ABSTRACT
Human noroviruses (HuNoV) are the leading cause of nonbacterial gastroenteritis worldwide. Similar to HuNoV, murine noroviruses (MNV) are enteric pathogens spread via the fecal-oral route and have been isolated from numerous mouse facilities worldwide. Type I and type II interferons (IFN) restrict MNV-1 replication; however, the antiviral effectors impacting MNV-1 downstream of IFN signaling are largely unknown. Studies using dendritic cells, macrophages, and mice deficient in free and conjugated forms of interferon-stimulated gene 15 (ISG15) revealed that ISG15 conjugation contributes to protection against MNV-1 both in vitro and in vivo. ISG15 inhibited a step early in the viral life cycle upstream of viral genome transcription. Directly transfecting MNV-1 RNA into IFN-stimulated mouse embryonic fibroblasts (MEFs) and bone marrow-derived dendritic cells (BMDC) lacking ISG15 conjugates bypassed the antiviral activity of ISG15, further suggesting that ISG15 conjugates restrict the MNV-1 life cycle at the viral entry/uncoating step. These results identify ISG15 as the first type I IFN effector regulating MNV-1 infection both in vitro and in vivo and for the first time implicate the ISG15 pathway in the regulation of early stages of MNV-1 replication.
IMPORTANCE Type I IFNs are important in controlling murine norovirus 1 (MNV-1) infections; however, the proteins induced by IFNs that restrict viral growth are largely unknown. This report reveals that interferon-stimulated gene 15 (ISG15) mitigates MNV-1 replication both in vitro and in vivo. In addition, it shows that ISG15 inhibits MNV-1 replication by targeting an early step in the viral life cycle, MNV-1 entry and/or uncoating. These results identify ISG15 as the first type I IFN effector regulating MNV-1 infection both in vitro and in vivo and for the first time implicate the ISG15 pathway in the regulation of viral entry/uncoating.
INTRODUCTION
Human noroviruses (HuNoV) are enteric pathogens responsible for the majority of epidemic, nonbacterial gastroenteritis worldwide (1–3). In the United States alone, approximately 23 million people a year are infected with HuNoV where most outbreaks occur in day care settings, nursing homes, and cruise ships and at catered events (1–4). HuNoV transmission occurs predominantly via the fecal-oral route by ingestion of contaminated foods or water (3). Following an incubation period of 12 to 48 h, HuNoV infection leads to a multitude of symptoms, including explosive vomiting and diarrhea, low-grade fever, headache, chills, and malaise (2, 4). Little is known about the molecular mechanisms mediating HuNoV pathogenesis, as there has been no immunocompetent small-animal or tissue culture system of HuNoV infection to date. The discovery of murine norovirus 1 (MNV-1) and the establishment of an MNV-1 animal model as well as a tissue culture system have significantly broadened our understanding of the host and viral determinants of NoV pathogenesis (5–15).
Murine noroviruses are positive-stranded, encapsidated viruses which enter cells using a pH-independent and cholesterol- and dynamin II-dependent endocytic pathway (16, 17). MNV-1 is sensitive to the antiviral effects of both type I and type II interferons (IFN), as mice lacking alpha IFN (IFN-α), IFN-β, and IFN-γ signaling succumb to MNV-1 infection (5, 7, 11–13). In tissue culture, MNV-1 replication in dendritic cells and macrophages is also sensitive to the antiviral effects of type I and type II IFN (7, 13, 18). In macrophages, type II IFN-mediated anti-MNV-1 defenses require signal transducer and activator of transcription 1 (STAT1), the autophagy protein complex Atg5-Atg12/Atg16L, and the transcription factor interferon regulatory factor 1 (IRF-1) (11, 13). However, neither IRF-1 nor the Atg5-Atg12/ATG16L complex is required for type I IFN-mediated restriction of MNV-1 replication (11, 13).
The type I IFN-mediated effectors that restrict MNV-1 replication are poorly characterized. Previous studies have shown that the IRF3 and IRF-7 transcription factors, along with the viral sensor melanoma differentiation-associated gene 5 (MDA5), play a critical role in inducing the type I IFN antiviral response to MNV-1 (12, 15, 19). Furthermore, the actions of type I IFN in bone marrow-derived dendritic cells (BMDC) are at least partially dependent on the presence of protein kinase R (PKR), RNase L, and/or Mx-1, as the effect of IFN treatment on triply deficient cells was less robust than in wild-type (WT) cells (18). While the type I IFN effectors that restrict MNV-1 replication are not well defined, utilization of IFN-α/βR conditional knockout (KO) mice revealed that type I IFN responses in dendritic cells and macrophages/neutrophils mitigate acute MNV-1 replication in vivo (12). Therefore, identifying additional IFN effectors in dendritic cells and macrophages will further our understanding of type I IFN-mediated regulation of MNV-1.
Interferon-stimulated gene 15 (ISG15) is an ubiquitin-like protein strongly induced by type I IFN, which functions as both an antiviral and an immunoregulatory molecule. Mice deficient in ISG15 are susceptible to numerous viruses, including influenza A and B virus, herpes simplex virus 1 (HSV-1), Sindbis virus, and Chikungunya virus (CHIKV) (20–24). Consisting of two ubiquitin-like domains separated by a peptide linker, ISG15 can covalently associate with numerous target proteins (ISGylation) via its C-terminal LRLRGG motif. It utilizes an E1 (UbE1L)-, E2 (UbcM6 and UbcM8)-, and E3 (Herc5, Herc6, HHARI, and TRIM25)-dependent conjugation cascade (25–30), all of the members of which are induced by type I IFN. Similarly to ubiquitination, ISGylation is a reversible process yielding unconjugated “free” forms of ISG15 found both inside and outside the cell (31–34). Recent work has shown that, depending on the virus, ISG15 conjugation to target proteins and the actions of free ISG15 can suppress viral replication and regulate host responses to infection (20–24).
While ISG15 has been implicated in type I IFN-dependent antiviral responses to numerous viruses, its mechanism(s) is not well defined. However, one study showed that ISG15 conjugation can inhibit influenza A virus protein expression in virally infected human cells (35). Studies using human immunodeficiency virus (HIV), Ebola virus (EBOV) virus-like particles (VLPs), and influenza A virus have implicated ISG15 in disruption of viral egress (36–39). ISG15 overexpression was shown to inhibit HIV Gag and Tsg101 ubiquitination, which is important for Gag-Tsg101 association and subsequent release of infectious virions from the cell (37). Tsg101 is also important for influenza A virus trafficking. Recent studies have shown that influenza A virus hemagglutinin transport is inhibited by type I IFNs in a Tsg101- and ISG15-dependent manner (39). Moreover, ISG15 was found to inhibit ubiquitination mediated by the Nedd4 ubiquitin ligase (36, 38). Disruption of Nedd4 activity inhibited EBOV VP40 ubiquitination and was shown to disrupt EBOV VLP release. Those studies demonstrated that ISG15 can act as an antiviral effector capable of disrupting viral protein translation and viral egress.
In this study, we identified ISG15 as a type I IFN-mediated effector molecule that restricts MNV-1 replication early in the viral life cycle. Utilizing mice and primary cells deficient in ISG15 and/or UbE1L, we showed that the ISG15 pathway contributes to type I IFN-mediated control of MNV-1. In both ISG15−/− and UbE1L−/− cells treated with IFN, we observed increased MNV-1 replication compared to that in WT cells, indicating that ISG15 conjugation mediates this antiviral activity. ISG15−/− and UbE1L−/− mice infected with MNV-1 also displayed increased viral replication early during the course of infection. Further analysis revealed that ISG15 conjugation inhibited an early step in the MNV-1 life cycle, preceding genome transcription. The ability to bypass this inhibition following the transfection of MNV-1 RNA into mouse embryonic fibroblasts (MEFs) and BMDC indicates that the MNV-1 entry/uncoating step in the life cycle is being inhibited by ISG15. These results identify ISG15 as an IFN effector protein regulating MNV-1 pathogenesis in a step preceding genome replication.
MATERIALS AND METHODS
Mice.
Mice were bred and housed at Washington University School of Medicine in accordance with all federal and university guidelines, under specific-pathogen-free conditions (40). All protocols were approved by the IACUC at Washington University. Wild-type C57BL/6 mice (catalog no. 000664) were purchased from Jackson Laboratory (Bar Harbor, ME). Mice were bred and maintained in our animal facilities. ISG15−/− mice (provided by Klaus-Peter Knobeloch, University Clinic Freiburg, Freiburg, Germany) and UbE1L−/− mice (provided by Dong-Er Zhang, University of California San Diego School of Medicine) were generated as previously described (41, 42). UbE1L−/− and ISG15−/− mice were fully backcrossed (>99% identity to C57BL/6 by congenic single nucleotide polymorphism [SNP] analysis through Taconic Laboratories [Hudson, NY]). ISG15−/− × UbE1L−/− (double-knockout [DKO]) mice were generated by breeding ISG15−/− and UbE1L−/− mice to obtain F1 progeny, which were then brother and sister mated.
Cells.
Bone marrow-derived macrophages (BMM) were generated as described previously (13) using BMM media supplemented with 2% supernatant derived from CMG14-12 cells, as a source of macrophage colony-stimulating factor (M-CSF) (43). Bone marrow-derived dendritic cells (BMDC) were produced by flushing bone marrow from femurs and lysing cells in red blood cell (RBC) lysis buffer (Sigma, Saint Louis, MO). Cells were seeded at 9 × 106 cells in T175 flasks and cultured for 7 days in BMDC media and 2% supernatant derived from granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing cells, which were a gift from Marco Colonna (Washington University, St. Louis, MO) (44). Cells were fed on day 4 and were harvested and plated for experiments on day 7. BMDC were rested for 24 h before MNV-1 infection or IFN stimulation. BMDC media were comprised of RPMI 1640 (Sigma, Saint Louis, MO) supplemented with 10% fetal bovine serum (Sigma, Saint Louis, MO), 1% penicillin (Cellgro, Manassas, VA), 1% streptomycin (Cellgro, Manassas, VA), 1% sodium pyruvate (Sigma, Saint Louis, MO), and 1% nonessential amino acids (Cellgro, Manassas, VA). Mouse embryonic fibroblasts (MEFs) were made as previously described (22) and utilized at passage 3 for experimentation.
Virus infections.
A concentrated stock of MNV-1.pCW3 (10), here referred to as MNV-1, was generated using RAW264.7 cells (American Type Culture Collection [ATCC], Manassas, VA) as described previously (10) and used in all experiments. Day 7 BMDC and BMM were plated at 1 × 106 cells per well of a 6-well plate and were rested for 24 h. Cells were then either left untreated or treated at the indicated doses with recombinant IFN-β or IFN-α4 (PBL, Piscataway, NJ) for 18 to 24 h before inoculation with MNV-1 at a multiplicity of infection (MOI) of 0.05 at 4°C for 30 min. The MNV-1 inoculum was removed, the cells were washed once with Dulbecco's modified Eagle's medium (DMEM; Cellgro, Manassas, VA), and unsupplemented medium or medium supplemented with type I IFN was added back onto cells. Supernatants and/or cells were collected at various times after infection, and MNV-1 titers were determined by a plaque assay (12).
Eight-to-10-week-old, sex-matched mice were orally inoculated with 25 μl of concentrated MNV-1 diluted in phosphate-buffered saline (PBS). Mice were sacrificed 1 to 7 days after inoculation, and tissues (lung, liver, spleen, mesenteric lymph node [MLN], distal illeum, cecum, and stool) were harvested as previously described (45) and frozen at −80°C until titers were determined.
MNV-1 RNA transfection.
Total RNA was isolated from 150 μl of concentrated MNV-1 using 1 ml of TRIzol (Ambion, Grand Island, NY) according to the manufacturer's instructions. MEFs and BMDC (5 ×104 cells in 24-well plates) were either left untreated or were treated with 100 U/ml IFN-α4 followed by transfection with 200 ng of MNV-1 RNA complexed with 2 μl of Lipofectamine 2000 (Invitrogen, Grand Island, NY) according to the manufacturer's instructions. Supernatant and cells were washed once with 1× PBS, and samples were collected at various times after transfection. MNV-1 titers were determined by a plaque assay.
Western blotting.
Cell lysates were harvested in radioimmunoprecipitation assay (RIPA) buffer supplemented with 150 mM phenylmethylsulfonyl fluoride (PMSF; Sigma, Saint Louis, MO), a 1:100 dilution of protease cocktail inhibitor (Sigma, Saint Louis, MO), 1 μg/ml okadaic acid (Merck, Damstadt, Germany), and a 1:100 dilution of Halt phosphatase inhibitor (Thermo Scientific, Logan, UT) for ISG15 and UbE1L protein expression studies. Protein concentrations in samples were determined using a DC protein assay kit (Bio-Rad, Hercules, CA), and 25 μg of protein was resolved on 4% to 15% Tris-HCl gradient gels (Bio-Rad, Hercules, CA). Proteins were transferred onto polyvinylidene fluoride (PVDF) membranes using a semidry transfer system (Fisher, Pittsburgh, PA). Membranes were blocked in 5% nonfat milk in 1× PBS (pH 7.5)–0.05% Tween-20 (PBST) and subsequently probed overnight at 4°C with primary antibodies against mUbE1L (Santa Cruz, Santa Cruz, CA) (1:500) and α-ISG15 (1:3,000) rabbit polyclonal serum (1551), as previously described (21), diluted in 5% nonfat milk. β-Actin (Sigma, Saint Louis, MO) (1:5,000) was probed as a loading control. Goat anti-mouse IgG-horseradish peroxidase (HRP) (Jackson ImmunoResearch, West Grove, PA) (1:20,000) and goat anti-rabbit IgG-HRP (Jackson ImmunoResearch, West Grove, PA) (1:200,000) secondary antibodies were also diluted in 5% nonfat milk and incubated with membranes for 1 h at room temperature. To detect specific proteins, membranes were incubated with an Immobilon chemiluminescent HRP substrate kit (Millipore, Saint Louis, MO) and exposed to film.
Northern blot analysis.
Total RNA from MNV-1-infected samples (1 × 106 cells in 6-well plates) were purified as described above, and 1 μg of RNA was resolved on a 1% formaldehyde gel and transferred to a Nytran SuPerCharge (SPC) membrane. Northern blot reagents were obtained from a NorthernMax kit (Ambion, Grand Island, NY). An antisense probe against the VP1 major capsid was used to detect the MNV-1 subgenomic RNA, as previously described (13). Membranes were also probed with GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as a loading control (13).
Statistics.
Graphs were created using Prism 5 software version 5.0b (Graphpad, San Diego, CA). Statistical analyses were determined by nonparametric analysis (Mann-Whitney test) using the Prism software.
RESULTS
ISG15 participates in type I IFN-mediated restriction of MNV-1 replication.
Type I interferon (IFN) signaling in dendritic cells and macrophages is important for restricting MNV-1 replication in vitro and in vivo (7, 12, 18). Since MNV-1 induces type I IFN (8, 19), we evaluated whether ISG15, one of the most robustly upregulated interferon-stimulated genes, functions as an IFN effector during infection. MNV-1 replication, along with ISG15 and UbE1L protein expression, was assessed 48 h after infection in WT and ISG15−/− cells that were either left untreated or IFN treated. In unstimulated BMDC, no differences in MNV-1 replication were detected between WT and ISG15−/− cells (Fig. 1A). Consistent with this phenotype, we observed little if any ISG15 expression in MNV-1-infected WT cells (Fig. 1C, lanes 7 to 9). IFN treatment induced expression of free ISG15, ISG15 conjugates, and UbE1L in infected WT BMDC (Fig. 1C, lanes 10 to 12). Under these conditions, MNV-1 replication was decreased in WT cells, with a nearly 3-log reduction in titers compared to untreated WT BMDC (Fig. 1A). In ISG15−/− BMDC, IFN treatment with either IFN-β or IFN-α resulted in less-effective inhibition, with ISG15−/− cells exhibiting a 10-fold increase in MNV-1 loads compared to similarly treated WT BMDC (Fig. 1A). A similar effect was also observed in ISG15−/− bone marrow-derived macrophages (BMM; Fig. 1B). These results indicate that ISG15 contributes to the type I IFN antiviral response during MNV-1 infection in dendritic cells and macrophages.
FIG 1.
ISG15 participates in type I IFN-mediated restriction of MNV-1 replication. (A and B) WT, ISG15−/−, and UbE1L−/− BMDC (A) and BMM (B) were left untreated or pre- and posttreated with the indicated doses of IFN-α4 or IFN-β and then infected with MNV-1 at an MOI of 0.05 for 48 h. Supernatants were harvested and titers were determined by an MNV-1 plaque assay. Data represent the compiled results from seven independent experiments in panel A and three independent experiments in panel B. P values were determined by the Mann-Whitney test. *, P < 0.05; **, P ≤ 0.01; ***, P < 0.001; N.S., not significant. (C) WT (W), ISG15−/− (15), and UbE1L−/− (U) BMDC were either left untreated or pre- and posttreated with 1,000 U/ml of IFN-α4 prior to infection with MNV-1 at an MOI of 0.05. Cell lysates were harvested 48 h postinfection and resolved by SDS-PAGE followed by Western blot analysis. Data represent the results of 3 independent experiments. The right inset represents the results of densitometry analysis of free ISG15 levels from 3 independent experiments. (D and E) WT, ISG15−/−, and UbE1L−/− BMDC were either left untreated or pre- and posttreated with 1,000 U/ml IFN-α4, and the cells were infected with MNV-1 at an MOI of 0.05. At various times postinfection, supernatants were harvested and titers were determined by a plaque assay. Data represent the results of 3 to 5 individual experiments. P values were determined by the Mann-Whitney test. *, WT versus ISG15−/−; #, WT versus UbE1L−/−; +, ISG15−/− versus UbE1L−/−. *, #, or +, P < 0.05; *** or ###, P < 0.001.
ISG15 conjugates participate in the type I IFN-mediated anti-MNV-1 response.
Both free ISG15 and ISG15 conjugates regulate host responses to viral infection (20–22, 24). To determine whether ISG15-mediated regulation of MNV-1 is conjugation dependent or independent, we next evaluated MNV-1 replication and ISG15 expression in UbE1L−/− cells. MNV-1 replication in unstimulated UbE1L−/− BMDC was comparable to that seen with WT and ISG15−/− BMDC (Fig. 1A). In contrast, untreated ISG15−/− and UbE1L−/− BMM displayed about a 5-fold increase in viral replication compared to WT cells, suggesting that ISG15 conjugation is induced in BMM during MNV-1 infection (Fig. 1B and data not shown). Following IFN stimulation, BMDC and BMM deficient in UbE1L did not form ISG15 conjugates and appeared to have increased levels of free ISG15 compared to WT BMDC, although this did not reach statistical significance (Fig. 1C, lanes 4 to 6 and 10 to 12, and data not shown). IFN treatment restricted MNV-1 replication in UbE1L−/− BMDC and BMM; however, similar to our observations in ISG15−/− cells, MNV-1 replication in IFN-treated UbE1L−/− cells was not inhibited to the same extent as seen in WT cells. UbE1L−/− cells had nearly 100-fold-higher titers in BMDC and 50-fold-higher titers in BMM than in IFN-treated WT cells (Fig. 1A and B). Interestingly, we consistently observed 2-to-5-fold more MNV-1 replication from UbE1L−/− cells than from ISG15−/− cells (Fig. 1A and B). IFN-stimulated ISG15−/− and UbE1L−/− BMDC also displayed elevated titers compared to WT cells at a multiplicity of infection (MOI) of 5 (data not shown). To further characterize the differences in viral replication that we observed in the knockout cells, we performed full analyses of growth curves in both untreated cells and cells pretreated with IFN. We observed no differences in viral replication at any of the time points analyzed among the three genotypes of cells that were not pretreated with IFN (Fig. 1D). With IFN pretreatment, in contrast, we observed increased viral replication in both the ISG15−/− and UbE1L−/− BMDC (Fig. 1E). This increase was detected as early as 12 h postinfection and persisted until 48 h postinfection (ISG15−/− and UbE1L−/− versus WT cells at 12 h [7.8-fold and 13.2-fold, respectively], 18 h [7.8-fold and 11.6-fold, respectively], and 48 h [16.6-fold and 24.3-fold, respectively] postinfection) (Fig. 1E). Since MNV has been previously reported to induce apoptosis, we also assessed whether the induction of apoptosis was altered in cells lacking ISG15 or UbE1L, which could contribute to the differences in MNV-1 titers observed between genotypes. However, no significant differences among the genotypes were observed during the course of infection in the percentage of late-stage apoptotic (annexin V-positive [annexin V+] propidium iodide-negative [PI−]), apoptotic (annexin V+ PI+), or necrotic (annexin V− PI+) cells (data not shown). Taken together, these results show that type I IFN-dependent induction of ISG15 conjugates restricts MNV-1 replication in dendritic cells and macrophages.
As seen in Fig. 1A and B, we did observe an increase in MNV-1 replication in the UbE1L−/− cells above that observed in the ISG15−/− cells in both the BMM and BMDC. Moreover, free ISG15 expression levels trended higher in UbE1L−/− BDMCs (Fig. 1C). Increased MNV-1 replication in UbE1L−/− cells beyond the replication observed in ISG15−/− cells could have been due to an ISG15-independent function of UbE1L or could have been due to the actions of free ISG15 and their effects on either the virus or the host cells. To determine if the increased replication observed in the UbE1L−/− cells was dependent on the presence of ISG15, we generated ISG15−/− × UbE1L−/− double-knockout (DKO) mice. BMDC generated from WT, ISG15−/−, UbE1L−/−, and DKO mice were either left untreated or pretreated with IFN, and viral replication was measured in the supernatants at 48 h postinfection as shown in Fig. 1A. As expected, levels of viral replication in unstimulated BMDC from all the genotypes were comparable (Fig. 2). Interferon treatment inhibited replication in WT cells by nearly 4 logs. In contrast, replication in both the ISG15−/− and UbE1L−/− cells was increased compared to that in WT cells (Fig. 2). Furthermore, as had been seen previously, the UbE1L−/− cells had nearly a 10-fold increase in viral replication beyond that observed in the ISG15−/− cells (Fig. 2). However, virus levels in the DKO BMDC were comparable to those observed in the ISG15−/− cells and differed significantly from those observed in the UbE1L−/− cells (Fig. 2). Thus, the increase in viral replication observed in UbE1L−/− cells, beyond what was observed in ISG15−/− cells, was dependent upon ISG15 and indicated that free ISG15 in the absence of conjugates increases MNV-1 replication in BMDC at 48 h postinfection.
FIG 2.
Free ISG15 increases MNV-1 titers in vitro. WT, ISG15−/−, UbE1L−/−, and DKO BMDC were stimulated with 1,000 U/ml of IFN-α4 as described for Fig. 1 and subsequently infected with MNV-1 at an MOI of 0.05. Viral titers were determined in the supernatants at 48 h postinfection (hpi) by an MNV-1 plaque assay. Data shown are the compiled results from 3 independent experiments. P values were determined by the Mann-Whitney test. *, P < 0.05; **, P < 0.01.
ISG15 conjugation contributes to the type I interferon-mediated control of MNV-1 in vivo.
We next assessed the role of ISG15 during MNV-1 infection in vivo. Wild-type, ISG15−/−, and UbE1L−/− mice were infected with MNV-1 perorally and monitored for clinical signs and survival for 14 days. Clinical signs monitored included hunched posture, ruffled fur, shivering, and weight loss. No significant weight loss or clinical signs were observed in any of the mice over this 14-day period, and none of the mice succumbed to infection. Upon tissue harvest, mice were inspected for diarrhea and none was observed. We therefore assessed whether WT, ISG15−/−, and UbE1L−/− mice displayed any differences in viral burdens following MNV-1 infection. Mice were inoculated with MNV-1, and viral replication was assessed in various organs at 1, 2, 3, and 7 days following infection. At 1 day postinfection, we observed a modest increase in viral replication in the ISG15−/− mice in both the distal ileum (7.9-fold) and stool (5.5-fold) (Fig. 3). At 2 days postinfection, we observed elevated viral loads in several organs in both the ISG15−/− and UbE1L−/− mice. Viral loads were increased in the distal ileum, MLN, and spleen in both the ISG15−/− mice (7.8-fold, 11.14-fold, and 11.3-fold, respectively) and the UbE1L−/− mice (13.7-fold, 14.8-fold, and 11.2-fold, respectively) compared to WT mice (Fig. 3B). This increased replication was noted in the same organs at 3 days postinfection in both the ISG15−/− and UbE1L−/− mice, with viral titers ranging between 8-fold and 17-fold higher than in WT mice (Fig. 3C). However, despite the increased viral replication noted in both the ISG15−/− and UbE1L−/− mice, they were able to clear the infection equally, since we detected no replicating virus in any of the organs analyzed in any genotype by 7 days postinfection (Fig. 3D). These results provide further evidence that ISG15, through its conjugation to target proteins, functions as an IFN-induced effector protein with activity against MNV-1.
FIG 3.
ISG15 conjugates control MNV-1 replication in vivo. WT, ISG15−/−, and UbE1L−/− mice were infected with 3 × 107 PFU MNV-1 perorally. Mice were sacrificed at days 1, 2, 3, and 7 postinfection, and the distal ileum, MLN, spleen, and stool were harvested and titers determined by a plaque assay. Data show the time course of infectious MNV-1 detected in distal ileum, MLN, spleen, and stool on day 1 (WT = 13; ISG15−/− = 9; UbE1L−/− = 10), day 2 (WT = 12; ISG15−/− = 9; UbE1L−/− = 11), day 3 (WT = 16; ISG15−/− = 13; UbE1L−/− = 11), and day 7 (WT = 10; ISG15−/− = 8; UbE1L−/− = 9). P values were determined by the Mann-Whitney test. *, WT versus ISG15, ^ ISG15−/− versus UbE1L−/−; #, WT versus UbE1L−/−. * or ^, P < 0.05; ** or ##, P < 0.01; ###, P < 0.001.
ISG15 regulates MNV-1 replication upstream of genome transcription.
To further characterize the antiviral actions of ISG15, we next assessed where in the viral life cycle it was acting. MNV-1 enters cells by a poorly characterized pH-independent, cholesterol- and dynamin II-dependent endocytic pathway (16, 17). Caliciviruses uncoat, releasing their genome into the cytoplasm, and undergo a pioneer translation step that produces the nonstructural polyprotein precursor. This polyprotein precursor is then cleaved, yielding the mature nonstructural proteins that form the replication complex, which is responsible for viral genome replication (46). The positive-strand genome is then transcribed, producing a negative-strand template, which is subsequently used to produce the genomic positive strand and the subgenomic strand (46). The latter transcripts are translated to produce the structural and nonstructural proteins that assemble into the mature virions that are released from the cell (47). Previous studies have shown that newly synthesized infectious virions are detected in BMDC lysates 9 to 12 h after MNV-1 infection (7). We first detected differences in viral titers in our growth curves at around 12 h postinfection (Fig. 1E), suggesting that ISG15 was impacting MNV-1 growth during a single round of viral replication. Since previous studies have implicated the ISG15 pathway in viral release (36–39), we first determined whether ISG15 was disrupting MNV-1 replication by impacting viral release from the cell. BMDC were infected as previously described, and the level of virus release into the supernatant, compared to the level of virus that remained cell associated, was measured 12 h after infection. As we had shown in Fig. 1, we observed increased MNV-1 titers in the supernatants of IFN-treated ISG15−/− and UbE1L−/− BMDC compared to WT cells, with viral titers being 10- to 50-fold higher than those seen with WT cells (Fig. 4A). Similar results were also obtained when we analyzed cell-associated virus from samples in which ISG15−/− and UbE1L−/− BMDC had significantly elevated titers in comparison to those seen with WT cells (Fig. 4B). In both cases, we observed no differences in viral load from cells that were not pretreated with IFN. Interestingly, at this time point we also noted no difference in MNV-1 replication between ISG15−/− and UbE1L−/− cells in either the supernatant or cell-associated samples, suggesting that effects of free ISG15 on infectious MNV-1 levels are not observed under conditions of a single round of replication. Together, these results suggest that ISG15 conjugation restricts MNV-1 replication early in the MNV-1 life cycle and does not impact MNV-1 release. Rather, it appears that ISG15 conjugates act at a step upstream of MNV-1 assembly, which distinguishes its role in this system from what has been observed for EBOV, HIV, and influenza A virus (36–38).
FIG 4.
ISG15 conjugation does not inhibit MNV-1 release. WT, ISG15−/−, and UbE1L−/− BMDC were stimulated with 1,000 U/ml of IFN-α4 and subsequently infected with MNV-1 at an MOI of 0.05 for 12 h. The results of MNV-1 replication in supernatant (A) and cells (B) are shown. Data represent the results of 4 to 5 independent experiments. P values were determined by the Mann-Whitney test. *, P < 0.05; ***, P < 0.001.
Since our results suggested that ISG15 conjugation impacts a step upstream of MNV-1 assembly and release, we next examined whether ISG15 conjugates restrict MNV-1 RNA synthesis by assessing the replication of subgenomic transcripts early during infection. We reasoned that if ISG15 conjugates impact a step upstream of genome replication, then the levels of subgenomic transcripts in ISG15−/− and UbE1L−/− BMDC would be greater than in WT cells, mimicking the viral infection phenotype that we observed at 12 h after infection. WT-, ISG15−/−-, and UbE1L−/−-infected BMDC, either left unstimulated or stimulated with IFN, were mock infected or infected with MNV-1. Total RNA was harvested at 0, 8, and 12 h after infection, and expression of subgenomic MNV-1 RNA was assessed by Northern blotting. Following MNV-1 infection alone, subgenomic fragments were detected by 8 h postinfection and these levels were sustained at 12 h, with no significant differences observed between the genotypes at any time point examined (Fig. 5, left panel, lanes 4 to 6 and 7 to 9). After IFN stimulation, subgenomic transcripts were detected at 8 h and 12 h in all infected genotypes (Fig. 5, right panel, lanes 4 to 6 and 7 to 9). However, densitometry analysis revealed that IFN-treated WT cells contained less subgenomic RNA than similarly treated ISG15−/− and UbE1L−/− cells at 8 and 12 h postinfection (Fig. 5, right panel, lanes 4 to 6 and 7 to 9). We also observed that IFN-treated ISG15−/− cells contained lower levels of MNV-1 subgenomic transcript than similarly treated UbE1L−/− cells (Fig. 5, left panel, lanes 5 to 6 and 8 to 9), indicating that the impact of free ISG15 on MNV-1 replication can be observed with a single round of replication at the level of MNV-1 transcription (Fig. 5, left panel, lanes 5 to 6 and 8 to 9) before its impact is seen on the release of infectious virus (Fig. 4). Together, these results mimic the viral infection phenotype that we observed in vitro, suggesting that ISG15 conjugation impacts a step further upstream in the MNV-1 life cycle.
FIG 5.
ISG15 regulates MNV-1 replication upstream of genome transcription. Data represent the results of Northern blot analysis of expression of MNV-1 subgenomic transcripts. WT, ISG15−/−, and UbE1L−/− BMDC were either left untreated or pre- and posttreated with 1,000 U/ml of IFN-α4 and were subsequently infected with MNV-1 at an MOI of 0.05. At 0, 8, and 12 h postinfection, cells were harvested for total RNA isolation and RNA was resolved by Northern blotting. Densitometry values are shown as percentages based on the controls for each experimental group. Data represent the results of three independent experiments. “SG” denotes the MNV-1-1 subgenomic fragment. The star denotes a nonspecific band.
ISG15 functions as a type I IFN effector early in the MNV-1 life cycle.
Since ISG15 impacts a step upstream of viral transcription, we next questioned whether ISG15 mitigates MNV-1 entry or uncoating. Mouse embryonic fibroblast (MEFs) cannot be infected with MNV-1 (presumably because they lack the receptor for virus entry) but can support a single round of viral replication following MNV-1 RNA transfection (7, 13, 18). This transfection strategy was utilized to demonstrate that type II IFN-dependent Atg5 responses occur at a step downstream of MNV-1 entry/uncoating (13). We reasoned that if ISG15 conjugates restrict virus entry or uncoating, then the levels of MNV-1 replication in WT, ISG15−/−, and UbE1L−/− MEFs transfected with MNV-1 RNA should be comparable following IFN stimulation. MEFs from WT, ISG15−/−, and UbE1L−/− mice were left untreated or were treated with IFN followed by transfection of purified MNV-1 RNA. Untreated MEFs displayed no MNV-1 replication differences at 12 or 24 h after transfection in either the supernatant or cells (Fig. 6). IFN stimulation significantly diminished MNV-1 replication in transfected cells; however, unlike what was observed in IFN-treated BMDC and BMM infected with MNV-1 (Fig. 1A and B and Fig. 2), no significant differences in MNV-1 replication were observed in any of the MEFs transfected with viral RNA at any of the time points examined (Fig. 6). These results support the hypothesis that ISG15 regulates MNV-1 replication by targeting viral entry or uncoating or both. However, since there may be cell-type-specific differences in the IFN responses between MEFs and either BMDC or BMMs, we assessed the impact of bypassing viral entry/uncoating by transfecting viral RNA into BMDC. We transfected WT, ISG15−/−, and UbE1L−/− BMDC with a dose of MNV-1 RNA, which resulted in a viral yield similar to what was observed following infection of BMDC at a low multiplicity of infection (MOI). Similarly to our transfection experiments with MEFs, no significant differences in supernatant or cell-associated MNV titers were observed in BMDC transfected with viral RNA at 12 or 24 h posttransfection (Fig. 6B). Taken together, these results suggest that ISG15 conjugates participate in an antiviral function observed only during MNV-1 infection and not observed following MNV-1 RNA transfection. Thus, ISG15 conjugation impacts an early step of the MNV-1 life cycle upstream of genome transcription and may potentially impede MNV-1 entry and/or uncoating.
FIG 6.
ISG15 conjugation-dependent effects on MNV-1 replication are lost in MEFs and BMDC upon bypassing viral entry/uncoating. WT, ISG15−/−, and UbE1L−/− MEFs (A) and BMDC (B) were either left untreated or pre- and posttreated with 100 U/ml of IFN-α4 followed by transfection with 200 ng of MNV-1 RNA complexed with Lipofectamine 2000. Supernatant and cell-associated virus were harvested 12 and 24 h postinfection for determination by an MNV-1 plaque assay. Data represent the compiled results of 3 to 5 independent experiments.
DISCUSSION
Type I interferons are important for controlling MNV-1 infection both in vitro and in vivo. In particular, IFN responses produced by dendritic cells and macrophages are critical for restricting MNV-1 replication (12). The ISGs downstream of type I IFN regulating MNV-1 infection are poorly defined; however, studies have shown that BMDC deficient in the MDA5 intracellular viral sensor display increased MNV-1 replication in comparison to wild-type cells at 24 to 48 h postinfection (19). IFN-β-treated BMDC triply deficient in PKR, RNase L, and Mx1 also have significantly increased viral titers compared to similarly treated WT cells at 24 to 72 h postinfection, but this does not account for the entire IFN effect, nor were these effects observed during early stages of infection (i.e., 12 h postinfection), suggesting that PKR, RNase L, and/or Mx1 impacts MNV-1 growth following multiple rounds of replication (18). To further understand how type I IFN mitigates MNV-1 replication, it is therefore important to identify the type I IFN effectors involved early after infection and determine their mechanism(s) of action.
In this study, we identified and characterized ISG15 as a type I IFN effector with anti-MNV-1 activity during early stages of infection. Our results show that the ability of ISG15 to inhibit MNV-1 replication within 12 h of infection is largely dependent upon protein conjugation. Moreover, they show that ISG15 conjugation also plays a role in vivo during MNV-1 infection. Surprisingly, unlike what has been observed in other viral systems, ISG15 conjugation did not target late events in viral replication such as protein translation or viral egress. Instead, ISG15 conjugation targeted the MNV-1 life cycle upstream of genome transcription, potentially impeding MNV-1 entry and/or uncoating.
Previous studies have suggested that type I IFN impacts multiple steps of the MNV-1 life cycle, including viral protein translation and steps preceding genome transcription (18). Given the vast array of ISGs induced following IFN-α/βR signaling, it is not surprising that IFN effectors impact multiple steps of the MNV-1 life cycle. Previous analyses of MNV-1 genome copies by quantitative reverse transcription-PCR (qRT-PCR) demonstrated that viral genome levels did not decrease within the first 8 h of infection in either untreated or IFN-β-treated RAW264.7 cells, indicating that IFN treatment does not induce degradation of the viral genome (18). Interestingly, between 6 and 8 h postinfection, viral genome copy numbers in untreated RAW264.7 cells were increased 10-fold over genome copy numbers in IFN-β-treated cells, indicating that a type I IFN effector response impacts a step upstream of genome replication (18). In our analysis of MNV-1 subgenomic RNA levels, we observed increased amounts of subgenomic transcripts in untreated samples in comparison to those seen with IFN-treated samples (Fig. 5), confirming that type I IFN can impact a step upstream of genome replication. From our studies, it appears that ISG15 is at least one of the effector proteins regulating MNV-1 replication at this stage since viral subgenomic transcript levels were increased in IFN-treated ISG15−/− and UbE1L−/− BMDC in comparison to WT cells (Fig. 5). Bypassing MNV-1 entry/uncoating by viral RNA transfection into MEFs and BMDC resulted in a loss of the viral replication phenotype (Fig. 6), suggesting that ISG15 may be impacting one or both of these early steps in the viral life cycle.
The mechanism(s) by which ISG15 conjugates regulate the early processes of the MNV-1 replication requires further investigation, as the pathways utilized by MNV-1 for cellular entry are still being characterized. Our results demonstrate that ISG15 conjugates regulate a step upstream of MNV-1 genome replication and are consistent with the notion that ISG15 regulates some aspect of virus entry/uncoating. However, ISG15 effects may not be direct, as ISG15 could in fact modulate the function of a host or viral protein important for viral entry or uncoating once the virion engages a receptive cell. Recent studies in MNV-1-infected RAW264.7 cells treated with the cholesterol-sequestering agent methyl-β-cyclodextrin and the dynamin inhibitor dynasore indicate that cholesterol and dynamin II are important for MNV-1 entry (16, 17). These studies suggest that cholesterol-rich microdomains or lipid rafts may be important for entry of MNV-1 into cells. Proteomic studies searching for ISG15-modified target proteins have revealed numerous potential interacting proteins involved in intracellular trafficking and transport. Of these, multiple members of the annexin family (i.e., AnxA1, AnxA2, and AnxA6) and β-actin have been implicated as ISG15 targets (28, 48). Annexins are calcium binding proteins involved in cholesterol homeostasis and membrane organization and repair (49). These proteins are also important for tethering the actin cytoskeleton near endosomal membranes undergoing vesicular trafficking (49). AnxA6 interacts with both cholesterols and phospholipids (50–54) and, along with AnxA2, has been found to be associated with the actin cytoskeleton (55–58). Interestingly, both AnxA6 and AnxA2 have been associated with lipid rafts and microdomain organization (59–61). It is therefore possible that ISG15 regulates annexin interactions with lipids rafts or binding to actin filaments and that it could therefore regulate intracellular trafficking of cholesterol-rich endosomes or other vesicles important in MNV-1 entry and trafficking. Future studies are needed to better define the MNV-1 entry pathway to further assess the mechanism by which ISG15 is regulating this process.
Interestingly, in our studies, IFN-treated UbE1L−/− BMDC had viral replication that was significantly increased above what was observed in similarly treated ISG15−/− cells at 48 h postinfection (Fig. 1A, B, and E and Fig. 2). Moreover, IFN-treated UbE1L−/− BMDC had increased subgenomic transcripts levels compared to ISG15−/− cells at 12 h postinfection (Fig. 5). In other viral models where the antiviral effects of ISG15 are conjugation dependent, no differences have been noted between ISG15−/−- and UbE1L−/−-infected cells or mice (20, 22). The increased levels of replication and subgenomic transcripts that were observed in UbE1L−/− versus ISG15−/− cells could be due to the effects of increased levels of free ISG15 on either the cell or virus or, alternatively, could be due to an uncharacterized function of UbE1L. The analysis of DKO cells lacking both ISG15 and UbE1L (Fig. 2) confirmed that the increased replication observed in the UbE1L−/− cells beyond the levels observed in ISG15−/− cells was indeed dependent upon the presence of ISG15, suggesting that free ISG15 was responsible for this effect. However, we did not observe an increase in viral loads in UbE1L−/− compared to ISG15−/− mice in vivo, suggesting that the impact of free ISG15 may be cell type specific. Mechanistically, how free ISG15 mediates increased MNV-1 replication in BMDC is currently unclear. Free ISG15 has been shown to impact host immune responses in both positive and negative manners. ISG15 can function as a cytokine in vitro to stimulate production of IFN-γ (33, 62) and has also been reported to function as a neutrophil chemoattractant (63). We have also shown that free ISG15 protects against lethal Chikungunya virus (CHIKV) infection (24). In this model, free ISG15 functions as a negative regulator of the immune response, since levels of the proinflammatory cytokines IL-1β, IL-6, and TNF-α were elevated in CHIKV-infected ISG15−/− pups compared to WT and UbE1L−/− pups (24). Additional studies will be required to determine the free ISG15 mechanism(s) of action and whether it plays a role in any other cell type during MNV-1 infection.
Analysis of ISG15 protein expression in BMDC revealed that very little ISG15 is expressed during MNV-1 infection (Fig. 1C). We have also observed a lack of ISG54 and ISG56 expression under similar conditions (data not shown). This likely explains why the levels of MNV-1 replication were similar in untreated WT, ISG15−/−, and UbE1L−/− BMDC. Previous work has shown that MNV-1 controls ISG expression with the help of the ORF4-encoded VF1 protein, as cells infected with a VF1-deficient virus (M1) induced CXCL10, ISG54, and ISG56 transcripts more quickly and to significantly higher levels than WT-infected cells (8). Moreover, M1-infected cells also had robust increases in IFN-β transcripts at 20 to 24 h postinfection which correlated with significant increases in IFN-β secretion (8). During MNV-1 infection, uninfected bystander cells exposed to IFN are able to upregulate ISGs, including ISG15. Mimicking these conditions in vitro, WT cells were found to express free ISG15, ISG15 conjugates, and UbE1L to levels comparable to those seen with IFN treatment alone (Fig. 1C, lanes 4 to 6 and 10 to 12), indicating that MNV-1 was not inducing ISG15 or UbE1L degradation. ISG15 expression is therefore induced and capable of mediating its effector function when IFN is present during MNV-1 infection.
In conclusion, we have identified ISG15 as an important type I IFN effector protein with activity against MNV-1. ISG15 is the first canonical type I IFN effector identified that regulates MNV-1 infection under conditions of a single round of replication. Both in vitro and in vivo, ISG15 conjugate-dependent effects were able to suppress MNV-1 replication. These studies have revealed a novel role for ISG15 impacting early steps of a viral life cycle which precede genome replication. Dissecting how ISG15 mitigates these early MNV-1 life cycle processes will further enhance our understanding of the diverse mechanisms by which ISG15 functions as a host antiviral molecule and should also provide additional insight into the MNV-1 entry process.
ACKNOWLEDGMENTS
We thank Anthony French and Herbert “Skip” Virgin for providing helpful discussions and advice for preparation of the manuscript. We also thank S. Takeshita for providing reagents.
This work was supported by NRSA T32 AR07279-30 to M.R.R. and by NIH grant RO1 A1080672 and a Pew Scholar Award to D.J.L. Experimental support was provided by the Speed Congenics Facility of the Rheumatic Disease Core Center (P30 AR048335).
Footnotes
Published ahead of print 4 June 2014
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