Axl and GAS6: apoptotic mimicry and NLRP-3 inhibition during Zika Virus infection

Zika Virus (ZIKV) is a positive sense RNA virus that belongs to the Flavivirus genus of the Flaviviridae family that includes other human pathogens including Hepatitis C Virus (HCV), Yellow Fever Virus (YFV), West Nile Virus, Dengue Virus (DENV), Tick Borne Encephalitis Virus (TBEV), and Japanese Encephalitis Virus (JEV).

Although being first isolated in 1947, until recently ZIKV was not associated with severe disease; following the introduction of ZIKV in the Americas however, foetal ZIKV infection became associated with neonatal cognitive defects, including viral Microcephaly as well as GBS in adult patients.

Like other flaviviruses such as DENV or JEV, ZIKV entry into host cells is mediated by several cell surface receptors that belong to the Tyro3-Axl-Mer (TAM) family of receptor tyrosine kinases, T cell immunoglobulin and mucin domain (TIM) phosphatidylserine (PS) and C-type lectin receptor families followed by endocytosis of the viral particle. As discussed below, activation of at least one of these receptors, Axl, by ZIKV might promote the inhibition of the secretion of pro-inflammatory cytokines.

Chloroquine: targeting multiple steps of viral infection?

To investigate if the degradation of viral and/or cellular proteins within the lysosome impacts the replication of ZIKV MR766, ZIKV PE/243 or ZIKV Recife, infected Vero cells were treated for 5 days or for 48 hrs with varying concentrations of Chloroquine (CQ) and viral replication was assessed by flow cytometry and indirect immunofluorescence analysis for the presence of the viral E protein as well as measuring viral titres. Results obtained from flow cytometry analysis of Vero cells infected either with ZIKV MR766 or ZIKV Recife 5 days p.i. and treated with 25 μM CQ exhibit a reduction of viral replication by 65% (ZIKV MR766) or 70% (ZIKV Recife) respectively concomitant with a decrease in viral titres and an increase in cell viability. In a similar way, treatment of ZIKV MR766 infected human brain microvascular endothelial cells (hBMEC), that serve as a model for the (human) blood –brain barrier, with up to 50 μM CQ reduces viral replication up to 45% whilst increasing cell viability.

As discussed before, the infection of human neural progenitor cells (hNPC) with ZIKV MR766, the infection of human neuroepithelial stem cells (NES) with ZIKV FSS13025 or the infection of foetal human neural progenitor cells with ZIKV PRV ABC59 or the infection of iPSC-derived human neural stem cells (NSC) or human astrocytes with ZIKV ArB41644 induces extensive Caspase-3 dependent apoptosis of infected cells. Similar to results obtained from Vero cells or hBMEC, ZIKV MR766 infected human NSC derived from human fibroblast cells are protected from ZIKV induced apoptosis upon treatment with CQ in addition to reduced viral replication. 

CQ mediated inhibition of viral replication can occur at various stages during the viral replication, either by inhibiting viral entry, release of the viral genome into the cytoplasm or at later stages of the viral replication such as inhibiting the degradation of PRR or STAT2, the degradation of TLR3 by viral induced autophagy or lipophagy. 

Figure: Chloroquine treatment inhibition of lipophagy

In addition, treatment of infected cells with CQ might also prevent the degradation of IFTIM-2 and IFTIM-3 positive endosomes that also contain the viral capsid or preventing the degradation of TLR-3 by autophagy following binding of viral RNA and thus promoting the induction of antiviral signalling. Indeed, the application of CQ from 30 min to 12 hrs p.i. decreases viral titres, indicating that several steps of the viral replication are sensitive to CQ treatment with viral entry being mostly affected.

Figure: Chloroquine treatment and TLR-3 signalling 

Axl and ZIKV entry: viral apoptotic mimicry

Flavivirus entry into susceptible cells is mediated by several receptors including Axl receptor tyrosine kinase which belongs to the TAM family, a group of tyrosine kinase receptors that are involved in the clearance of apoptotic cells by recognizing and binding to Phosphatidylserine (PS) which is located on the cell surface of apoptotic cells, a process which has been previously discussed for Ebola Virus. In general, the binding of viral particles that contain PS is mediated by bridging molecules such as Gas6 followed by clathrin mediated entry of viral particles by endocytosis and subsequent formation of early endosomes and subsequent release of the viral genome.

In the case of HCV, DENV, YFV and WNV the ectopic expression of either TIM and/or TAM receptors not only enhances viral replication but PS is also incorporated into the membrane of DENV particles, suggesting that apoptotic mimicry is being used for the infection of TAM and TIM expressing cells by members of the Flaviviridae in a Gas6 and ProteinS (PROS) dependent manner.

In the case of cells that support the replication of ZIKV, the expression of known Flavivirus entry factors including Axl is necessary to support viral replication; indeed, the expression of siRNA targeting Axl or the pretreatment of with Axl neutralizing antibodies of dermal reduces the replication of ZIKV MR766 in primary human dermal fibroblast cells. Accordingly, hNPC, hNSC, human astrocytes, human microglial cells and human radial glial cells of the developing neocortex express high levels of Axl mRNA whereas the expression of Axl mRNA in human placental cells varies with gestational age, correlating with the ability to support ZIKV replication. Like placental cells, microglial cells of the ventricular zone (VZ) and subventricular zone (SVZ) -GFAP+ cells such as radial glial progenitor cells at the ventricular border- derived from a foetus at 20 gestational weeks (GW) express high levels of Axl as indicated by indirect immunofluorescence staining whereas at 26 GW, Axl can only be detected in residual GFAP+ cells, thus explaining the absence of ZIKV E protein in mature neuronal cells.

Experimentally, the infection of an immortalized microglial cell line (CHME-3) as well as human and murine astrocytes but not hNPC with ZIKV HD78788 (an African isolate) can be inhibited both a polyclonal antibody against Axl as well as by MYD1 (an Axl decoy receptor) that sequesters Gas6, indicating that the entry of ZIKV HD78788 is dependent on both Axl and Gas6, suggesting that ZIKV is (at least in part) dependent on Axl and Gas6, utilizing viral apoptotic mimicry similar to DENV. Accordingly, the infection of induced pluripotent stem cell (iPSC) derived NPC and cerebral organoids with ZIKV PRVABC59 is not dependent on the presence of Axl since genetic ablation of Axl using CRISPR does not prevent ZIKV replication and ZIKV induced and Caspase-3 dependent apoptosis nor ZIKV dependent decrease of cell proliferation, whereas the replication and entry of both ZIKV H/PF/2013 and ZIKV HD78788 is abrogated in CHME-3 Axl^-/- cells.

As mentioned above, Flavivirus entry is dependent on clathrin mediated endocytosis. Consequently, the downregulation of either clathrin heavy chain (CLTC) or dynamin-2 (DNM-2) by transfecting siCLTC or siDNM-2 into CHME-3 cells and Hela-Axl cells impairs ZIKV HD78788 replication. Further analysis using GFP-tagged Rab5GTPase and Rab7GTPase WT and dominant negative (DN) constructs suggest that viral particles are targeted to the early endosome (EEA) since the expression of Rab5GTPase DN and Rab7GTPase WT but not Rab7GTPase DN reduces ZIKV HD78788 replication. In this scenario, the expression of a dominate negative mutant of Rab5 prevents the formation of the EEA whereas the expression of Rab7 WT promotes the formation of late endosomes and subsequent degradation of viral particles in the lysosomes. 

Figure: Rab5GTPase DN and Endosome formation: inhibition ? 

Furthermore, Rab5GTPase DN might also prevent the formation viral replication complexes, whereas the overexpression of Rab7GTPase WT might inhibit viral replication by promoting the formation of autolysosomes. So far it is not clear if the expression of Rab5GTPase DN stabilizes IFITM-2 and/or IFTIM-3 and thus contributes to IFITM-3 dependent inhibition of the fusion of the viral membrane and instead promotes the degradation of the viral content as it is the case for Influenza A. Also, so far it is not clear if the release of the ZIKV genome is dependent on the formation of the late endosome as it is the case for YFV and JEV.

Figure: Rab5GTPase DN and Rab7GTPase WT: IFITM-2/-3 stabilization ? 

Axl and the immune response: role of Gas6 in NLRP-3 inflammasome inhibition

The interaction of Gas6 with Axl and other members of the TAM family of receptor tyrosine kinases has been shown to lead to severe hepatic injury in Gas6 ^-/- mice upon the induction of liver injury by treating Gas6 ^-/- mice with CCl₄.

Recently published results showed that in murine P388D1 macrophages, Axl undergoes autophosphorylation of two Tyr residues (Tyr815 and Tyr 860) within the cytoplasmic domain upon treatment with GAS6 for 24 hrs. The autophosphorylation of Axl is followed

by an increase in LC3-II positive autophagic vesicles and increased autophagic flux which is accompanied by the induction of Atg5, Beclin-1 and LC3 expression.

GAS6/Axl induced autophagy however is not induced by the mTOR pathway since the neither the treatment of PD3881 cells with Rapamycin nor starvation inhibits the formation of autophagosomes by Axl; in contrast, treatment of PD3881 cells with SB203580 (MAPK 11/14 inhibitor) as well as the transfection with shMAPK14 abolishes the formation of autophagosomes, indicating that autophagy induced by GAS6/Axl is indeed induced by MAPK 14 mediated formation of autophagosomes.

Autophagy is involved in several biological processed such as the clearance of organelles and misfolded proteins. Recent observations suggest that autophagy is also involved in the maturation of the NLRP-3 inflammasome, which because of being induced in a two-step process first by a Toll-like receptor (TLR)/nuclear factor (NF)-κB pathway that induces the expression of NLRP-3 and then by PAMPs and DAMPs which induces the assembly of a multi-protein complex that consists of the linker protein ASC that binds both pro-Caspase-1 via the CARD domain and NLRP. Subsequent activation of Caspase-1 cleaves pro-IL-1β into the mature form of IL-1β. 

Figure: Formation of the NLRP-3 Inflammasome

Upon treatment with GAS6, Axl ^+/+ macrophages exhibit decreased levels of cleaved (mature) IL-1β, suggesting that the activation of Axl inhibits NLRP-3 mediated activation of Caspase-1 similar to glibenclamide treatment. Most importantly however, the treatment of autophagy deficient Atg7 ^fl/fl conditional knockout macrophages with GAS6 does not prevent the maturation of IL-1β, indicating that Axl induced autophagy inhibits the NLRP-3 inflammasome.

In the context of viral infections, GAS6 coated HIV-1 and WNV not only bind to and activate Axl but also inhibits the Type-I Interferon response, suggesting that GAS6 not only facilitates viral entry but also inhibits antiviral signalling.

In the case of ZIKV HD78788 infected CHME-3 wt cells treated with R428 –an Axl inhibitor- or the infected CHME-3 Axl KO cells, infection not only increases Interferon-β and SOCS-1 mRNA levels but also in increased expression of TNF-α, IL-6, and IL-1β, suggesting that the binding of ZIKV particles to GAS6 and Axl does induce the inhibition of NLRP-3. If this inhibition however is mediated by the induction of autophagy has not been demonstrated yet.

Figure: GAS6/Axl and ZIKV: inducing degradation of NLRP-3 via autophagy and inhibition of TLR-3 mediated signalling pathways ? 

In conclusion, the treatment of susceptible cells with Chloroquine might inhibit ZIKV infection at various stages of viral entry. One possibility is the stabilization of IFTIM-2 and -3 by preventing lysosomal degradation. Another possibility is that Chloroquine stabilizes the NLRP-3 inflammasome and thus increases the expression of cytokines by preventing the degradation of NLRP-3. Further studies are however needed to determine the role of NLRP-3 inhibition and IFITM-2/-3 during ZIKV infection of susceptible foetal cells and abnormal foetal development. 

Apart from Axl, ZIKV uptake might also be mediated by other receptors, including TIM and Tyro-3. The presence of a co-receptor therefore might allow not only ZIKV entry but also the induction of TLR mediated activation of NLRP-3. Axl induced formation of autophagosomes therefore might prevent the activation of NLRP-3 by TLR and thus prevent the recruitment of Caspase-1 to (inactive) NLRP-3 monomers.

The expression of Rab5GTPase DN therefore might not only stabilize IFTIM-2 and -3 but also prevent the induction of autophagy following the binding of the GAS6/ZIKV complex to Axl without affecting viral entry (unless it is dependent on Rab5GTPase). Viral RNA recognized by PAMPs induces the NLRP-3 in cells expressing Rab5GTPase DN and thus the secretion of pro-inflammatory cytokines. Further experiments are however needed to validate this hypothesis.

Figure: Inhibition of viral entry and Axl mediated induction of autophagy by Rab5GTPase DN ? 

A screen for inhibitors of ZIKV infection of U20S, human brain microvascular cells (HBMC) and human placental trophoblast cells (JEG-3) with ZIKV MR766, ZIKV Mex2-81 or ZIKV FSS13025 lead to the identification of 19 (U2OS), 12 (HBMC) and 16 (JEG-3) compounds that inhibit ZIKV replication, with Nanchangmycin the most potent inhibitor in all cell types (in addition to Vero cells) bar HBMC tested. Cells infected with ZIKV and treated for 4 hrs with Nanchangmycin exhibit decreased viral replication as determined by indirect immunofluorescence for the viral E protein at 24 hrs p.i. In a similar way, the inhibition of Axl by treating infected cells with Cabozantinib during the first 4 hrs following ZIKV infection decreases viral replication as well, indicating that the RTK activity of Axl is indeed necessary for viral entry.  

A fluorescence based uptake assay determined that both Nanchangmycin and Cabozantinib block the uptake of viral particles rather than inhibiting processes such as the induction of autophagy or the formation of EEA. In this assay, Nanchangmycin or Cabozantinib pre-treated U2OS cells were infected with ZIKV Mex2-81 in the presence of either drug at 4°C to synchronise the infection and then released for 3 hrs at 37°C to allow the entry of viral uptake prior to removal of the medium containing the drug and treating the infected cells with NH4Cl to prevent further viral uptake. Like U2O2 cells, both Cabozantinib and Nanchangmycin also block the entry of ZIKV FSS13025 into human primary uterine microvascular endothelial cells (UtMEC), HUVEC as well as primary placental fibroblast cells; in addition, Nanchangmycin also inhibits viral uptake of ZIKV Mex2-81 into a mixed population of murine neurons.



Furthermore, both Nanchangmycin and Cabozantinib also inhibit the entry of other viruses, including DENV, WNV, and CHIKV, that use clathrin mediated endocytosis to enter cells. 

Further reading

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