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. Author manuscript; available in PMC: 2014 Aug 15.
Published in final edited form as: Cell Host Microbe. 2013 Aug 14;14(2):136–147. doi: 10.1016/j.chom.2013.07.005

Enveloped Viruses Disable Innate Immune Responses in Dendritic Cells by Direct Activation of TAM Receptors

Suchita Bhattacharyya 1,5, Anna Zagórska 2,5, Erin D Lew 2, Bimmi Shrestha 3, Carla V Rothlin 4, John Naughton 1, Michael S Diamond 3, Greg Lemke 1,2,*, John AT Young 1,*
PMCID: PMC3779433  NIHMSID: NIHMS509158  PMID: 23954153

SUMMARY

Upon activation by the ligands Gas6 and Protein S, TAM receptor tyrosine kinases promote phagocytic clearance of apoptotic cells and downregulate immune responses initiated by Toll-like receptors and type I interferons (IFNs). Many enveloped viruses display the phospholipid phosphatidylserine on their membranes, through which they bind Gas6 and Protein S and engage TAM receptors. We find that ligand-coated viruses activate TAM receptors on dendritic cells (DCs), dampen type I IFN signaling, and thereby evade host immunity and promote infection. Upon virus challenge, TAM-deficient DCs display type I IFN responses that are elevated in comparison to wild-type cells. As a consequence, TAM-deficient DCs are relatively resistant to infection by flaviviruses and pseudotyped retroviruses, but infection can be restored with neutralizing type I IFN antibodies. Correspondingly, a TAM kinase inhibitor antagonizes the infection of wild-type DCs. Thus, TAM receptors are engaged by viruses in order to attenuate type I IFN signaling and represent potential therapeutic targets.

INTRODUCTION

Virus infection of vertebrate cells elicits innate immune responses that are triggered by type I interferons (IFNs) and proinflammatory cytokines (Zuniga et al., 2007). These immune responses are initially induced by the recognition of virus nucleic acids by host pattern recognition receptors, which include Toll-like receptors (TLRs), RIG-I-like receptors, and cytosolic DNA sensors (Thompson et al., 2011). After virus engagement, these sensors activate signal transduction pathways that induce type I IFNs (multiple IFNα and IFNβ), which, in turn, stimulate the production of antiviral cellular restriction factors in order to control virus replication (Yan and Chen, 2012). Consequently, pathogenic viruses have evolved specific countermeasures for evading or interfering with these protective host responses (Yan and Chen, 2012).

The TAM receptor tyrosine kinases (RTKs) Tyro3, Axl, and Mer (Lai and Lemke, 1991; Lemke, 2013; Lemke and Rothlin, 2008) and their cognate ligands Protein S and Gas6 (Stitt et al., 1995) are negative regulators of the innate immune response to microbial infection. They are activated at the end of this response (Rothlin et al., 2007) and exert their immunosuppressive functions through two interlinked mechanisms. First, they promote the rapid phagocytic clearance of apoptotic cells (ACs) by macrophages, dendritic cells (DCs), and other dedicated phagocytes (Lemke and Burstyn-Cohen, 2010; Lemke and Rothlin, 2008). Both Protein S and Gas6 have a γ-carboxylated “Gla domain” at their amino termini that allows them to bind to phosphatidylserine (PtdSer), which is displayed on the surface of ACs. This phospholipid is among the most common and potent of the “eat me” signals through which ACs are recognized by phagocytes (Ravichandran, 2011), given that PtdSer is normally confined to the inner leaflet of the plasma membrane bilayer in healthy cells. Then, through their carboxy termini, Protein S and Gas6 bind and activate TAM receptors that are expressed on the surface of phagocytes, thereby “bridging” the phagocyte to the AC that it will engulf. In a second, mechanistically-linked action, the binding of Gas6 or Protein S to TAM receptors on macrophages or DCs activates a negative feedback loop that inhibits innate immune responses initiated by TLR and type I IFN signaling pathways (Lemke and Lu, 2003; Lemke and Rothlin, 2008; Lu and Lemke, 2001; Rothlin et al., 2007). In DCs, this negative feedback is achieved through Axl-mediated induction of the genes encoding the suppressor of cytokine signaling (SOCS) proteins 1 and 3 (Rothlin et al., 2007; Yoshimura et al., 2012).

Several gain-of-function studies have implicated TAM receptor-ligand interactions in promoting infection by enveloped viruses. Ectopic expression of one or more TAM receptors into infection-resistant cell lines (e.g., human embryonic kidney [HEK] 293T cells) has been found to potentiate infection by both filoviruses (e.g., Ebola virus) and HIV-derived model lentiviruses (Brindley et al., 2011; Hunt et al., 2011; Morizono et al., 2011; Shimojima et al., 2007; Shimojima et al., 2012; Shimojima et al., 2006). A recent study extends these findings to TAM potentiation of infection by Dengue (DENV) and West Nile (WNV) viruses (Meertens et al., 2012)—two flaviviruses that are global health concerns (Bhatt et al., 2013; Suthar et al., 2013). TAM receptor facilitation of viral infection has been interpreted generally in the context of the TAM ligand bridging activity outlined above for ACs, given that many enveloped viruses—including WNV, DENV, HIV-1, Ebola, Marburg, Amapari, Tacaribe, Chikungunya, and Eastern Equine Encephalitis viruses, among others—also display PtdSer on the external leaflet of their membrane envelopes (Jemielity et al., 2013; Mercer, 2011). For example, PtdSer on the surface of DENV virions can be detected by PtdSer-specific antibodies and by the PtdSer-binding protein annexin V, and preincubation with annexin V diminishes DENV infectivity (Meertens et al., 2012). Similarly, PtdSer on the HIV-1 envelope is a cofactor for the infection of monocytes, and HIV-1 can be purified with annexin V (Callahan et al., 2003).

These observations have led to the hypothesis that viruses attach and gain access to cells via the imitation of ACs in a PtdSer-dependent process termed “apoptotic mimicry” (Jemielity et al., 2013; Mercer and Helenius, 2008, 2010). For the TAM system, this mimicry does not involve a direct interaction of TAM receptor with virus but rather an interaction between TAM receptor and virions that are opsonized with a TAM ligand (Meertens et al., 2012; Mercer, 2011). For this mechanism to operate, enveloped viruses must be exposed to an environment that contains Protein S or Gas6, and the target cell must express one or more TAM receptors. Given that Protein S is present at ~300 nM in the vertebrate bloodstream (Burstyn-Cohen et al., 2009; García de Frutos et al., 2007), it is likely that many blood-borne enveloped viruses carry this TAM ligand in vivo.

The action of the TAM system during enveloped virus infection has been thought to be restricted to the facilitation of virus attachment and entry into the target cell (Brindley et al., 2011; Frei et al., 2012; Hunt et al., 2011; Meertens et al., 2012; Morizono et al., 2011; Shimojima et al., 2007; Shimojima et al., 2012; Shimojima et al., 2006) (Figure 1A). However, we reasoned that ligand-coated virions should also act as potent TAM agonists because of the multimerization, concentration, and immobilization of Gas6 or Protein S on the virion surface and that activation of TAM receptors by ligand-coated viruses should, in turn, inhibit the type I IFN response in target cells (Rothlin et al., 2007). Accordingly, we hypothesized that cells deficient in TAM receptor expression or signaling should be relatively resistant to virus infection. In this report, we test these predictions. We show that TAM-deficient DCs are less susceptible to infection by retroviruses and flaviviruses, that reduced infectivity is due to elevated type I IFN production, and that a small-molecule TAM kinase inhibitor attenuates virus infection of wild-type (WT) DCs. These results identify a general immune evasion mechanism by which enveloped viruses engage TAM receptors in order to inhibit type I IFN signaling.

Figure 1. Gain- and Loss-of-Function Studies for Evaluating the Impact of TAM Receptors on Virus Infection.

Figure 1

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(A) Existing model of TAM receptor-ligand facilitation of enveloped virus entry. PtdSer, phosphatidylserine.

(B) Immunoblots showing expression of individual TAM receptorsin HEK 293T cells either untransfected (293) or stably transfected with individual TAM receptors.

(C) Levels of HIV-1 viral DNA produced 24 hr postinfection of the indicated HEK 293T transformants (from B) by pseudotyped viruses bearing envelope glycoproteins from VSVg, Ebola, Marburg, or MLV-A viruses. Levels shown were normalized to those obtained with untransfected HEK 293T cells for each pseudotype (defined as 100%, dashed blue line).

(D) Immunoblots showing constitutive activation of all three types of TAM receptor in HEK 293T cells that ectopically express Axl. First, TAM receptors were immunoprecipitated with the indicated antibodies and subjected to SDS-PAGE before being immunobloted with receptor-specific antibodies (IB: Tyro3, Axl, or Mer) or with a phosphotyrosine-specific antibody (IB: pY).

(E) BMDCs generated from WT or specific TAM receptor knockout mice were infected with HIV-1-derived virus pseudotyped with the indicated viral glycoproteins, and the levels of reverse transcribedHIV-1 DNA were measured at 24 hr postinfection. Levels shown were normalized to those seen with WT BMDCs for each pseudotype (100%, dashed blue line). A, Axl; M, Mer; T, Tyro3; KO, single knockout; DKO, double knockout; TKO triple TAM receptor knockout. *p < 0.05; **p < 0.01; ***p < 0.001. Error bars are SEM of samples from three independent experiments. VSVg, EbGP, MARVGP, and MLV-A denote the glycoproteins of vesicular stomatitis, Ebola, Marburg, and amphotropic murine leukemia virus, respectively. See also Figure S1.

RESULTS

Ectopic Expression of TAM Receptors Enhances Virus Infection

First, we employed a gain-of-function approach similar to those used previously (Brindley et al., 2011; Hunt et al., 2011; Meertens et al., 2012; Morizono et al., 2011; Shimojima et al., 2007; Shimojima et al., 2012; Shimojima et al., 2006). Tyro3, Axl, or Mer were each expressed stably in HEK 293T cells, which are not readily infected by HIV-1-derived retroviruses. Using a western blot, we confirmed that the three TAM receptors were each highly expressed in the stably transfected HEK 293T cell lines (Figure 1B). These blots also demonstrated that parental HEK 293T cells express low and moderate levels of endogenous Mer and Tyro3, respectively, but do not express Axl (Figure 1B).

The clonal HEK 293T cell lines were exposed to a single-cycle, replication-incompetent, HIV-1-derived virus vector that was pseudotyped with one of four different viral glycoproteins (GPs)—Ebola virus GP, Marburg virus GP, vesicular stomatitis virus GP (VSVg), or amphotropic murine leukemia virus Env—as model enveloped viruses. Infection experiments were conducted with cell culture medium supplemented with 10% fetal bovine serum (FBS), which contained ~30 nM of the TAM ligand Protein S. (As detailed below, we confirmed that these HIV-1-derived viruses bind Protein S in a Ca+2-dependent manner.) Virus infection was monitored by measuring reverse transcribed HIV-1 DNA produced at 24 hr postinfection (König et al., 2008) (Figure 1C).

Cells ectopically expressing individual TAM receptors were 2-to 10-fold more susceptible to infection by all four pseudotyped viruses than WT HEK 293T cells. Because the viruses used have different particle:infectivity ratios, we conclude that receptor-specific effects on virus infection are largely independent of viral glycoprotein type (Figure 1C). We attempted to determine whether one of more of the TAM receptors were activated (tyrosine phosphorylated) during virus challenge—an assessment that has not yet been made for any viral infection. However, we found that the high-level expression of one receptor (e.g., Axl in 293Axl cells) led to the constitutive activation of all TAM receptors in the same HEK 293T cell, even in the absence of virus (Figure 1D). These data highlight the difficulties associated with using gain-of-function approaches to assign specific functions to individual TAM receptors during virus infection.

TAM Receptors Are Required for Efficient Lentiviral Infection of Dendritic Cells

Therefore, we conducted loss-of-function studies with bone-marrow-derived DCs (BMDCs) prepared from WT or specific TAM gene knockout mice (Lu et al., 1999). WT BMDCs express both Axl and Mer but only express very low levels of Tyro3 (Rothlin et al., 2007). We found that infection by all four pseudotyped viruses was dependent upon BMDC expression of TAM receptors (Figure 1E). As for the HEK 293T cell gain-of-function experiments, these BMDC loss-of-function studies were conducted with serum-supplemented (e.g., Protein S-containing) culture medium. Protein S is a ligand for Mer and Tyro3, but it does not activate Axl (Lemke and Rothlin, 2008; Prasad et al., 2006; Stitt et al., 1995). Serum did not contain appreciable levels of Gas6, the pan-TAM ligand (Lemke and Rothlin, 2008; Stitt et al., 1995; Ekman et al., 2010), and BMDCs did not secrete detectable levels of this protein.

Consistent with Mer expression in BMDCs and this receptor’s ligand specificity, virus infection of BMDCs was strongly dependent on Mer: the low levels of infection seen with TAM triple knockout (TKO) animals were recapitulated with cells derived from either Mer single knockout or Axl-Mer double knockout (AM DKO) mice (Figure 1E). In comparison to Mer and Tyro3 KO, loss of Axl, which, in other cell types, has been found to be a cofactor for virus entry (Brindley et al., 2011; Hunt et al., 2011; Shimojima et al., 2006) and a facilitator of DENV infection (Meertens et al., 2012), had minimal effect on the efficiency of BMDC infection by the viral pseudotypes (Figure 1E). This was consistent with the fact that Gas6, the ligand required for Axl activation and the bridging of Axl to PtdSer on the virus membrane, was not present in these BMDC experiments.

TAM Receptors Potentiate a Postentry Step of Enveloped Virus Replication

To determine whether TAM receptors are associated with increased pseudotyped virus entry into BMDCs, we employed an established HIV-1 reporter system for assaying cytosolic delivery that used a viral core protein (Vpr) fused to β-lactamase (BLAM) (Barnard et al., 2004; Cavrois et al., 2004). Entry-mediated delivery of Vpr-BLAM to the cytosol cleaved a cytoplasm-trapped fluorescent substrate (CCF2) that was loaded into target cells, abolishing its fluorescence energy transfer and resulting in a shift in peak emission wavelength from 518 nm (green) to 447 nm (blue) after excitation at 409 nm. These studies were performed at either 4°C (to block virus entry) or 37°C (to permit entry). They revealed identical levels of HIV-1 reporter virus entry into WT and AM DKO BMDCs at 4 hr postinfection (Figure 2A). Therefore, we concluded that the increased level of pseudotyped virus seen with WT versus TAM-deficient BMDCs (Figure 1E) was due to TAM receptor regulation of events that are subsequent to delivery of the viral nucleoprotein core complex to the cytosol.

Figure 2. TAM Activity Stimulates Postentry Events during Virus Infection.

Figure 2

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(A) BMDCs prepared from wild-type (WT, blue) or Axl−/−Mer−/− (AM DKO, red) mice were infected with VSVg-pseudotyped HIV-1-derived viruses carrying a Vpr-BLAM fusion protein (see the Experimental Procedures) and maintained at either 4°C or 37°C, as indicated, for 4 hr. Then, cells were loaded with the fluorescent β-lactamase (BLAM) substrate CCF2, and BLAM activity was assayed. Error bars are SD of six samples. Results are representative of three independent experiments.

(B) Axl-transfected (293 Axl) and kinase-dead Axl-transfected (293 AxlKD) HEK 293T cells were treated with (+) or without (−) 10 nM recombinant mouse Gas6, and lysates from these cells were immunoprecipitated with anti-HA. (The cDNA expression constructs for both Axl proteins were HA-tagged.) Protein equivalents of the IPs were immunoblotted with anti-pY (top blot) or anti-HA (bottom blot).

(C) Wild-type (293 WT, panels 1–3), Axl-transfected (293 Axl, panel 4), and kinase-dead Axl-transfected (293 AxlKD, panel 5) stable HEK 293T cell lines were infected with WNV at moi = 10, and the presence of internalized WNV was scored by confocal immunofluorescence microscopy for WNV E protein (E16, red, panels 1 and 3–5) at 4 hr postinfection. Panel 2 is a control staining with a monoclonal antibody (CHKV-152) against chikungunya virus. Nuclei of HEK 293T cells are visualized with DAPI (blue). One representative of three independent experiments is shown.

(D) Axl-transfected (Axl, blue) and kinase-dead Axl-transfected (293 AxlKD, red) stable HEK 293T cell lines were infected with WNV at moi = 0.01 and scored for virus replication as plaque-forming units (PFU; see the Experimental Procedures) at 4 and 24 hr postinfection. Error bars are SD of three samples. Results are representative of three independent experiments.

Additional support for this conclusion came from experiments in HEK 293T cells in which we asked whether Axl tyrosine kinase activity was required for TAM potentiation of either initial entry or subsequent replication of WNV. Similar gain-of-function studies have demonstrated that Axl facilitation of DENV entry does not require Axl kinase activity, but that potentiation of subsequent DENV replication required a catalytically competent Axl kinase (Meertens et al., 2012). We generated stable HEK 293T cell lines that expressed approximately equivalent levels of either hemagglutinin (HA)-tagged WT or kinase-dead mutant Axl (AxlKD, a D666A mutation in the catalytic domain; Figure 2B). As before (Figure 1B), ectopic expression of WT Axl in HEK 293T cells lead to ligand-independent Axl activation (Figure 2B). However, ectopically expressed AxlKD was catalytically inactive, even in the presence of added Gas6 (Figure 2B). These two HEK 293T cell lines were infected with WNV and assayed for virus entry at 2 and 4 hr postinfection with immunohistochemistry and flow cytometry with antibodies to the WNV E protein. We found that WNV entered Axl-expressing and AxlKD-expressing HEK 293T cells equally well (Figure 2C). In parallel experiments, we also measured WNV replication by titering the virus present in extracellular supernatants at 4 and 24 hr postinfection. In keeping with the earlier DENV results (Meertens et al., 2012), we found that WNV titers in AxlKD HEK 293T cells were markedly reduced at 24 hr postinfection relative to HEK 293T cells expressing WT Axl (Figure 2D). These results also argue that TAM tyrosine kinase activity is required for enhancing a postentry step of WNV replication but has no impact on virus entry. We conclude that, although TAM receptors can promote virus infection by providing a docking site on cells and facilitating initial entry, as described by others, they also operate as signaling proteins that render target cells more susceptible to postentry replication steps and productive infection.

LIGAND-DEPENDENT ACTIVATION OF TAM RECEPTORS DURING ENVELOPED VIRUS ENTRY

Given that TAM receptors must be catalytically active in order to promote viral infection, we considered the possibility that viruses themselves might function as TAM activators. We hypothesized that TAM tyrosine kinase activity might be stimulated—directly and immediately—by ligand-coated virus particles and that this activation might, in turn, inhibit innate immune responses that would otherwise antagonize infection. To determine whether endogenous TAM receptors are activated upon virus challenge, WT BMDCs were challenged with a VSVg-pseudotyped virus in either the presence or absence of purified Protein S or Gas6. For these experiments, the culture medium used during virus production and challenge was depleted of all Gla domain containing proteins, including Protein S, with sodium citrate and barium chloride precipitation (Souri et al., 2005). The addition of purified Protein S or Gas6 alone led to a rapid (5 min) dose-dependent activation of Mer or Axl, respectively (Figures 3A and 3B, left panels, lanes 1–4). Autophosphorylation of Axl, for example, was first detectable at 0.5 nM Gas6 (Figure 3B).

Figure 3. Enveloped Virus Potentiates Ligand-Dependent TAM Receptor Activation.

Figure 3

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(A) Left, WT BMDCs were incubated with increasing concentrations (0.5–2 nM) of purified human Protein S (Pros1), in either Gla-depleted cell culture medium alone (medium) or along with enveloped VSVg-pseudotyped HIV-1-derived virus that was produced in Gla-depleted medium (VSVg). Cell lysates were prepared for immunoblot 5 min after virus or Pros1 challenge. Mer was specifically immunoprecipitated from these samples, subjected to SDS-PAGE, and immunoblotted with a phosphotyrosine-specific antibody (IB: pY). Immunoblot analysis was used to confirm equal amounts of Mer in each sample (IB: Mer), and the amount of protein in each lysate was assessed by immunoblot for Gapdh (IB: Gapdh). Right, virus potentiation of Mer autophosphorylation in the presence of 1 nM Pros1 for 5 min is seen for the enveloped VSVg-pseudotyped virus, but not with WT MAV-1, a nonenveloped virus.

(B) Left, the same experiments as in (A, left) except that WT BMDCs were incubated with 0.25–1 nM of recombinant mouse Gas6 (Gas6), and the tyrosine autophosphorylation of Axl was monitored (IB: pY). Right, Virus potentiation of Axl autophosphorylation in the presence of 1 nM Gas6 was seen only with the enveloped VSVg-pseudotyped virus and not with the nonenveloped MAV-1.

(C) Microtiter wells, precoated with purified BSA, Gas6, or Pros1 (10 μg/ml ON for each protein) and incubated with equivalent amounts of VSVg-HIV (50 ng/ml p24 concentration) at 37°C for 2 hr with or without 5 mM EDTA, and the amount of bound virus was measured by ELISA for p24 protein (see Experimental Procedures). Error bars are SD of three samples. Results are representative of three independent experiments. See also Figure S2.

The addition of the VSVg-pseudotyped virus along with the ligands potentiated the stimulatory effects of both Gas6 and Protein S, shifting their dose response curves to lower protein concentrations (Figures 3A and 3B, left panels, lanes 5–8). Strong autophosphorylation of Axl, for example, was now observed at the same 0.5 nM concentration of Gas6, at which activation was barely detectable in the absence of virus (compare lanes 3 and 7 in Figure 3B). Virus-mediated potentiation of Protein S- or Gas6-triggered TAM ligand signaling was not seen with mouse adenovirus type1(MAV-1), a nonenveloped virus that does not display PtdSer and, therefore, should not bind either ligand (Figures 3A and 3B, right panels). Correspondingly, MAV-1 challenge of WT and AM DKO BMDCs resultedin a comparable induction of IFNα4, IFNβ, tumor necrosis factor a (TNFα), and SOCS1/3 messenger RNAs (mRNAs) (data not shown), and the efficiency of MAV-1 infection in BMDCs was not influenced by the presence or absence of TAM receptors (Figure S1A available online).

These results suggest that virus-associated PtdSer is required for TAM activation. To assess this further, we asked whether TAM ligands can bind to PtdSer on the virion surface and whether this binding was required for the viral enhancement of TAM activation. PtdSer binding by Gla-domain-containing proteins is Ca+2 dependent (Huang et al., 2003). Consistent with this dependence, purified Gas6 and Protein S immobilized onto microtiter wells bound VSVg-pseudotyped virus in a Ca+2-dependent manner (Figure 3C). In addition, incubation of VSVg-pseudotyped virus with a limiting concentration (100 nM) of the PtdSer-binding protein annexin V significantly attenuated VSVg-HIV potentiation of the Gas6 dose response curve for Axl activation in BMDCs (Figure S1B). Finally, we generated “bald” HIV-1 virions lacking any viral glycoprotein. As expected, these bald virions were noninfectious in BMDCs (Figure S2A). However, they were as competent as VSVg-pseudotyped virions at potentiating Gas6-induced Axl activation (Figure S2B). Thus, the ability of the pseudotyped virus to activate TAM receptors was dependent on the presence of PtdSer but independent of any specific viral glycoprotein. We hypothesize that Protein S and Gas6 bound to PtdSer on the enveloped virus surface act as virion-tethered “superligands” that rapidly activate TAM receptors upon contact with the target cell surface.

Virus-Mediated TAM Receptor Activation Blunts the Cellular Antiviral Response

TAM receptor activation in DCs leads to the inhibition of signaling by host cytokines, including type I IFNs (Rothlin et al., 2007). Given that the latter are potent antiviral cytokines, we assessed whether virus-mediated activation of TAM receptors blunts the cellular antiviral response. BMDCs derived from either WT or TAM TKO mice (Lu et al., 1999; Lu and Lemke, 2001; Rothlin et al., 2007) were challenged with the four pseudotyped HIV-1-derived viruses, and the expression of type I IFN mRNAs was monitored from 1–24 hr after virus addition. Virus infection of WT BMDCs resulted in only minimal induction of mRNAs encoding the type I IFNs IFNβ and IFNα4 (blue bars in Figures 4A and 4B, respectively).

Figure 4. Enveloped Virus Infection Abrogates the Cellular Antiviral Response in a TAM-Dependent Manner.

Figure 4

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(A–C) WT BMDCs (blue bars) or TAM TKO BMDCs (red bars) were challenged with the pseudotyped viruses of Figure 1B. Expression levels of IFNα4 (A), IFNβ (B), and SOCS1 (C) mRNAs were measured by qRT-PCR at the indicated time points after virus addition. The levels of the indicated mRNAs were normalized to control β-actin mRNA. *p < 0.05; **p < 0.01; ***p < 0.001. Results comparable to those for IFNα4 and IFNβ mRNAs were seen for measurements of TNFα, IRF-5, and IRF-7 mRNAs (see Figure S3). Error bars are SEM of samples from three independent experiments. See also Figure S3.

In marked contrast, the same challenge led to a robust induction of these mRNAs, ranging from 20- to 80-fold in TAM TKO BMDCs (red bars Figures 4A and 4B). Virus infection also led to a significant elevation of TNFα, interferon regulatory factor 5 (IRF-5), and IRF-7 mRNAs in TAM TKO BMDCs, but not in WT BMDCs (see Figures S3B–S3D). The reciprocal effect was seen for mRNAs encoding the cytokine signaling inhibitor SOCS1 (Figure 4C) and, to a lesser extent, SOCS3 (Figure S3A), which were more upregulated after retrovirus infection in WT BMDCs than in TAM TKO BMDCs. Altogether, these results indicate that virus-triggered TAM receptor activation in DCs is associated with a marked inhibition in type I IFN signaling.

TAM Receptor Antagonism of Type I IFN Signaling Promotes Virus Replication in DCs

To determine the contribution of TAM-dependent type I IFN inhibition to the efficiency of lentivirus infection, we used neutralizing antibodies that bind to IFNα and IFNβ (see the Experimental Procedures) to block the action of type I IFNs during virus challenge of BMDCs. The antibody cocktail had no impact on the level of infection seen with WT BMDCs (Figure 5A, blue bars), a result consistent with the minimal type I IFN induced in these cells after virus challenge (Figures 4A and 4B, blue bars). However, these antibodies restored the level of infection seen with TAM TKO BMDCs nearly to that seen with WT BMDCs (Figure 5A, red bars). Therefore, a primary effect of virus-mediated TAM activation in DCs appears to be inhibition of the antiviral type I IFN response.

Figure 5. The Primary Effect of TAM Receptor-Ligand Interactions on Virus Infection in BMDCs Is to Inhibit the Cellular Antiviral Response.

Figure 5

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(A) WT BMDCs and TAM TKO BMDCs were incubated with VSVg-pseudotyped virus in either the absence (−) or presence (+) of 100 μg each of neutralizing anti-mouse IFNα and IFNβ antibodies, and the levels of HIV-1 DNA were measured at 24 hr postinfection. Results were normalized to the level of viral DNA seen with WT BMDCs incubated with no antibody (100%, dashed blue line). ***p < 0.001. Error bars are SEM of samples from three independent experiments.

(B) WT BMDCs were preincubated for 30 min with or without BMS-777607 and stimulated with Gas6 for 10 min. Receptor activation was monitored with the immunoprecipitation and immunoblotting protocol shown in Figure 3; this protocol employed a phosphotyrosine-specific antibody (pY). Immunoblot was used to confirm equal amounts of Mer and Axl in each sample.

(C) WT BMDCs and TAM TKO BMDCs were incubated with EbGP-pseudotyped virus in the absence (−) or presence (+) of 300 nM BMS-777607, and the levels of HIV-1 viral DNA were measured at 24 hr postinfection. Results were normalized to those seen with WT BMDCs (100%, dashed blue line). **p < 0.01. Error bars are SEM of samples from three independent experiments.

(D) WT BMDCs (blue bars), AM DKO BMDCs (red bars), and IFNAR KO BMDCs (green bars) were incubated with WNV in the absence (−) or presence (+) of 1 μM BMS-777607, and the levels of infectious virus (PFU per ml) were measured at 24 hr postinfection. ***p < 0.001. Error bars are SEM of three independent samples. Results are representative of three independent experiments. BMS-777607 effects are not due to cytotoxicity (see Figure S4).

One implication of these data is that antagonists targeting TAM receptors, such as small-molecule TAM tyrosine kinase inhibitors, might confer an antiviral effect through their ability to block virus-induced TAM-dependent attenuation of type I IFN signaling. To test this idea, we used BMS-777607, an ATP mimetic that targets the c-Met, TAM, and Ron tyrosine kinases, which are closely related in structure (Schroeder et al., 2009). This orally administered compound has already been used in human clinical trials for the treatment of advanced or metastatic solid tumors and is most potent as an inhibitor for Axl (IC50 = 1.1 nM in vitro) among all kinases assayed (Schroeder et al., 2009). BMS-777607 strongly inhibited the ligand-dependent activation of both Axl and Mer in cultured cell assays, effectively blocking Gas6-triggered receptor activation at concentrations ranging from 30–3600 nM (Figure 5B). WT BMDCs treated with BMS-777607 were much less susceptible to virus infection than untreated cells (Figure 5C, blue bars). Importantly, the loss of infectivity seen with TAM TKO BMDCs (Figure 1E) was not reduced further by the addition of BMS-777607 (Figure 5C), suggesting that the drug reduces virus infection in WT BMDCs by blocking the activation of TAM receptors, as opposed to other RTKs, or through nonspecific cytotoxic effects. (We used a luminescent cell viability assay [see the Experimental Procedures] and detected no BMDC cytotoxicity for BMS-777607 from 10–1,000 nM [Figure S4].) It should be noted that BMS-777607 potently blocks TAM kinase activity without diminishing either Axl or Mer protein expression (Figure 5B; Axl and Mer immunoblots). This suggests that virus attachment to an inactive TAM receptor on a target cell is not sufficient to promote infection in this system.

TAM Receptor Antagonism Reduces WNV Infection in a Type I IFN-Dependent Manner

Given that gain-of-function studies (Meertens et al., 2012) have shown that TAM facilitation of virus infection extends to flaviviruses (DENV and WNV), we asked whether our loss-of-function results with HIV-1-derived pseudoviruses also extend to WNV, a WT virus that (unlike DENV) can infect mouse DCs. We infected either WT or AM DKO BMDCs with WNV and measured virus levels 24 hr later. We observed a 10-fold reduction in infectivity in the AM DKO BMDCs relative to WT BMDCs (Figure 5D). Similarly, treatment with BMS-777607 reduced WNV infection of WT BMDCs by approximately 10-fold, but this treatment had no additional effect on the reduced WNV infectivity seen for AM DKO BMDCs (Figure 5D). This also suggests that the antiviral activity of BMS-777607 is specific to its action as an inhibitor of TAM receptors as opposed to other RTKs.

To examine the contribution of type I IFN signaling to the effect of TAM inhibition on WNV replication, we generated BMDCs from Ifnar1−/− mice, which lack the R1 chain of the type I IFN receptor and are signaling incompetent (Müller et al., 1994). WNV is very sensitive to type I IFNs and replicates to much higher levels in cells and mice lacking type I IFN signaling (Samuel and Diamond, 2005, 2006). Consistent with this, WNV titers were ~100-fold higher in Ifnar1−/− BMDCs than WT cells (Figure 5D). However, in contrast to WT BMDCs, the BMS-777607 TAM inhibitor had no effect on WNV replication is these Ifnar1−/− cells (Figure 5D). Combined with our results showing no effect of TAM receptors on initial virus entry steps (Figures 2A–2C) and the type I IFN dependence of the inhibitor on pseudovirus infection (Figure 5A), these findings suggest that BMS-777607 inhibits pseudovirus and WNV infection of DCs by blocking the ability of virus-activated TAM receptors to downregulate the cellular type I IFN response. The fact that there was no impairment of virus infection by BMS-777607 in Ifnar1−/− BMDCs (Figure 5D) argues against the possibility that this inhibitor acts through a nonspecific cytotoxic mechanism.

DISCUSSION

We describe a mechanism of enveloped virus evasion of host innate immune responses that is based on TAM receptor activation through apoptotic mimicry. We propose that PtdSer—exposed on the surface of an enveloped virus—binds, concentrates, and reduces the free diffusion of extracellular Protein S and Gas6, which, therefore, appear to act as superligands for activating cell surface TAM receptors during initial contact with the target cell surface. In DCs, and perhaps other cell types, TAM receptor activation leads to the downregulation of host TLR and type I IFN signaling pathways (Figure 6). Indeed, this appears to be the main mechanism behind TAM-receptor-stimulated infection of BMDCs, given that type I-IFN-neutralizing antibodies reversed the pseudovirus infection defect that was seen with TAM-deficient BMDCs (Figure 5A) and a pharmacological TAM inhibitor blocked WNV infection of WT, but not Ifnar1−/−, DCs (Figure 5D).

Figure 6. Virus-Ligand Activation TAM of TAM Receptors Inhibits the Antiviral Immune Response.

Figure 6

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A model showing that the major effect of TAM receptor-ligand interactions on enveloped virus infection of dendritic cells is at the level of inhibiting the cellular innate immune response, including TAM inhibition of type I IFN signaling.

These results do not rule out the possibility that TAM receptor-ligand interactions, including interactions that rely on the activation of TAM kinase activity, also play an important role in promoting virus uptake and initial entry into other cell types (Brindley et al., 2011; Meertens et al., 2012; Morizono et al., 2011; Shimojima et al., 2007; Shimojima et al., 2012; Shimojima et al., 2006). However, it should be noted that the Axl cytoplasmic domain and its tyrosine kinase activity are dispensable for the initial uptake of DENV particles into HEK 293T cells that ectopically express this receptor but are required for Axl potentiation of DENV proliferation, which occurs over the next 48 hr (Meertens et al., 2012), in these calls. This is the only published study in which the requirement for TAM kinase activity has been examined in the context of initial virus entry versus later stages of infection, although other studies have similarly shown that an active TAM tyrosine kinase is required for TAM potentiation of infectivity measured 48 hr after virus challenge (Shimojima et al., 2007; Shimojima et al., 2012). This is the case even though kinase-dead mutants of both Axl and Tyro3 have been found to bind ligand-opsonized virus particles as effectively as their WT counterparts (Shimojima et al., 2007; Shimojima et al., 2012). Our gain-of-function studies with WNV (Figures 2B and 2C) demonstrate that Axl tyrosine kinase activity is dispensable for WNV entry into HEK 293T cells (which express endogenous Tyro3 and Mer) but is required for Axl potentiation of subsequent events in WNV infection.

Our studies were conducted with pseudotyped HIV-1-based viruses and WNV as model enveloped viruses. Previously, PtdSer on pseudotyped lentiviruses and DENV was shown to bind TAM ligands (Meertens et al., 2012; Morizono et al., 2011). The presence of exposed PtdSer is also a feature of WT retroviruses as well as arenaviruses, poxviruses, filoviruses, and alphaviruses (Callahan et al., 2003; Jemielity et al., 2013; Mercer and Helenius, 2008). Given that PtdSer exposure seems to be relatively common, it is possible that many different enveloped viruses use TAM receptor-ligand interaction as a mechanism for counteracting the antiviral response of TAM-positive cells.

Our findings suggest a potential antiviral strategy based upon inhibiting the virus- and ligand-dependent activation of TAM-receptor-associated signaling pathways (Figures 5C, 5D, and 6). This strategy is illustrated with BMS-777607, a small-molecule kinase inhibitor that led to a marked reduction in virus infectivity for both HIV-1-derived lentivirus and WNV. This compound not only inhibits the activation of TAM receptors but also blocks the activities of other closely related tyrosine kinase receptors, including c-Met and Ron (Schroeder et al., 2009). However, the fact that BMS-777607 reduced virus infection in TAM-receptor-expressing WT, but not TAM KO, BMDCs suggests that its antiviral function is mediated specifically through TAM receptor inhibition. Although an antiviral approach employing TAM receptor antagonists may not be suitable for the long-term treatment of chronic virus infections because of the risk of undesirable side effects, such as autoimmunity (Rothlin et al., 2007; Rothlin and Lemke, 2010), we anticipate that shorter-term treatments with such inhibitors during the acute phase of infection might be useful for promoting the clearance of enveloped viruses.

EXPERIMENTAL PROCEDURES

HEK 293T Stable Cell Lines and Mouse BMDC Cultures

Complementary DNAs (cDNAs) for mouse Tyro3 or Axl were subcloned into a pcDNA6 expression vector (Invitrogen) containing a blasticidin resistance gene. The construct was transfected into HEK 293T cells, and a stable cell line was generated by growing cells in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS(SAFC Biosciences), 100 U/ml penicillin and 100 μg/ml streptomycin (PenStrep), and 10 μg/ml blasticidin. A mouse Mer cDNA was subcloned into a pIRES puro3 vector (Clontech) containing a puromycin resistance gene and transfected into HEK 293T cells, and a stable cell line was generated by growing cells in medium supplemented with 2.5 μg/ml puromycin.

Bone marrow cells were isolated from tibias and femurs of 6- to 8-week-old mice according protocols approved by the International Animal Care and Use Committee (11-00051), as described previously (Harding et al., 2010), and were grown for 7 days in RPMI containing 20 ng/ml mouse GM-CSF (Akron Biotech), 5% FBS, and antibiotic-antimycotic cocktail (Invitrogen) in order to generate BMDCs. Before stimulation with purified Gas6 or Protein S, cells were starved overnight (ON) in serum-free RPMI medium.

Virus Production and Infection

Pseudotyped viruses were produced as described previously (Bhattacharyya et al., 2011). Viruses were treated with 40 U/ml DNase I (Roche) for 1 hr before being added to cells. HEK 293T cells and HEK 293T cells stably expressing TAM receptors were plated at 105 cells per well of a 12-well plate and challenged with 500 μl of each virus stock. DNA was harvested from cells with quantitative PCR (qPCR) lysis buffer (10 mM Tris–HCl [pH 8.0], 1 mM EDTA, 0.2 mM CaCl2, 0.001% Triton X-100, 0.001% SDS, and 1 mg/ml proteinase K) at 24 hr postinfection and subjected to qPCR with TaqMan Fast PCR (Applied Biosystems) with primers specific for HIV-1 reverse transcribed HIV-1 DNA products or for the cellular porphobilinogen deaminase (PBGD) gene, as described previously (König et al., 2008) (Table S1).

HIV-1 DNA levels from TAM-expressing HEK 293T cells were normalized to PBGD before they were compared to those seen in untransfected HEK 293T cells. BMDCs (105) were spinoculated with 500 μl of each virus at 1,200 × g for 1 hr followed by incubation at 37°C. HIV-1 DNA products were measured 24 hr postinfection by qPCR and normalized to cellular β-actin DNA prior to normalization to WT BMDCs for each pseudotype. Oligonucleotide primers and TaqMan probes used for qPCR are listed in Table S1. For measuring HIV-1 DNA products, a standard curve was generated with six 10-fold serial dilutions of HIV-1 plasmid DNA. For β-actin, a standard curve was generated with six 10-fold serial dilutions of uninfected BMDCs or HEK 293T cells.

WNV (strain New York 2000) was produced in C6/36 Aedes albopictus cells as described previously (Diamond et al., 2003) and was used to infect WT and AM DKO BMDCs at a multiplicity of infection (moi) of 0.01. WNV titers in culture medium were measured at 24 hr postinfection by plaque assay on BHK21-15 cells as described previously (Beasley et al., 2002; Diamond et al., 2003). For adenovirus infection, 105 BMDCs were infected with MAV-1 at an moi of 1. Cells were lysed 24 hr postinfection, and the expression of adenovirus E1A was assayed by qRT-PCR and normalized to endogenous cyclophilin A.

BMDCs were pretreated with 0 or 100 μg of neutralizing anti-mouse IFNα and IFNβ antibodies (PBL, IFNα and IFNβ) for 3 hr and then spinoculated with VSVg-pseudotyped virus at 1200 × g for 1 hr followed by incubation at 37°C with or without antibody. Reverse transcribed HIV-1 DNA products were measured 24 hr postinfection. For BMS-777607 studies, WT or TAM TKO BMDCs were incubated with EbGP-pseudotyped virus ± 300 nM BMS-777607, and the levels of HIV-1 DNA were measured at 24 hr postinfection. The same protocol was followed for WNV experiments, except that BMS-777607 was used at 1 μM, and mutant BMDCs were prepared from AM DKOs.

Immunoprecipitations and Immunoblotting

HEK 293T cells were grown to 80% confluency in DMEM with 10% FBS and PenStrep. Cells were washed in ice cold PBS and lysed in buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, and 1% Triton X-100 supplemented with protease and phosphatase inhibitor cocktails (Roche). BMDCs were incubated with serum-free medium ON and then for 5 min with increasing amounts of either human Protein S (Haematologic Technologies) or mGas6 with or without a VSVg-pseudotyped virus or MAV-1. Cells were washed with cold PBS and lysed on ice in a buffer containing 50 mM Tris–HCl (pH 7.5), 1 mM EGTA, 1 mM EDTA, 1% (w/v) Triton X-100, 0.27 M sucrose, 0.1% β-mercaptoethanol, and protease and phosphatase inhibitors.

For western blots, equal amounts of protein (5 μg) in lithium dodecyl sulphate (LDS) sample buffer (Invitrogen) were subjected to electrophoresis on a polyacrylamide gel and transferred to polyvinylidene fluoride (PVDF) membranes. For immunoprecipitations, cell lysates were incubated ON at 4°C with antibodies against Tyro3 (goat polyclonal, C-20, Santa Cruz Biotechnology), Axl (goat polyclonal, M-20, Santa Cruz), and Mer (Prasad et al., 2006), respectively. Protein G or A Sepharose was added for 2 hr, and immunoprecipitates (IPs) were washed twice with 1 ml of lysis buffer containing 0.5 M NaCl and once with 1 ml of 50 mM Tris–HCl (pH 7.5). IPs were eluted in LDS buffer, separated on polyacrylamide gels, and transferred to PVDF membranes. Membranes were blocked in TBST (50 mM Tris–HCl [pH 7.5], 0.15 M NaCl, and 0.25% [v/v] Tween-20) containing 5% (w/v) BSAand immunoblotted ON at 4°C with antibodies diluted 1,000-fold in blocking buffer. Immunoblotting antibodies were anti-Tyro3 and anti-Axl (described above), anti-Mer (goat polyclonal, AF591, R&D Systems), anti-GAPDH (mouse monoclonal, clone 6C5, Millipore); and anti-pTyr (pY, mouse monoclonal, clone 4G10, Millipore). Blots were washed in TBST and incubated for 1 hr at room temperature with secondary horseradish-peroxidase-conjugated antibodies (GE Healthcare) diluted 5,000-fold in 5% (w/v) skim milk in TBST. After repeating the washes, signal was detected with enhanced chemiluminescence reagent.

Protein S and Gas6 Binding to Virus Particles

Nunc MaxiSorp plates were coated ON with 10 μg/ml of hProS or mGas6. Wells were washed five times with a buffer containing 10 mM HEPES (pH 7.4), 150 mM NaCl, 2.5 mM CaCl2, and 0.1% v/v Tween-20, blocked with 10% w/v BSA for 2 hr at room temperature, and washed as above. Virus was diluted in DMEM with 5% BSA to p24 concentration 50 ng/ml, and 100 μl was added to wells with or without 5 mM EDTA and incubated at 37°C for 2 hr. Wells were washed as above, and bound virus was lysed and measured with a p24 ELISA Kit (ZeptoMetrix).

Vpr-BLAM BMDC Infection and Assay

VSVg-HIV Vpr-BLAM virus stocks were prepared by transfection of HEK 293T cells (Barnard et al., 2004; Cavrois et al., 2004). Virus entry was assayed by infection with virions containing Vpr-BLAM and by loading target cells with CCF2. Upon entry, CCF2-AM was cleaved by esterases, and, in the absence of BLAM, excitation at 409 nm (405 nm filter) results in fluorescence resonance energy transfer (FRET) of the intact CCF2 and peak emission at 518 nm (535 nm filter). In the presence of BLAM, cleavage of CCF2 disrupts FRET, and excitation at 409 nm produces a blue fluorescence (465 nm filter). WT and AM DKO BMDCs were starved ON in serum-free media. Next, 5 × 105 cells were spinoculated at 4°C with 0.25 ml of virus stock and incubated in the presence of virus at 37°C for 4 hr. Control cells were kept for 4 hr at 4°C in order to prevent virus entry. Virus was washed away with Hank’s balanced salt solution (HBSS); cells were resuspended in HBSS and loaded with CCF2 dye for 3 hr with the LiveBLAzer FRET CCF2 Loading Kit (Invitrogen). Fluorescence was measured using 405 nm excitation and 465 nm and 535 nm emission filters. After background subtraction, a 465:535 ratio was calculated. Response ratio was calculated by dividing 465:535 ratios of experimental samples by the same ratio of the no-virus control.

Quantitative RT-PCR

BMDCs were infected by spinoculation with each pseudotyped virus at 1,200 g for 1 hr followed by incubation at 37°C for the indicated time points up to 24 hr. RNA was isolated from the cells with the RNeasy Mini Kit (Qiagen) and cDNA was prepared with the QuantiTect Reverse Transcription Kit (Qiagen). qPCR reactions were performed to measure mRNA expression levels with the oligonucleotide primers listed in Table S1 with Power SYBRGreen PCR Master Mix (Applied Biosystems). A dissociation curve analysis was performed for each reaction with the SDS (Applied Biosystems). A standard curve was generated with six 10-fold dilutions of samples from uninfected BMDCs.

Adenovirus Infection

Purified MAV-1 was a generous gift from C. Powers and C. O’ Shea (Salk Institute). MAV-1 was grown on mouse 3T6 cells, concentrated from supernatant with PEG precipitation, and purified on a CsCl gradient. Purified virus was dialyzed in 10% glycerol, 10 mM Tris (pH7.5), 150 mM NaCl, and 1 mM MgCl2 and titered by plaque assay on 3T6 cells. 105 BMDCs were infected with MAV-1 at MOI 1. Cells were lysed at 1, 2, 4, 6, and 24 hr postinfection, and mRNA was isolated with a RNeasy Kit with DNase digestion.

Purification of Recombinant Mouse Gas6

A mammalian expression vector was engineered to encode full-length mouse Gas6 followed by a C-terminal TEV-cleavable His6 tag. This vector was transfected into HEK 293T cells, and cells stably expressing the construct were selected in DMEM supplemented with 10% FBS, PenStrep, 0.25 mg/ml G418, and 100 μg/ml hygromycin B. For expression studies, cells were grown in serum-free medium containing 10 μM vitamin K2, and conditioned medium was collected after 72 hr. Gas6 was isolated with affinity chromatography with Ni-NTA beads followed by purification on a HiTrap Q FF ion exchange column (GE Healthcare). The protein was eluted in 20 mM Tris (pH 8) with a sodium chloride gradient up to 1 M.

Cytotoxicity Assay

BMS-777607 cytotoxicity in WT BMDCs was measured with a CellTiter-Glo Luminescent Cell Viability Assay (Promega).

Supplementary Material

1

Acknowledgments

We thank C. Powers and C. O’Shea (the Salk Institute) for providing the purified MAV-1 virus stock and S. Buhrlage and N. Gray at Harvard Medical School for alerting us to BMS-777607 and for sharing other candidate TAM receptor antagonists with us. We also thank members of the Young and Lemke labs and the Nomis Center for helpful discussions. This research was supported by grants from the National Institutes of Health (PO1 AI090935 and UO1 AI074539 to J.A.T.Y.; R01 AI077058 and R01 AI101400 to G.L.; R01 R01AI089824 to C.V.R., U54 AI081680 to M.S.D., and P30 CA014195-38 to the Salk Institute), the Nomis and Auen Foundations, the James B. Pendleton Charitable Trust (to J.A.T.Y.), a Salk Institute innovation grant (to G.L. and J.A.T.Y.), and postdoctoral fellowships from the Human Frontiers Science Program (to A.Z.) and the Leukemia and Lymphoma Society and Nomis Foundation (to E.D.L.).

Footnotes

SUPPLEMENTAL INFORMATION

Supplemental Information contains four figures and one table and can be found with this article online at http://dx.doi.org/10.1016/j.chom.2013.07.005.

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