The present study adds significant insights into the molecular interactions that occur between DV and the host cell during viral entry. We show that TIM and TAM proteins, two receptor families involved in apoptotic cell recognition and clearance, mediate DV infection. TIM- and TAM-mediated enhancement of virus infection is dependent on PtdSer associated with DV particles. This supports a model by which DV use a strategy of “apoptotic mimicry” to infect target cells (Laliberte and Moss, 2009
; Mercer and Helenius, 2008
We provide strong evidence indicating that TIM and TAM receptors are cellular factors mediating DV binding and infection. First, ectopic expression of TIM and TAM rendered human cells susceptible to DV infection. Second, Ab against TIM and TAM receptors inhibited DV infection in cells naturally expressing these proteins. Third, in analyses of DV infectivity, evidence of loss-of-function was obtained using RNA interference. Finally, binding studies revealed that TIM and TAM molecules interact with DV particles and enhance virus endocytosis. However, our data suggest that TIM and TAM proteins do not directly promote DV internalization. We observed that the TIM-1 cytoplasmic tail is dispensable for DV entry, indicating that TIM-mediated enhancement of infection relies mainly on the receptor ectodomain. We propose that TIM-1 may act primarily as a tethering/attachment receptor that binds and enhances DV infection without any obligatory role in virus endocytosis. This is consistent with the proposed role of TIM receptors during the engulfment of apoptotic cells (Park et al., 2009
; Toda et al., 2012
). Our results also suggest that TIM-mediated enhancement of DV infection relies on still uncharacterized cellular receptors. It is possible that TIM receptors pass the virus in cis
to another molecule that is ultimately responsible for virus endocytosis, as we proposed for DC-SIGN-mediated DV entry (Lozach et al., 2005
). Alternatively, TIMs could also directly associate with unknown molecules to form an entry complex that coordinates virus endocytosis. Our data suggest that TIM and TAM receptors may play distinct roles during DV infection. We found that tailless and kinase-dead AXL variants are still able to enhance DV endocytosis. However, and as shown for Ebola and Lassa virus entry (Shimojima et al., 2007
), both mutated AXL molecules are unable to enhance DV infection, indicating that tyrosine kinase activity is important for AXL potentiation of viral infection. These data suggest that AXL function is not restricted to enhance virus endocytosis but also to initiate signaling that facilitates infection of the target cells. Of note, Gas6 binding to TAM receptors has been shown to inhibit innate inflammatory immune responses (Lemke and Rothlin, 2008
), including signaling downstream of TLRs (Rothlin et al., 2007
). It is thus tempting to speculate that TAM ligation by DV during entry may initiate signal transduction events that may modulate host immune responses to potentiate a postendocytic step of the DV life cycle.
In cells expressing both TIM-1 and AXL (such as A549 and Vero), DV infection was partly reduced by anti-TIM-1 or anti-AXL Ab administrated alone and completely inhibited by the two Ab in combination. Thus, TIM-1 and AXL may cooperate to mediate optimal DV infection. TAM receptors are known to physically associate with non-TAM receptors by heterotypic dimerization (Linger et al., 2008
; Rothlin et al., 2007
). One can speculate that they may recruit TIM and/or additional receptors to form a viral entry complex required for DV uptake and infection (). DVs use the clathrin-mediated endocytosis as a major entry route for internalization (Krishnan et al., 2007
; van der Schaar et al., 2008
). It will be worth further assessment of the role of the clathrin-dependent pathway and other endocytic routes during TIM- and TAM-mediated enhancement of DV infection.
Model of PtdSer Acquisition by DV Particles and the TIM and TAM Receptor-Mediated Enhancement of DV Infection
The role played by TIM and TAM proteins during DV pathogenesis in vivo is currently unknown. In vivo, TIM and TAM receptors are expressed in many cell types relevant to DV infection. AXL is widely expressed (Lemke and Rothlin, 2008
) with prominent expression in macrophages, dendritic cells, and vascular endothelia, and we show here that DV infection of cultured epithelial cells and astrocytes is AXL-dependent. TIM-1 is abundant on Th-2 T cells, mucosal epithelial cells, and mast cells; the last have been recently identified as important DV target cells (Freeman et al., 2010
; Kondratowicz et al., 2011
; St John et al., 2011
). TIM-4 is present exclusively on antigen-presenting cells (Kobayashi et al., 2007
; Wong et al., 2010
), which are sensitive to DV infection and thought to play an important role in DV pathogenesis. Our data indicate that TIM and TAM usage is not sufficient to explain DV tropism. Indeed, we have shown that DVs can infect cells lacking these proteins, suggesting that other cellular receptors that dictate virus tropism remain to be characterized. However, our results suggest that TIM and TAM receptors contribute to enhance infection of relevant target cells, which may have important consequences for virus spread.
We reveal an unexpected role for PtdSer during DV infection. Our data suggest that PtdSer is displayed on the surface of DV particles and is important for TIM- and TAM-mediated infection. In agreement with the binding of apoptotic cells to PtdSer receptors (Ravichandran, 2011
; Ravichandran and Lorenz, 2007
), we describe a dual mode of recognition of DV by TIM and TAM receptors. TIM molecules directly interact with DV. Our muta-genesis study of TIM strongly suggests that PtdSer associated with DV particles binds to the MILIBS of the molecule. Indirect recognition of DV is mediated by a TAM ligand such as Gas6, which recognizes through its Gla domain viral PtdSer and bridges DV virions to AXL and TYRO3. Together, these observations suggest that, by mimicking apoptotic bodies, DVs subvert the apoptotic clearance function of TIM and TAM receptors to enhance infection.
TIM and TAM proteins mediate the entry of other viruses (Feigelstock et al., 1998
; Kondratowicz et al., 2011
; Morizono et al., 2011
; Shimojima et al., 2012
). AXL and TIM-1 are receptors for Ebola virus, which, like DV, infects a broad range of cell types and causes hemorrhagic fever (Kondratowicz et al., 2011
; Shimojima et al., 2006
). TIM-1-mediated Ebola infection depends on a direct interaction between the viral glycoprotein GP through residues outside the MILIBS (Kondratowicz et al., 2011
), indicating that DV and Ebola virus may use distinct TIM-1 regions. A recent study also identified Gas6 and ProS as “1bridging factors” that link PtdSer expressed on the viral envelope of lentiviral pseudotypes and vaccinia virus to AXL expressed on the target cell (Morizono et al., 2011
). Collectively, these data suggest that TIM and TAM facilitation of viral infection may represent a general mechanism exploited by viruses that incorporate PtdSer in their membrane for optimal infection.
An interesting direction for future work is to identify how DVs acquire this lipid and how PtdSer becomes accessible to TIM and TAM receptors. The DV membrane is derived from the ER of the infected cell upon budding. The ER membrane is known to contain PtdSer in the lumenal leaflet (Leventis and Grinstein, 2010
), which suggests an obvious mechanism through which PtdSer may be incorporated into DV virions (). However, the viral membrane does not appear to be readily exposed to receptors at the virion surface. This is shown by structural studies, which have revealed that a closed icosahedral shell of the envelope protein completely encloses the viral membrane in mature DV particles (Kuhn et al., 2002
). It is plausible that TIM and TAM molecules or other yet-to-be identified receptors may display weak interactions with the E protein that trigger opening of the icosahedral shell, leading to exposure of viral membrane, as recently suggested by studies with Ab complexes (Cockburn et al., 2012
). Also, recent studies have indicated that the majority of the DV particles released from infected cells are partially mature virions, which display a mixture of immature and mature surfaces (Junjhon et al., 2010
; Pierson and Diamond, 2012
; Plevka et al., 2011
). These immature-like regions could expose membrane patches, such that PtdSer would be accessible for TIM and TAM interactions. Partially mature virions result in an incomplete cleavage of the envelope glycoprotein prM. Additional studies are thus required to evaluate whether modulation of the efficiency of the prM cleavage impacts TIM and TAM receptor usage.