Defining the distinct, intrinsic properties of the novel type I interferon, IFNϵ

The type I interferons (IFNs) are a family of cytokines with diverse biological activities, including antiviral, antiproliferative, and immunoregulatory functions. The discovery of the hormonally regulated, constitutively expressed IFNϵ has suggested a function for IFNs in reproductive tract homeostasis and protection from infections, but its intrinsic activities are untested. We report here the expression, purification, and functional characterization of murine IFNϵ (mIFNϵ). Recombinant mIFNϵ (rmIFNϵ) exhibited an α-helical fold characteristic of type I IFNs and bound to IFNα/β receptor 1 (IFNAR1) and IFNAR2, but, unusually, it had a preference for IFNAR1. Nevertheless, rmIFNϵ induced typical type I IFN signaling activity, including STAT1 phosphorylation and activation of canonical type I IFN signaling reporters, demonstrating that it uses the JAK–STAT signaling pathway. We also found that rmIFNϵ induces the activation of T, B, and NK cells and exhibits antiviral, antiproliferative, and antibacterial activities typical of type I IFNs, albeit with 100–1000-fold reduced potency compared with rmIFNα1 and rmIFNβ. Surprisingly, although the type I IFNs generally do not display cross-species activities, rmIFNϵ exhibited high antiviral activity on human cells, suppressing HIV replication and inducing the expression of known HIV restriction factors in human lymphocytes. Our findings define the intrinsic properties of murine IFNϵ, indicating that it distinctly interacts with IFNAR and elicits pathogen-suppressing activity with a potency enabling host defense but with limited toxicity, appropriate for a protein expressed constitutively in a sensitive mucosal site, such as the reproductive tract.

The type I interferons (IFNs) are a family of cytokines with diverse biological activities, including antiviral, antiproliferative, and immunoregulatory functions. The discovery of the hormonally regulated, constitutively expressed IFN⑀ has suggested a function for IFNs in reproductive tract homeostasis and protection from infections, but its intrinsic activities are untested. We report here the expression, purification, and functional characterization of murine IFN⑀ (mIFN⑀). Recombinant mIFN⑀ (rmIFN⑀) exhibited an ␣-helical fold characteristic of type I IFNs and bound to IFN␣/␤ receptor 1 (IFNAR1) and IFNAR2, but, unusually, it had a preference for IFNAR1. Nevertheless, rmIFN⑀ induced typical type I IFN signaling activity, including STAT1 phosphorylation and activation of canonical type I IFN signaling reporters, demonstrating that it uses the JAK-STAT signaling pathway. We also found that rmIFN⑀ induces the activation of T, B, and NK cells and exhibits antiviral, antiproliferative, and antibacterial activities typical of type I IFNs, albeit with 100 -1000-fold reduced potency compared with rmIFN␣1 and rmIFN␤. Surprisingly, although the type I IFNs generally do not display cross-species activities, rmIFN⑀ exhibited high antiviral activity on human cells, suppressing HIV replication and inducing the expression of known HIV restriction factors in human lymphocytes. Our findings define the intrinsic properties of murine IFN⑀, indicating that it distinctly interacts with IFNAR and elicits pathogen-suppressing activity with a potency enabling host defense but with limited toxicity, appropriate for a protein expressed constitutively in a sensitive mucosal site, such as the reproductive tract.
The type I interferons (IFNs) are a family of cytokines comprising ϳ20 members, including 14 ␣ subtypes and one of each ␤, , , ⑀, , , and (1), that are critical in regulating innate and adaptive responses to infection and tumorigenesis. They induce this protection by a myriad of effects on cells, including the activation of antiviral and antibacterial states and regulation of cell proliferation, migration, and survival. In addition, the wellcharacterized "conventional" type I IFNs, such as IFN␣ subtypes and IFN␤, can regulate the development and activation of virtually every effector cell of the innate and adaptive immune response (2). Members of the type I IFN family of cytokines can promote survival of activated T and B cells (3,4), activate natural killer (NK) 5 cells (5), induce MHC-I up-regulation (6), and provide signals for dendritic cell maturation (7,8). Their importance in host defense is underscored by the conservation of a multicomponent, species-specific type I IFN family found throughout vertebrates.
We identified the gene encoding the newest member of the type I IFN family of cytokines, IFN⑀, in the IFN locus on human chromosome 9 and the syntenic mouse chromosome 4 (9). We also showed it was unique in being constitutively expressed in the female reproductive tract and regulated by hormones but not by pathogens (10). Using IFN⑀ Ϫ/Ϫ mice, we demonstrated that this new IFN was important in protection from herpes simplex virus 2 and Chlamydia infections of the reproductive tract (10). However, the mechanism of action was unclear in these studies because the intrinsic properties of IFN⑀ protein had not been elucidated. Although some studies have proposed antiviral protection by IFN⑀ constructs in mucosal immune responses, no protein product was characterized (10 -12).
Therefore, to complement in vivo studies and to facilitate further work in murine models to understand the functions of this distinct protein, we undertook to define the intrinsic properties of murine IFN⑀. Here, we report the identification and characterization of the mature form of a mammalian IFN⑀, specifically the production and purification of recombinant murine (rm) IFN⑀, and have profiled its physicochemical and biological properties. rmIFN⑀ showed the same broad range of biological activities (antiviral, antiproliferative, and immunoregulatory) as conventional IFNs ␣ and ␤, but its potency was significantly lower. Consistent with this, we found that rmIFN⑀ had a low affinity for binding IFNAR components relative to conventional type I IFNs. Another clear difference between rmIFN⑀ and conventional type I IFNs was its high activity on human cells, which confirms its distinct interaction with the IFNAR receptor, a property that will make it suitable for study in humanized mouse models of disease. Indeed, we demonstrate here that rmIFN⑀ induces HIV restriction factors and inhibits HIV replication in human T cells. Thus, we present new and critical data on the range and potency of a novel cytokine, murine IFN⑀, with unique characteristics fit for purpose as it functions to regulate mucosal immunity in the female reproductive tract.

Expression and physicochemical characterization of rmIFN⑀
As a first step in characterizing the physicochemical and biological properties of murine IFN⑀, it was essential to elucidate where the signal peptide of this protein was cleaved to generate the mature, secreted protein as is the case with previously characterized type I IFNs. The Ifne1 gene was expressed under the control of a CMV promoter and transiently transfected into HEK293 cells. Supernatants from these cells were found to contain a protein of ϳ20 kDa detected by SDS-PAGE and immunoblotting with an anti-IFN⑀ monoclonal antibody (Fig. 1A, left  panel). Immunoprecipitation of IFN⑀ from these supernatants led to the visualization of a band at ϳ20 kDa on Coomassiestained SDS-PAGE that was not seen when immunoprecipitation was carried out with an isotype control antibody (Fig. 1A, right panel). Amino-terminal sequencing of this 20-kDa protein identified six amino acid residues, LEPKRI, representing residues 22-27 of the rmIFN⑀ protein (RefSeq accession number NP_796322) (Fig. 1B). This result indicated that the mature IFN⑀ polypeptide began at leucine 22 of the published sequence for rmIFN⑀ (RefSeq accession number NP_796322) and, therefore, that the mature protein has a theoretical molecular mass of 20,006 Da (13).

Insect cell-expressed rmIFN⑀ has an ␣-helical fold
For physicochemical and biological characterization, rmIFN⑀ was produced in a baculovirus expression system and purified

Properties of novel type I interferon, IFN⑀
using an immunoaffinity chromatography column coupled with an anti-IFN⑀ monoclonal antibody. Analysis of the purified protein by SDS-PAGE (Fig. 1C, left panel) and Western blotting (Fig. 1C, right panel) revealed the presence of a protein at the size expected for rmIFN⑀ (ϳ20 kDa) that was detected with an anti-IFN⑀ antibody (clone H3). The purified protein was subjected to circular dichroism (CD) spectral analysis to demonstrate the overall protein fold. As can be seen in Fig. 1D, the mean residue ellipticity showed minima at 208 and 222 nm, a profile characteristic of ␣-helical proteins, such as IFN␣ and IFN␤. These data suggest that the ϳ20-kDa protein expressed and purified from insect cell culture had an ␣-helical fold typical of other type I IFNs.

rmIFN⑀ has lower affinity for its cognate receptors
We used microscale thermophoresis (MST) to assess the binding affinities between rmIFN⑀ and recombinant forms of the extracellular domains (ECDs) of mIFNAR1 and mIFNAR2 and compared these results with those obtained with other type I IFNs, rmIFN␣1 and rmIFN␤ (Table 1 and Fig. S1). Our results revealed that rmIFN⑀ had a lower binding affinity for mIF-NAR2-ECD than both rmIFN␣1 and rmIFN␤. The affinity of the rmIFN␣1-mIFNAR2-ECD interaction was 2.18 nM (mean of three independent experiments), similar to previously published studies (14), whereas the mIFN⑀-mIFNAR2-ECD interaction was measured to be 6.58 M, showing ϳ3000-fold lower affinity for mIFNAR2-ECD than rmIFN␣1. For the interaction with mIFNAR1-ECD, we measured the affinity of rmIFN⑀ to be 589 nM (mean of three independent experiments), which is around 46-fold lower compared with the rmIFN␤-mIFNAR1-ECD interaction at 12.7 nM (mean of three independent experiments) but ϳ4-fold higher than the affinity of the rmIFN␣1-mIFNAR1-ECD interaction. These results suggest that rmIFN⑀ has different binding affinities for IFNAR1 and IFNAR2 compared with rmIFN␣1 and rmIFN␤.

Signaling
Following receptor engagement, an early step in IFN signaling is activation of signal transducers and activators of transcription (STAT) proteins, which enter the nucleus to bind interferon-stimulated response elements (ISREs) in the promoters of interferon-regulated genes (IRGs). Therefore, we next investigated whether the rmIFN⑀ would induce activation of STAT1 and whether STAT1 would bind ISRE and IRG promoter-driven signaling reporters.

rmIFN⑀ induces STAT1 phosphorylation
We sought to determine whether rmIFN⑀ activated STAT1 like other type I IFNs. STAT1 phosphorylation on tyrosine 701 was apparent after stimulation of RAW264.7 cells with as little as 3 pmol/ml rmIFN⑀ and was found to increase in a dose-dependent manner ( Fig. 2A). That rmIFN␤ induced phosphorylation of STAT1 at 0.3 pmol/ml, a lower dose than rmIFN⑀, suggested that rmIFN⑀ is less active than rmIFN␤ (Fig. 2B). To investigate whether or not the kinetics of STAT1 activation were different between rmIFN⑀ and rmIFN␤, samples were taken 5, 15, 30, 60, and 120 min following stimulation with a 10 pmol/ml concentration of either IFN. STAT1 phosphorylation occurred as early as 5 min after rmIFN⑀ stimulation, peaking 15-30 min after stimulation and decreasing after 60 -120 min (Fig. 2C). Similarly, rmIFN␤ stimulation resulted in peak STAT1 phosphorylation 5 min after treatment and was found to decrease by 120 min (Fig. 2D) as published previously (15). These results demonstrate that rmIFN⑀ can induce the rapid activation of STAT1, although a higher dose is required to achieve a similar level of activation as seen following stimulation with rmIFN␤.

rmIFN⑀ can activate ISRE and IRG promoter-reporters
To determine the consequence of the aforementioned STAT phosphorylation, we used the STAT-dependent ISRE-luciferase reporter transfected into mouse embryonic fibroblasts (MEFs) to ascertain the relative ability of rmIFN⑀ to drive conventional type I IFN-induced transcription. Our results show that rmIFN⑀ induced an ISRE-luciferase response in a dose-dependent manner, consistent with its induction of STAT1 (Fig.  2E). To confirm this result, another luciferase reporter was utilized. This reporter consisted of a cloned promoter 700 bp upstream of the transcription start site of the IRG Isg15. rmIFN⑀ was also able to achieve a similar dose-dependent luciferase induction with this construct, albeit more weakly than rmIFN␤ (Fig. 2F). These data suggest that rmIFN⑀ can induce STAT1 phosphorylation and signaling and the transcription of canonical IRGs via ISRE promoter elements.

Biological activities of rmIFN⑀
Type I IFNs are well-characterized in regard to their abilities to induce antiviral, antiproliferative, and immunoregulatory states in cells (16). We investigated the ability of rmIFN⑀ to induce these responses in cells in comparison with rmIFN␣1 or rmIFN␤ in vitro. Table 1 Specific activities and properties of rmIFN␣1, rmIFN␤, and rmIFN⑀ The concentrations at which the IFNs exhibit 50% of the maximal response (EC 50 or IC 50 as indicated) for each of the antiviral, antiproliferative, and antibacterial responses on mouse cells are shown. The dissociation constant for binding to recombinant forms of mouse IFNAR1-ECD and IFNAR2-ECD are also given.

Interferon
Specific activity a

rmIFN⑀ demonstrates antibacterial activity against Chlamydia
Mice lacking IFN⑀ have increased susceptibility to Chlamydia infection in the reproductive tract, and treatment with rmIFN⑀ in vivo protects against this infection (10). To ascertain the ability of rmIFN⑀ to directly (i.e. not via immune cell activation) exert antibacterial effects on epithelial cells, we treated a mouse epithelial cell line (LA4 cells) with rmIFN⑀ or rmIFN␤ before infecting the cells with Chlamydia muridarum in vitro. The proportion of cells with chlamydial inclusions was significantly reduced in a dose-dependent manner following pretreatment with rmIFN⑀ and rmIFN␤ (Fig. 3C). The IC 50 for rmIFN⑀ was 382 pmol/ml compared with the more potent rmIFN␤ (IC 50 ϭ 3.156 pmol/ml). (Fig. 3C). Thus, rmIFN⑀ has direct anti-Chlamydia activity, although it is ϳ100-fold less potent than rmIFN␤ as for its other properties examined above.

Properties of novel type I interferon, IFN⑀ rmIFN⑀ shows immunoregulatory activity on immune cells
Type I IFNs have a well-documented ability to activate immune cells. They have been shown to induce the survival and proliferation of T cells, induce isotype switching of B cells, and potently activate NK cells (3)(4)(5). To investigate the immunoregulatory activities of rmIFN⑀, we stimulated spleen cells from mice ex vivo with increasing doses of rmIFN⑀, rmIFN␣1, or rmIFN␤ for 24 h and measured the cell-surface expression of the lymphocyte activation marker CD69 (19) on several immune cell types. All three IFNs showed a dose-dependent activation of CD4 and CD8 T cells, B cells, and NK cells (Fig. 4,  A and B). The potency of rmIFN⑀ to up-regulate the expression of CD69 on CD4 ϩ , CD8 ϩ , B220 ϩ , and NK1.1 ϩ cells was 100 -1000-fold less than rmIFN␣1 and rmIFN␤, which were similar in potency to each other (Fig. 4B). Up-regulation of CD69 following stimulation by rmIFN⑀ was greatest on B cells and NK cells (Fig. 4A).

Murine IFN⑀ demonstrates activity on human cells
One of the hallmarks of the conventional biological activities of type I IFNs is that they are highly species-specific (20) because the IFNs of one species do not bind to IFNAR receptors of other species. We therefore investigated whether rmIFN⑀ was similarly restricted.

rmIFN⑀ shows antiviral activity and induces an ISRE-luciferase reporter in human cells
We analyzed the ability of rmIFN⑀ and rmIFN␤ to protect WISH (Wistar Institute, Susan Hayflick) cells from infection with encephalomyocarditis virus (EMCV) using the CPE inhibition assay. Surprisingly, rmIFN⑀ demonstrated high antiviral activity in human cells, exhibiting an IC 50 of 0.36 pmol/ml (Fig.  5A), equivalent to a specific antiviral activity of 2.4 Ϯ 0.6 ϫ 10 7 IU/mg on human cells and remarkably 100-fold higher than its activity on mouse cells. By contrast, rmIFN␤, as expected, had no detectable activity on human cells in the same assay (21). To further characterize the cross-species activity of rmIFN⑀, we next performed ISRE-luciferase assays in human HEK293 cells. rmIFN⑀ stimulation was found to produce a strong luminescence signal that increased in a dose-dependent manner after 16 h of stimulation (Fig. 5B). As expected, rmIFN␤ administration to the same cells did not result in a luminescence signal (Fig. 5B).

Murine IFN⑀ restricts HIV infection in human cells in vitro
Our in vivo mouse studies showed that IFN⑀ protected the female reproductive tract from viral infection (10), and more recently, we have also shown that, in vitro, human IFN⑀ restricts HIV infection at multiple stages of the viral cycle (22). Following from these results, in this study, we tested the ability of rmIFN⑀ to protect human cells from HIV infection. First, we demonstrated the antiretroviral activity of this novel IFN using a reporter assay to demonstrate that rmIFN⑀ inhibits HIV replication in a human Sup-T1 cell line (Fig. 5C) and in primary human peripheral blood lymphocytes (PBLs; Fig. S2A) in a dose-dependent manner. Next, we demonstrated the ability of rmIFN⑀ to induce the expression of HIV restriction factors, namely TRIM5, IFITM3, MX2, APOBEC3G, HERC5, and BST2 (tetherin) mRNA, in a human Sup-T1 cell line (Fig. 5D) and primary human PBLs (Fig. S2B). Together, these data demonstrate that rmIFN⑀ has high antiviral activity on human cells.

Discussion
The functions of the type I IFN family have been studied extensively over the last five decades to elucidate the pleiotropic biological roles of the prototypic ␣ and ␤ subtypes. However, the newest type I IFN to have been discovered, IFN⑀ (9), is unique because it is constitutively expressed and hormonally regulated. These properties, together with our studies of IFN⑀deficient mice, suggest a distinct function for IFN⑀. However, there were no data on the intrinsic properties of this protein.
The data we present here represent critical specifications on the range and potency of the intrinsic properties of a novel cytokine, murine IFN⑀. These data will complement our interpretation of the functions of IFN⑀ inferred from experiments in IFN⑀ Ϫ/Ϫ mice. The availability of a recombinant murine form of this novel cytokine and knowledge of the nature and potency of its intrinsic actions will help elucidate its mechanism of action and potential therapeutic applications based on preclinical murine models of disease.

Properties of novel type I interferon, IFN⑀
As murine IFN⑀ had not been purified from biological tissues, it was important to determine the precise sequence of the mature protein. The only available information on the mature amino terminus was via prediction software (9,23), which showed conflicting results in defining the IFN⑀ signal peptide. We therefore expressed IFN⑀ in mammalian cells and performed amino-terminal sequencing. Identification of leucine 22 as the first residue of the mature mIFN⑀ is as predicted (23) and aligns with the amino-terminal residue identified for canine IFN⑀ (24) and with the first residue of other mature IFNs, including IFN␤ from numerous species. It is, however, different from IFN␣s that tend to have a signal peptide of 23 residues in length and begin at residue 24 of the proprotein (Fig.  S3). Having identified the amino-terminal sequence of mature mIFN⑀, we constructed a baculovirus expression construct for the production of a recombinant form of this protein. Antibody affinity chromatography yielded a preparation that was highly pure, endotoxin-free, and, according to CD spectral analysis, folded into an ␣-helical secondary structure in line with that of other type I IFN family members (25,26).
The type I IFN system represents an interesting paradigm among cytokines whereby a myriad of biological activities are elicited by about 20 distinct but related proteins signaling via a common cell-surface receptor complex. The subtleties that govern these responses remain unclear (27)(28)(29)(30)(31). Sequence alignment of mIFN⑀ with mIFN␤, mIFN␣1, and hIFN␣2 reveals the degree of homology of the IFNs within the known IFNAR1-and IFNAR2-binding interfaces, suggesting differences in the way these IFNs might interact with their receptors (Fig. S3). Analysis of this multiple alignment reveals that mIFN⑀ is most similar at the amino acid level to mIFN␤ and that over-all, between the IFNs, residues are more conserved within the receptor interface regions than in other portions of the IFNs (Fig. S3). Although we have reported previously that mIFN⑀ signaling required both IFNAR1 and IFNAR2, we did not determine direct interactions. In the present study, we assessed direct interactions and determined the binding affinity of IFN⑀ for IFNAR components using MST. This analysis unexpectedly demonstrated that mIFN⑀ bound mIFNAR1-ECD with higher affinity than mIFNAR2-ECD, different from data reported for the IFN␣, which has a higher affinity for IFNAR2 (29). As expected, the affinity of the mIFN␤-IFNAR1 interaction measured by MST was consistent with our previous report using surface plasmon resonance (32). Notably, the affinity of the mIFN␤-IFNAR1 interaction was higher than and the mIFN␤-IFNAR2 interaction lower than these interactions reportedly measured using human proteins (29). In line with their secondary structure conservation, the relative affinities for IFNAR1 and IFNAR2 suggest that mIFN⑀ is more similar to mIFN␤ than to mIFN␣. Nevertheless, we have reported a non-canonical IFN signaling pathway mediated by interactions between murine IFN␤ and IFNAR1 in the absence of IFNAR2 (27), providing a precedent for selective interaction of IFNs with specific receptor chains.
These distinct properties of the IFN⑀-IFNAR interaction are also evidenced by the cross-species reactivity of rmIFN⑀ we have demonstrated here on human cells. This feature contrasts with conventional type I IFNs whose interactions with their cognate receptors are such that murine IFN␣ and -␤ do not bind human IFNARs. This feature of IFN⑀ is consistent with studies of canine IFN⑀, which was also shown to exhibit crossspecies activities (24). This cross-species activity of murine IFN⑀ might also have a practical advantage. For example, it may

Properties of novel type I interferon, IFN⑀
be useful in "humanized" mouse models established to study HIV, which have not been utilized to examine IFN⑀ previously (33). To this end, we demonstrated that rmIFN⑀ protects human PBLs and T cell lines from HIV infection. Consistent with this finding, this new IFN induced the expression of several HIV restriction factors that are active at different stages of the HIV replication cycle. Therefore, humanized mouse models could be used in the future to dissect the role of endogenous IFN⑀ in the early stages of infection by HIV.
In addition to the unique differential interactions of rmIFN⑀ with IFNAR1 and IFNAR2 components, these interactions exhibited 100 -1000-fold lower affinity than interactions with rmIFN␤ and rmIFN␣, respectively. Although it may seem unusual that a biologically important protein had such a low affinity for receptors, there are parallels in IFN␣ subtypes that also vary in affinity and activity to a similar extent (29). The low affinity of IFNAR binding is consistent with the low biological activities of IFN⑀ relative to conventional type I IFN␣ and/or -␤ (Table 1). All signaling and bioactivity measurements for rmIFN⑀, which we showed to activate classical JAK-STAT pathways, including STAT1 phosphorylation, IRG reporter transactivation, and antiviral, antibacterial, and immunoregulatory activities, were at least 100-fold lower than conventional IFNs (Table 1). This is consistent with the activities of human  (TRIM5␣, MX2, HERC5, IFITM3, APOBEC3G, and BST2 (tetherin)) and normalized to 18S rRNA. Results were expressed as a relative change using the ⌬⌬Ct method. The Sup-T1 cells data shown are a mean of three independent experiments for each performed in duplicate (error bars represent S.D.). No significant differences between cell lines were found at the gene expression baseline levels. Significance of responses was calculated using one-way ANOVA with Tukey's multiple comparison testing (*, p Ͼ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001; ****, p Ͻ 0.0001). US, unstimulated. (11) and canine (29) IFN⑀ expressed in bacterial systems, which were characterized to have similarly low specific activity.

Properties of novel type I interferon, IFN⑀
Although IFN⑀ activities are relatively low for a type I IFN, they are obviously sufficient to have protective effects against viral and bacterial infections in vivo (10). As such, it is important to note that we have demonstrated for the first time that murine IFN⑀ indeed does have significant intrinsic activities to protect cells from viral or bacterial infection and activates T, B, and NK cells. Presumably, it is the combination of these actions of IFN⑀ and/or its constitutive, compartmentalized expression that provides a unique, tissue-specific type I IFN profile sufficient for critical in vivo efficacy.
Indeed, the low affinity of receptor interaction for IFN⑀ may be an advantage by limiting the potential toxicity associated with conventional IFNs. Furthermore, this low affinity may enable IFNe to be constitutively expressed without causing internalization of the IFNAR receptor that would render these cells refractory to conventional type I IFNs, a critical component of host defense. Such a distinct mechanism of action may be of particular biological importance in a site, such as the female reproductive tract, that must be protected from infection yet remain tolerant to implantation of a semiautologous embryo during reproduction. Indeed, IFN⑀ expression is tightly regulated during the estrous cycle and pregnancy in mice and is lowest at the time of embryo implantation (10). It is therefore likely that the unique nature of IFN⑀ engagement with the type I IFN receptor that we demonstrate here results in tailored biological activities that are appropriate in nature, strength, and duration for its location and functions.

Identification of amino-terminal cleavage site
The mouse Ifne1 gene was amplified from C57Bl/6 genomic cDNA and cloned downstream of a human CMV promoter to generate pCMV-Ifne-IRES-mCitrine. 2 g of pCMV-Ifne1-IRES-mCitrine was transfected into 1 ϫ 10 6 HEK293 cells using FuGENE 6 according to the manufacturer's instructions (Promega, Sydney) and incubated for 72 h. The culture supernatant was harvested, and endogenous immunoglobulins were cleared from the medium with Protein G beads (GE Healthcare). mIFN⑀ was immunoprecipitated with 60 g of anti-IFN⑀ monoclonal antibody (clone H3; generated in-house; see below) and Protein G beads. PBS-washed Protein G beads were boiled in the presence of 5ϫ Laemmli buffer, and proteins were separated by 15% (v/v) SDS-PAGE. Proteins were transferred to PVDF membrane (Merck Millipore) and stained with Coomassie Blue R-250 (Sigma-Aldrich). The 20-kDa protein band of interest was excised, and the amino-terminal sequence was determined by amino-terminal sequencing at the Monash University Proteomics Facility.

Plasmid construction for insect cell expression
The Ifne1 nucleotide sequence was codon-optimized for expression in insect cells and corresponds to amino acid residues 22-192 of murine IFN⑀ (RefSeq accession number NP_796322). The 522-bp codon-optimized Ifne1 sequence was cloned into a modified pFastBac TM vector (Life Technologies) containing the honeybee melittin signal peptide (MSP), hereafter referred to as pFB-MSP. pFB-MSP was a kind gift from Kathryn Hjerrild (Hudson Institute of Medical Research). pFB-MSP-Ifne1 was transformed into JM109 cells, and colonies were screened for inserts using M13 forward (5Ј-GTACAATTGGAACCAAAGCGCA) and reverse (5Ј-GCAAGCTTTCATGGGTCAGGGTCT) primers. Positive clones were sequence-verified using the polyhedrin sequencing primer (5Ј-AAATGATAACCATCTCGC).

Generation of recombinant bacmid and baculovirus and expression of recombinant IFN⑀
The generation and PCR screening of recombinant bacmid and baculovirus were carried out as described previously (35). Briefly, PCR-positive colonies were expanded, and recombinant bacmid was isolated using an EndoFree Maxi-Prep kit according to the manufacturer's instructions (Qiagen). Recombinant baculovirus was generated by transfection of the purified bacmid into Sf9 insect cells, and high-titer baculovirus was generated as described previously (35). All recombinant protein expressions were carried out as described previously (35). The construct was designed so that rmIFN⑀ would be expressed as a soluble protein and secreted into the culture medium.

Preparation of antibody affinity column
Standard laboratory protocols were used to scale up the monoclonal antibody production. The hybridoma clone, designated H3, was tested for potential Mycoplasma contamination, and upon confirming it was negative, the clones were adapted

Properties of novel type I interferon, IFN⑀
to low (Ͻ2%) FBS and Gibco Hybridoma serum-free medium (Thermo, catalog number 12045076). After sufficient cell density was achieved, a 10-liter working volume Wave bioreactor (GE Healthcare) was inoculated. Culture was scaled up to 5.5 liter, and feeding with glucose, GlutaMAX-I (Thermo, catalog number 35050061), and Phytone (BD Biosciences, catalog number 292450) was initiated. Culture was harvested when cell viability dropped below 50%. Cells and cell debris were removed by centrifugation followed by a 0.2-m filtration. Monoclonal antibodies were captured on HiTrap MabSelect Xtra columns (GE Healthcare; 4 ϫ 5-ml columns) with the bound protein eluting at low pH (0.1 M citrate, 0.1 M NaCl, pH 3) with immediate neutralization with 3 M Tris, pH 8.1. Analytical size exclusion chromatography indicated that the antibody eluted at the expected volume with no aggregate detected. Ten milligrams of the purified monoclonal antibody was then coupled to AminoLink Plus resin according to the manufacturer's instructions (Thermo Scientific). The resin was poured into the supplied column for use as a monoclonal antibody affinity chromatography column. Each column was used five times before being discarded and replaced with freshly coupled resin.

Purification of recombinant IFN⑀
Insect cell culture supernatants were clarified of cells by centrifugation as described (35) and supplemented with phenylmethanesulfonyl fluoride (PMSF) at a final concentration of 1 mM before dialysis against Tris-buffered saline (TBS; 10 mM Tris-HCl, 150 mM NaCl, pH 8.0) overnight at 4°C using 12.5-kDa-cutoff dialysis tubing (Sigma-Aldrich). Particulates were removed by filtration of the dialysate through a 0.8-m syringedriven filter (Sartorius). The filtrate was applied to the anti-IFN⑀ monoclonal antibody affinity chromatography column prepared above, and the column was washed with five column volumes (CVs) of TBS to remove nonspecifically bound proteins. Bound rmIFN⑀ was eluted with 0.1 M glycine, pH 3.0, in 0.5ϫ CV fractions. Collected fractions were immediately neutralized with 0.1 CV of 1 M Tris-HCl, pH 8.0, and buffer-exchanged by addition of 10ϫ TBS (100 mM Tris-HCl, 1.5 M NaCl, pH 8.0). Protein-containing fractions, as determined by absorbance at 280 nm, were further supplemented with 10% (v/v) glycerol. Purified fractions were filter-sterilized and stored at 4°C or snap frozen in liquid nitrogen for long-term storage at Ϫ80°C.

Determination of protein concentration and endotoxin levels
Protein concentrations were determined by a standard Bradford colorimetric assay. Endotoxin levels were determined as described previously (35) using the ToxinSensor Endotoxin Assay kit according to the manufacturer's instructions (Genscript). Endotoxin concentrations were calculated as endotoxin units/g of protein.

CD spectral analysis
The secondary structure of IFNs was determined on a Jasco J815 CD spectrophotometer at room temperature. Proteins were scanned at a concentration of 130 g/ml in TBS, and triplicate scans between 190 and 260 nm were recorded. Data were converted to mean residue ellipticity by the equation of Correa and Ramos (37).

Microscale thermophoresis
For MST, mIFNAR1-ECD and rmIFN␤ were expressed and purified from mammalian cell and insect cell culture, respectively, as described previously (35), and mIFNAR2-ECDC94S and rmIFN␣1 were expressed and purified from mammalian cell culture also as described previously (27). Affinity measurements using MST were carried out with a Monolith NT.115 instrument (NanoTemper Technologies) as described previously (38,39). mIFNAR1-ECD and mIFNAR2-ECDC94S were labeled using the NHS RED NanoTemper labeling kit according to the manufacturer's instructions. For the assay, 5 l of labeled protein was mixed with 10 l of the unlabeled IFNs (rmIFN␣1, rmIFN␤, and rmIFN⑀) at various concentrations and 5 l of 0.05% (w/v) Tween 20. All experiments were incubated for 30 min before applying samples to Monolith NT standard treated capillaries (NanoTemper Technologies). Thermophoresis was measured at 25°C with laser off/on/off times of 5/30/5 s. Experiments were conducted at 20% light-emitting diode power and 40% MST IR laser power. Data from three independently performed experiments were fitted to the single binding model (NT.Analysis software version 1.5.41, NanoTemper Technologies) using the signal from thermophoresis ϩ T-jump.

Luciferase assays
Luciferase assays were performed in immortalized Ifnar1 Ϫ/Ϫ MEFs or in HEK293 cells that were either stably transfected with ISRE-luciferase reporter as described previously (34) or transiently transfected with an Isg15-luciferase reporter containing the upstream 700 bp of the Isg15 transcriptional start site. The Isg15-luciferase construct was kindly provided by Prof. Paula Pitha. Briefly, 2 ϫ 10 4 cells/well in a 24-well plate were transfected with 30 ng of ISRE-luciferase, 100 ng of thymidine kinase-Renilla reporter, 1 ng of mIFNAR1, 1 ng of mIFNAR2 (for HEK293 cells only), and up to 0.5 g of pEF-BOS with FuGENE 6 according to the manufacturer's instructions (Roche Applied Science). The cells were incubated for 24 h before being stimulated with IFN for 16 (HEK293 cells) or 8 h

Antiviral assays
Antiviral assays were performed using the CPE inhibition assay as described previously on mouse L929 cells challenged with Semliki Forest virus (17) or WISH cells challenged with EMCV. Activities were normalized against National Institutes of Health reference standards (mouse, GU-02-901-511; or human, GA23-901-532) where 1 international unit (IU) is the amount of IFN required to provide protection to 50% of virusexposed cells (IC 50 ).

C. muridarum infection
LA4 (mouse lung epithelial) cells were infected as described previously (40). In brief, cells were plated at 3 ϫ 10 4 on 10-mm glass coverslips in a 48-well culture plate in antibiotic-free Dulbecco's modified Eagle's medium/F-12 supplemented with 10% heat-inactivated FBS, 25 mM HEPES, and L-glutamine until Ͼ80% confluent (ϳ48 h). Cells were incubated for 24 h in the presence of various concentrations of rmIFN⑀, rmIFN␤, or PBS (untreated control). When monolayers were Ͼ80% confluent, cells were washed and then infected with Chlamydia (multiplicity of infection, 5/20) for 3 h. Cells were washed, and one of rmIFN⑀, rmIFN␤, or PBS was added again for a further 16 h. Chlamydial inclusions were stained using a Chlamydia Cel LPS kit according to the manufacturer's instructions (CelLabs). Intracellular chlamydial inclusions were labeled with fluorescein isothiocyanate, and cell nuclei were counterstained with rhodamine. The numbers of cell-associated inclusions per field were determined from each treatment with an average of 10 fields determined per coverslip and three to six coverslips per group at 40ϫ magnification using a fluorescence microscope (Zeiss Axio Imager M2). Each condition was run in at least triplicate. IC 50 was calculated using the nonlinear regression function in GraphPad Prism software and reported as pmol/ml of IFN required to inhibit formation.

HIV luciferase reporter infection of Sup-T1 cells and PHA-activated PBL cells
HIV luciferase reporter virus was produced by cotransfection of HIV envelope construct (pNLA1) and HIV pNL4-3 Luc Rev(Ϫ) construct at a ratio of 1:4 into 293T cells using polyeth-ylenimine (PEI; Sigma). At 48 h post-transfection, viruses were harvested, cleared, and concentrated as described previously (22). Virus concentration was estimated with the HIV p24 CA antigen capture assay according to the manufacturer's instructions (Xpress Bio). 24 h before infection, Sup-T1 cells or PHAactivated PBL cells were treated with different concentrations of rmIFN⑀ or hIFN␤. 500 or 48 ng of HIV p24 capsid proteinequivalent virus particles were used to infect PBL cells (100,000/well) and Sup-T1 cells (100,000/well), respectively. 72 h postinfection, luciferase activity was measured using a Fluoroskan Ascent FL luminometer (Bright-Glo Luciferase Assay System, Promega). The amount of detectable luciferase activity reflected the relative levels of viral infectivity.

HIV infection of HeLa-based TZM-bl cells
TZM-bl cells were infected with NL4-3 WT virus produced via transfection of the pNL4-3 WT plasmid into 293T cells with PEI. Virus harvest, purification, concentration, and quantification were as described above. TZM-bl (10,000/well) cells were treated with 12.5 or 125 nmol of rmIFN⑀ or 0.0125 or 0.125 nmol of hIFN␤, and luciferase activity was measured in a Fluoroskan Ascent FL luminometer (Bright-Glo Luciferase Assay System) 48 h postinfection (41).

Extraction of RNA and cDNA synthesis for quantitative real-time PCR
To evaluate gene expression by quantitative real-time PCR, following treatment with 100 nmol of rmIFN⑀, RNA was extracted using the RNeasy kit (Qiagen) from Sup-T1 or PBL cells at different time points (0, 2, 4, 8, 12, 18, and 24 h posttreatment). RNA was treated with DNase (Promega), and cDNA was synthesized using Moloney murine leukemia virus and random hexamers (Promega). Reverse transcription products of TRIM5␣, MX2, HERC5, IFITM3, APOBEC3G, BST2 (tetherin), and 18S were quantified using previously published primers (22,42). RT-quantitative PCR was performed using SYBR reagents (ABI). Results were normalized to 18S rRNA and expressed as relative change using the ⌬⌬Ct method.