We identified a Gas6-mediated entry mechanism which also revealed the molecular mechanism by which envelope PtdSer facilitates viral replication. This mechanism acts to provide an alternative pathway for viral entry that is not limited by specific interaction between viral envelope proteins and cell receptors. This entry pathway seems to play a major role in viral binding when native binding between virus and cells is weak. Thus, Gas6 and protein S will broaden the tropisms of viruses. Indeed, ectopic expression of Axl and TYRO3 was previously shown to render unsusceptible cells susceptible to infection with Ebola virus by an unknown mechanism (Shimojima et al., 2006
). Axl- and TYRO3-mediated infection of Ebola virus might also involve bProtein S in FCS used in cell cultures.
Our data showed that lentiviral vectors pseudotyped with various envelope proteins can be enhanced by hGas6 to differing extents. The differences might be explained by varying affinities and/or avidities of interaction between virus and target cells. Binding by hGas6 can result in drastic increases in infectivity when virus does not bind target cells efficiently ( and Figure S2B
). Conversely, if viral binding is very efficient due to high affinity and/or avidity interaction between virus envelope proteins and target cells, the contribution of hGas6 to virus binding is negligible. However we cannot rule out the possibility that these differences may be due to differing amounts of exposed PtdSer dependent upon the pseudotyped envelope protein.
Among the viruses known to use envelope PtdSer for its replication, vaccinia virus is the only one clearly shown to use PtdSer for its binding to target cells. This led us to test the effects of hGas6 on vaccinia virus replication. The role of PtdSer in vaccinia virus entry was shown by blocking replication of vaccinia virus using ANX5 and reconstituting the membrane of detergent-extracted virions by PtdSer (Laliberte and Moss, 2009
; Mercer and Helenius, 2008
; Mercer et al., 2010
). These investigations designated PtdSer-mediated virus entry as apoptotic mimicry because exposed PtdSer was known to be a surface marker of apoptotic cells. However, these studies did not show whether the same PtdSer recognition mechanism is used for both phagocytosis of apoptotic cells and entry of vaccinia virus. Our study showed that virus entry can be mediated by the same molecular mechanism used for recognition of dead cells. The differing degrees of enhancement observed between the IMV and EEV preparations of vaccinia virus could be due to several possibilities. 1) IMV and EEV preparations have different envelope proteins on their surfaces, resulting in different affinities of the virus for target cells. 2) IMV and EEV obtain their envelopes from different cytoplasmic organelles, potentially varying the amount of exposed PtdSer. 3) EEV preparations are much less infectious for HMVEC than IMV. Thus, as seen with lentiviral vector pseudotypes, the degree of enhancement is greater when native infectivity is lower. One previous study demonstrated the role of PtdSer in vaccinia virus entry using the IMV form (Mercer and Helenius, 2008
). We observed much less of an effect of hGas6 on IMV, potentially due to different cell types used. 4) the EEV outer membrane is fragile, so rupture of its outer membrane may expose its luminal side which contains abundant PtdSer. Although the fragility of the outer envelope of EEV makes analysis difficult, detailed biochemical analysis of both the IMV and EEV envelope will be necessary to address these possibilities.
Gas6 and protein S belong to the family of VKD proteins that include proteins involved in the coagulation system (McCann and Ames, 2009
). The proteins of this family undergo γ-carboxylation, a post-translational modification of glutamic acid residues, by VKD γ-glutamylcarboxylase. We found that the Gla domain of hGas6 binds to virus. Interestingly, the Gla domain of factor X was also shown to bind adenovirus (Munch et al., 2007
). Although only a few proteins (less than 15) are known to belong to the VKD protein family, four (protein C and Factor VII, IX, and X) were shown to mediate binding of non-envelope virus (adenovirus) (Parker et al., 2006
) and two (Gas6 and protein S) mediate binding of envelope virus. These results suggest a common evolutionally convergence where replication of diverse viruses is enhanced by VKD proteins.
Gas6 shares 43% amino-acid sequence homology with Protein S, but there are differences in their expression and functions (Fernandez-Fernandez et al., 2008
; Hafizi and Dahlback, 2006
). Protein S is mainly produced in the liver, but expression of Gas6 is much lower in the liver than the heart, kidney, and lung. hProtein S has an anticoagulant effect but hGas6 does not. Functions of protein S and Gas6 are also species-dependent. For example, while human and bovine protein S share 82% amino acid sequence identity, only the bovine variant is shown to activate human TYRO3 (Evenas et al., 2000
PtdSer is known to be exposed not only on vaccinia virus envelope, but also on Pichinde virus (a model of Lassa fever virus) and HIV (Callahan et al., 2003
; Soares et al., 2008
). Although these studies did not indicate which step of viral replication is supported by PtdSer, both studies clearly demonstrated that exposed PtdSer contributes to viral replication. PtdSer of the plasma membrane is usually present in the inner leaflet, while PtdSer of the membrane of cytoplasmic organelles is present on both leaflets (Boon and Smith, 2002
). Asymmetrical distribution of PtdSer in the plasma membrane is maintained by lipid transporters, flippases. When apoptosis or necrosis occurs, flippases are inactivated and scramblase, which can transport PtdSer in both directions, is activated, resulting in exposure of PtdSer. There are three possible mechanisms that can expose PtdSer on the viral envelope: 1) viral infection induces apoptosis of infected cells and budding captures the exposed PtdSer (Soares et al., 2008
); 2) virions do not contain flippases, so it cannot maintain asymmetrical distribution of PtdSer (Denard et al., 2009
); and 3) virus obtains envelope from cytoplasmic organelles (Chertova et al., 2006
). TAM receptors belong to the receptor tyrosine kinase family that contains protein tyrosine kinase activity within the cytoplasmic domain (Lemke and Rothlin, 2008
). Signaling via TAM receptors is reported to activate various signaling molecules, including PI3K, STAT3, and Src. This signaling may facilitate endocytic and phagocytic activity of cells, potentially affecting entry of virus. We tested the activity of a tyrosine kinase inhibitor (SKI-606) on hGas6-mediated transduction and observed a 2-fold reduction (data not shown); however, because SKI-606 is a broad tyrosine kinase inhibitor, we are unsure as to the significance of this observation. TAM receptors are expressed on many cell types, including hematopoietic cells, bone-marrow stromal cells, and retinal pigment epithelial cells. Murine macrophages and dendritic cells have been shown to express Axl and TYRO3 in vivo
(Seitz et al., 2007
). Since envelope PtdSer has been shown to enhance HIV replication in human macrophages (Callahan et al., 2003
), we prepared human macrophages by in vitro
differentiation of monocytes using GM-CSF (Lee et al., 1999
) to study the effects of hGas6 on HIV replication. Unfortunately, we could not detect the expression of Axl and TYRO3 on the macrophages. However, monocyte-derived macrophages do not necessarily represent all types of macrophages in vivo
(Krutzik et al., 2005
). It was shown that usage of IFN-α for human dendritic cell differentiation from monocytes induces expression of Axl while other differentiation conditions do not (Scutera et al., 2009
). Thus, different conditions for macrophage differentiation could induce expression of Axl.
The serum concentration of hGas6 (200 pM) is higher than the concentration required for viral transduction enhancement (13pM) (Hafizi and Dahlback, 2006
). However all hGas6 in normal human serum has been shown to be bound with sAxl so it would not be able to mediate bridging of virus to cells (Ekman et al., 2010b
). Viral transduction enhancement by hGas6 is expected to be suppressed in normal human serum. We investigated whether human serum can enhance viral transduction using two different lots of human sera and we observed only very weak enhancing activity (data not shown). Increased plasma concentrations of Gas6 have been found in many disease states, including malignancy and inflammation (Ekman et al., 2010a
). Importantly, Axl expression on endothelial cells and vascular muscle is upregulated after vessel injury and inflammation (Korshunov et al., 2006
; Sharif et al., 2006
). It is well known that the virus can efficiently enter into the body via the skin and mucosa when injury and/or inflammation occur (Norkin, 2010
). We theorize that injury and inflammation will facilitate virus entry not only by breaks of the barrier function of the skin and mucosa, but also by the upregulation of Axl and Gas6 expression to enhance virus binding. This pathway could be particularly important for viral binding if the expression of native virus receptors is low at portal sites for viruses.
These studies began with efforts to identify factors responsible for nonspecific infectivity of redirected lentiviral vectors. Those efforts focused on both eliminating original receptor-binding regions of the viral protein and conjugating virus with ligands that specifically bind target cells and tissues. Here, we demonstrate that a lipid can mediate binding of virus, which should be considered when eliminating original tropism of viral vectors and/or conferring new tropism by modification of envelope lipids.
Because PtdSer is likely to be a common molecule among many types of envelope viruses, further investigation of the entry pathway mediated by PtdSer may lead to preventive and therapeutic approaches for viral diseases.