. (C) Cell death at 9 hpi was monitored by LDH release into conditioned medium relative to the freeze-thawed samples. (D) Bacterial invasion was determined by counting the CFU of infected cell lysates plated on LB agar. Bars represent means ± the SD of triplicate samples. The dashed line represents the limit of detection. BLD, below limit of detection. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

MHV68 infection reduces IL-1β transcripts.

Having observed the decrease in IL-1β production and secretion by MHV68-infected BMDMs after extrinsic stimulation, we examined levels of the proforms and mature forms of inflammasome components by immunoblotting. The levels of procaspase-1 and pro-IL-18 proteins did not change upon infection at 9 hpi (Fig. 6A). In addition, there was no difference in cleavage of the proforms of these inflammasome components between infected and uninfected cells upon LPS and ATP treatment. However, as seen for 25 hpi, infection reduced the levels of both proform and mature IL-1β compared to uninfected cells treated with LPS and ATP (Fig. 6A). The ∼2.5-fold reduction in pro-IL-1β occurred even upon stimulation with only the first priming signal, LPS. Thus, the virus does not impair NLRP3 inflammasome activation but instead reduces the production of pro-IL-1β. We assessed the changes in the transcript levels of il1β, caspase-1, il18, or tnfα in the infected cells upon LPS stimulation. MHV68 infection led to an ∼3.5-fold reduction in il1β transcripts in response to LPS stimulation, while the transcript levels of caspase-1 and the other NF-κB responsive cytokines il18 and tnfα were not changed by MHV68 infection (Fig. 6B). Thus, MHV68 infection interferes with inflammasome responses in part by impairing the induction of il1β transcripts.

FIG 6.

MHV68 infection impairs IL-1β at the transcript level. WT C57BL/6 BMDMs were infected with MHV68 at an MOI of 10 for 6 h, followed by treatment with LPS for 2 h and LPS alone or LPS plus ATP for an additional 1 h. (A) Proform and mature cleaved forms of the indicated proteins at 9 hpi were detected by immunoblotting and normalized by the GAPDH loading control. Values are the protein levels relative to either the uninfected cells treated with LPS and ATP or the uninfected cells treated with LPS alone. (B) Transcripts of inflammasome components and inflammatory cytokines were analyzed by reverse transcription-quantitative PCR and the use of ΔΔC_T. The HPRT gene served as the housekeeping gene for normalization. **, P < 0.01; ***, P < 0.001.

MHV68 repression of IL-1β upon extrinsic stimulation is independent of the viral transactivator RTA.

Having observed that MHV68 infection impairs il1β transcript levels, we sought to identify what aspect of viral infection mediated this reduction. To determine whether tegument protein was responsible, we prevented de novo gene expression by UV inactivating MHV68 prior to infection. MHV68 that was UV inactivated by 6 orders of magnitude did not trigger caspase-1 cleavage and failed to inhibit LPS-induced IL-1β production (data not shown). These data are consistent with a role for some component of the virion tegument in this blockade. However, the levels of tegument protein ORF75C delivered to the cell were also reduced upon UV treatment (data not shown), leaving open the possibility that the virions may have been damaged such that less virus entered the cell. Thus, we used a genetic approach to identify the class of viral genes that might contribute to this blockade of intrinsic activation. MHV68 gene orf50 encodes RTA, a conserved immediate-early viral transactivator that is necessary and sufficient for early viral gene expression (30, 58–61). Mutations in MHV68 orf50 severely limit viral gene expression and block virus replication (30, 62). RTA has additional functions in host signaling pathways, including the degradation of NF-κB signaling proteins, impairment of cytokine production, and interference with TLR signaling (63–65). To examine whether RTA or RTA-dependent gene expression drives suppression of IL-1β, we infected BMDMs with a recombinant MHV68-ORF50stop virus (30) prior to LPS and ATP treatment and evaluated the intracellular and secreted levels of IL-1β at 9 hpi. BMDMs infected with MHV68-ORF50stop had a reduction in intracellular and secreted IL-1β upon stimulation with LPS alone or in combination with ATP, respectively, as observed with WT infection (Fig. 7A). TNF-α secretion was not reduced in cultures infected with either WT or MHV68-ORF50stop virus (data not shown). Levels of the tegument protein ORF75C delivered to infected BMDMs were similar between WT and ORF50stop virus at 4 hpi, prior to immediate-early gene expression by MHV68 (Fig. 6B, left) (66), and demonstrate comparable levels of particle delivery. By 9 hpi, there were slightly increased levels of ORF75C in WT-infected BMDMs compared to ORF50stop-infected cells (Fig. 6B, right), a finding consistent with de novo gene expression driven by RTA transactivation. Expression of the immediate-early gene LANA that was ORF50 independent was observed in both WT and ORF50stop virus-infected cells. These data indicate that the ORF50stop virus led to early infection events comparable to WT virus, and the infection impaired IL-1β independently of RTA. This function is likely mediated by a tegument factor delivered by the virion or a newly expressed viral factor or virus-induced host factor that is independent of the major lytic gene transactivator RTA.

FIG 7.

MHV68 impairment of IL-1β is independent of ORF50 function. WT C57BL/6 BMDMs were infected with WT MHV68 or MHV68-ORF50stop at an MOI of 10 for 6 h, followed by treatment with LPS for 2 h and LPS alone or LPS plus ATP for an additional 1 h. (A) The intracellular level (left panel) or the secreted level (right panel) of IL-1β was analyzed by ELISA. Bars represent the means ± the SD of triplicate samples. The dashed line represents the limit of detection. BLD, below limit of detection. **, P < 0.01; ***, P < 0.001. (B) BMDMs were infected with WT MHV68 or MHV68-ORF50stop at an MOI of 10, and the expression of the indicated viral proteins at 4 and 9 hpi was detected by immunoblotting. GAPDH was used as a loading control.

DISCUSSION

In this study we examined how impairment of the inflammasome impacts gammaherpesvirus pathogenesis and, in turn, how a gammaherpesvirus impacts the inflammatory response of the host. We determined that the absence of caspase-1 and caspase-11 does not impact virus replication or pathogenesis of WT MHV68 in mice. Consistent with this observation, the inflammasome was not activated upon de novo infection of BMDMs. MHV68 infection of BMDMs repressed IL-1β production upon extrinsic activation by LPS and ATP or coinfection with Salmonella enterica serovar Typhimurium. The inhibition of IL-1β occurred at the transcript level early during infection and was independent of the viral immediate-early transactivator protein RTA. Our findings indicate that MHV68 evades inflammasome activation in BMDMs and impairs inflammatory cytokine responses to extrinsic stimuli, likely contributing to immune evasion during the initial stages of gammaherpesvirus infection.

During EBV infection, activated caspase-1 cleaves BPLF1, a viral ubiquitin and NEDD8-specific deconjugase, to promote BPLF1 nuclear localization and facilitate DNA replication (39). However, MHV68 replication in primary fibroblast or BMDM cells isolated from Caspase-1/11^−/− mice was not impaired by the absence of caspase-1 (Fig. 1). Lung tissue is more restrictive for the replication of mutant viruses that are only slightly attenuated for replication in culture (67, 68). However, the kinetics and peak level of MHV68 replication were not changed in the lungs of mice lacking caspase-1 and caspase-11 (Fig. 1). Although these data do not rule out a possible decrease in replication that is counterbalanced by a loss of immune control, the absence of any change in replication in primary fibroblasts or BMDMs suggests that caspase-1 does not influence virus replication at the cellular level.

Acute MHV68 replication in the lungs of mice is enhanced in mice lacking either NF-κB signaling components or IFN responses (63, 69, 70), including the cGAS cytoplasmic DNA sensor, which typically activates IRF3 signaling (71). The inflammasome is a powerful proinflammatory signaling pathway that restricts multiple pathogens (17, 41–43). During the preparation of the manuscript, it was reported that mice lacking the inflammasome components NLRP3 or ASC maintain higher levels of MHV68 viral DNA in the spleen during late infection (18). However, our studies found that mice lacking the inflammasome effector caspase-1 and caspase-11 maintained levels of MHV68 similar to WT (Fig. 2). This discrepancy may stem from inflammasome-independent functions for the upstream molecule NLRP3 and ASC. Activation of ASC, also known as TMS1, has been implicated in the induction of cell death (72). The loss of ASC could dysregulate apoptosis in response to infection and lead to an increase in viral load. A distinction between the pathogenesis studies in the NLRP3^−/− and ASC^−/− mice reported by Sun et al. (18) compared to the Caspase-1/11^−/− pathogenesis data reported here is the methodology for measuring levels of virus infection. Sun et al. (18) quantified total viral DNA in disrupted spleen tissues, wherein an increase in viral load might involve an increase in viral episome copy number per cell or a slight increase in lytic replication. Here, the Caspase-1/11^−/− studies measured the frequency of intact splenocyte and peritoneal exudate cells that harbor the viral genome and reactivate virus. Changes in copy number per cell will not skew the frequency determination and preformed infectious virus during lytic productive infection is monitored and, in these experiments, was not a contributing factor. Regardless, these phenotypic differences point to possibly subtly different roles for upstream inflammasome components, the sensor NLRP3 and the adaptor ASC, compared to the executioner caspase-1 and caspase-11 that require further investigation.

Having observed no significant change in pathogenesis in mice lacking caspase-1 and caspase-11, we examined MHV68 inhibition of inflammasome responses in vitro. MHV68 infection did not trigger inflammasome activation, as evidenced by lack of caspase-1 cleavage upon infection of BMDMs at an MOI of 10 (Fig. 3). These data agree with a recent report that an extremely high MOI of 100 to 1,000 is required to detect levels of inflammatory cytokine production by MHV68 levels that do not approximate the robust induction by HSV-1 and MCMV at much lower MOIs of 3 and 1, respectively, in dendritic cells (18). The HSV-1 capsid has been reported to be ubiquitinated and degraded en route to the nucleus, exposing the viral DNA for recognition by IFI16 (73). Interestingly, mutation of ORF64, a deubiquitinase of the MHV68 tegument, leads to the mislocalization of the viral genome in newly infected dendritic cells and augments the secretion of type I IFN and IL-1β in dendritic cells; the increase in IFN-α is lost in cells lacking the cytosolic DNA sensor STING (18). KSHV encodes ORF63, a tegument protein that impairs NLRP1 and NLRP3 inflammasome activation (24). Further investigations of both the host sensors that detect gammaherpesviruses and the roles of tegument proteins in counteracting inflammasome responses is critical to fully understand how gammaherpesviruses evade and subvert the innate immune system (74).

The downregulation of genes for costimulatory signaling molecules and inflammatory cytokines was reported for the THP-1 macrophage cell line latently infected with KSHV, but the mechanism remains undefined (75). In the present study, we uncovered a novel outcome of MHV68 infection in LPS-stimulated primary macrophages: a reduction in il1β at the transcript level (Fig. 6). The lytic transactivator RTA has been reported to inhibit multiple cell signaling pathways, including the TLR, MAVS, and NF-κB signaling pathways (63–65). However, the reduction in IL-1β at 9 hpi did not require RTA or RTA-dependent viral genes. Interestingly, LPS-induction of il18 and tnfα mRNA levels was not impacted at this early time point. Taken together, MHV68 does not globally repress the TLR4/MyD88/NF-κB axis early during BMDM infection, but instead it impairs il1β transcript levels via a different mechanism driven by a virion component or RTA-independent gene.

Although il1β and il18 transcripts are both induced and stabilized by LPS treatment (76–78), il1β mRNA, but not il18 mRNA, contains an AU-rich destabilization element in the 3′ untranslated region (79). Like KSHV, MHV68 encodes an alkaline nuclease (ORF37/vSox) that mediates the reduction of a large number of host genes and influences virion protein composition (80–82). vSox may play a role in the general repression of cytokines observed late during infection (Fig. 3). The mechanism by which vSox would selectively target il1β mRNA, but not il18 and tnfα mRNAs, early after infection is unclear. Future screens of viral mutants coupled with transcriptome profiles in primary macrophage cells will likely reveal several layers of viral interference with host sensing and innate immune effector responses such as inflammatory cytokine production.

Gammaherpesvirus infection must avoid clearance by both the adaptive and innate immune systems. To reach the lymphoid reservoir of latency, gammaherpesviruses infect multiple cell types, including dendritic cells and macrophages (83, 84). Some of the mechanisms by which the gammaherpesviruses impair and subvert the inflammatory response have only recently come to light, and the inhibition of il1β mRNA in response to extrinsic LPS stimulation and bacterial coinfection reveals a novel mechanism of viral interference with inflammatory signaling in a reservoir of infection in the host. Gaining a better understanding of the interplay of gammaherpesviruses with the innate immune system will help identify molecules critical for viral evasion mechanisms that could be targeted to reduce the burden of gammaherpesvirus infection.

ADDENDUM IN PROOF

While this manuscript was in revision, MacDuff et al. (eLife 4:e04494, 2015, http://dx.doi.org/10.7554/eLife.04494) published a study demonstrating that MHV68 infection of Caspase-1/11^−/− mice prevents lethality upon secondary infection with Listeria monocytogenes. The impact of the loss of caspase-1/11 on MHV68 pathogenesis was not reported.