Impairment of neurogenesis in ZIKV infected neuronal cells: strain specific ? Asian/American v. African strains

During the current Zika Virus (ZIKV) outbreaks in the Americas, an increased number of cognitive malformations including but not limited to microcephaly in foetuses and neonates of mothers who had been infected with ZIKV during pregnancy, lead to the conclusion that ZIKV might be neuroteratogenic,  a hypothesis that has been supported by findings that various ZIKV strains –including isolates from Asia such ZIKV SZ 01, ZIKV FSS13025 and H/PF/2013 as well as isolates from the Americas such as ZIKV Mex 1-144,  ZIKV PRV ABC059 and ZIKV BR Paraiba 2015 or the (original) African isolate ZIKV MR766- not only infect and replicate in neuronal cells in vitro and in vivo, but also induces apoptosis of infected and non-infected cells. These results suggest that ZIKV may cause abnormal neuronal development of the foetal brain by inducing cell death of infected cells via intrinsic apoptosis as well as bystander apoptosis of non-infected cells via the secretion of pro-inflammatory cytokines.

As described before, ZIKV MR766 infected human neuronal progenitor cells (hNPC), not only undergo apoptosis, but also are arrested at G2/M phase of the cell cycle as measured by single parameter flow cytometry, which is supported by findings that in ZIKV Mex 1-144 infected foetal brain tissue of mice a decrease in Ki67 positive and Histone H3-P (Ser-10) positive cells can be observed, suggesting that ZIKV infection arrests infected cells in G2 phase of the cell cycle. Further research however is need if ZIKV infection of proliferating neuronal cells also induces aberrant mitosis like Coronavirus infected Vero and primary chicken cells. Moreover, the infection of human i90c16 (induced pluripotent human neural stem cell that are derived from IMR-90 human lung fibroblast cells with ZIKV H/PF/2013 may induce genotoxic stress resulting in the formation of γH2AX (H2AX-Ser19) positive DNA damage repair foci; since in ZIKV MR766 infected hNPC genes encoding for proteins that are involved in the repair of DNA damage are downregulated, sustained presence of DNA damage might result in the activation of the G2 checkpoint, thus preventing mitotic entry. Change of the expression of genes related to the cellular DNA Damage Response (DDR), apoptosis and neurogenesis may be induced in a TLR-3 dependent manner since the infection of h9 derived human embryonic stem cells (hESC) with ZIKV MR766 induces the activation of TLR-3 and thus TLR-3 mediated pathways that downregulate the expression of genes related to neurogenesis and upregulation of genes related to apoptosis.

ZIKV BR AB_ES v. ZIKV MR766 induced apoptosis and the cell cycle 

In the original study published by Tang et al. in 2016, the authors infected hNPC derived from dermal fibroblasts with ZIKV MR766, the original ZIKV isolate from Uganda (1947) which was extensively passaged in mice in 1950s, and analysed the changes in the expression of genes at 72 hrs p.i. . As described in extensio before, ZIKV MR766 downregulates the expression of genes encoding proteins involved in the induction of DNA damage response pathways (HR, NHEJ and FA) as well as in the initiation of DNA replication such as MCM-6 and the progression of the cell cycle from G1 to S, S phase progression and mitosis. These results were confirmed in a study in which dermal derived hNPC were infected with ZIKV MR766 or ZIKV FSS13025 with gene expression analysed at 64 hrs p.i.. Interestingly, the latter study also identified genes involved in the repair of damaged DNA, DNA replication and cell cycle progression that are only downregulated in ZIKV FSS 13025 infected hNPC as well as genes that specifically upregulated in either ZIKV MR766 or ZIKV FSS 13025 infected hNPC, suggesting that different strains might alter the expression of a subset of genes in a strain specific manner. Indeed, only ZIKV strains of the Asian lineage, ZIKV FSS 13025 and ZIKV H/PF/2013 so far have been shown to induce p53.

The ZIKV strains that are currently circulating in the Americas are derived from the Asian ZIKV lineage with ZIKV circulating in Brazil being 97-100% similar to Asian isolates. The infection of hNPC derived from dermal fibroblasts with ZIKV BR AB_ES for 72 hrs results in widespread apoptosis concomitant with the activation of Caspase-3 and a reduction of the number of cells expressing the neuronal markers Sox-2 and HUC/D, indicating reduction in the growth of neurospheres, thus depleting the pool of neural progenitor cells. In contrast to ZIKV MR766 infected hNPC however, the infection of hNPC with ZIKV BR AB_ES does not induce a pronounced arrest in G2 phase of the cell cycle, suggesting that ZIKV BR AB_ES -in contrast to ZIKV H/PF/2013 infected hNPC- might induce apoptosis independent of cell cycle arrest. Further experiments are however needed to determine if this is truly the cases since it might be possible that infected cells arrest at G2 prior 72 hrs p.i.. Based on gene expression data, both ZIKV MR766 and ZIKV BR AB_ES downregulate the expression of Cyclin E2 suggesting that both in ZIKV MR766 and ZIKV BR AB_ES infected hNPC the assembly of the pre-replication complex at the DNA and the G1/S transition might be affected. In addition, infection of hNPC with either ZIKV isolate downregulates the expression of MCM-6, suggesting that DNA replication might be inhibited. In contrast to ZIKV MR766, ZIKV BR AB_ES infection of hNPC upregulates the expression of components of the DDR, namely FANCD2, BRCA1, and MRE11A as well inducing the expression of DRAM-1 (probably via the induction of p53), suggesting that at least some DDR pathways might be not affected by ZIKV BR AB_ES.

Figure: Differences between ZIKV BR AB_ES and ZIKV MR766: DNA replication and cell cycle 

In addition to differences in the expression pattern of genes related to the control of the cell cycle and the DDR, genes that have proposed to be involved in viral replication are upregulated in hNPC infected with ZIKV BR AB_ES compared with previously published data obtained from hNPC ZIKV MR766 infected cells.

Figure:  Differences between ZIKV BR AB_ES and ZIKV MR766: genes proposed to be involved in viral replication 

In conclusion, the infection of hNPC with ZIKV BR AB_ES or ZIKV MR766 induces apoptosis of hNPC at 72 hrs p.i. probably because of inducing either a G1/S arrest and/or G2 arrest of the cell cycle although mitotic abnormalities at least in a subset of infected cells cannot be ruled out.   These changes in cell cycle progression are accompanied by a downregulation of genes involved in the onset and progression of S phase and DNA replication, suggesting that both isolates from the African and the Asian ZIKV lineage induce either stalled replication forks or prevent the formation of DNA replication complexes. Indeed, CldU pulse labelling experiments of ZIKV Mex 1-144 infected foetal (mice) NPC display an extension of S phase and in ZIKV BR AB_ES infected hNPC the expression of CDKN1A is upregulated suggesting that the CyclinE-cdk2 complex is inhibited although this has not been tested using a H1-kinase assay.

The downregulation of the expression of several genes involved in the progress of mitosis in ZIKV MR766 infected hNPC suggests that the infection of hNPC with ZIKV might also prevent or prolong mitotic exit, promoting aberrant cytokinesis. ZIKV FB-GWUH-2016 and ZIKV H/PF/2013 infected hNPC exhibit mother centrioles that lack appendages as well as one to the triplet microtubular blades at the distal ends, potentially resulting in multiple centrosomes during mitosis and consequently in aberrant mitosis. Indeed the hNPC infected with ZIKV BR/Bahia exhibit abnormal chromosome number (aneuploidy 12 + 17, mono-somy 12/17 and trisomy 12/17) as well as  multi- and bipolar cell division followed by    formation of micronuclei. 

Figure Abnormal centrosomes in ZIKV AS/AM infected hNPC

In addition, the downregulation of Centriolin in ZIKV BR AB_ES and ZIKV MR766 infected hNPC suggests that infected hNPC might either undergo apoptosis due to arrested cytokinesis or be arrested in G1 phase of the cell cycle. This supported by findings that both ZIKV FB-GWUH-2016 and ZIKV H/PF/2013 infected) but not ZIKV MR766 infected hNPC exhibit impaired neurogenesis due to perturbed centrosomes. 




Similar to centrinone treated hTERT-RPE1 immortalized retinal pigment epithelial cells (RPE1 cells), the infection of hNPC with either ZIKV H/PF/2013, ZIKV FB-GWUH-2016 or ZIKV BR AB_ES might trigger G1 arrest via the induction of 53BP1 in a p53 dependent manner; interestingly, BRCA-1 has also been implicated in mediating G1/S arrest as well as Bax dependent apoptosis, suggesting that ZIKV might induce cell cycle arrest and apoptosis via multiple pathways.

   

Figure: Asian and American ZIKV isolates induce cell cycle arrest by different pathways 

Different infectious profiles of ZIKV strains were previously described for African (ZIKV ArB41644) and Asian (ZIKV H/PF/2013) infected human iPSc derived neural stem cells (NSC), dermal fibroblast derived hNPC infected with either ZIKV MR766 or ZIKV FSS 13025. So far however, no studies are available that analyse individual viral genes nor extensive studies that examine the progression of the cell cycle in synchronised cells infected with different ZIKV strains. Also, it remains to be seen if primary isolates of African strains are similar to ZIKV MR766 or resemble Asian isolates. Finally, while experiments in hNPC are important, similar experiments are needed in mosquitoe cells.


Further reading 

Li H, Saucedo-Cuevas L, Shresta S, & Gleeson JG (2016). The Neurobiology of Zika Virus. Neuron, 92 (5), 949-958 PMID: 27930910 

van den Pol AN, Mao G, Yang Y, Ornaghi S, & Davis JN (2017). Zika virus targeting in the developing brain. The Journal of neuroscience : the official journal of the Society for Neuroscience PMID: 28123079 

Li H, Saucedo-Cuevas L, Regla-Nava JA, Chai G, Sheets N, Tang W, Terskikh AV, Shresta S, & Gleeson JG (2016). Zika Virus Infects Neural Progenitors in the Adult Mouse Brain and Alters Proliferation. Cell stem cell, 19 (5), 593-598 PMID: 27545505 

Tang H, Hammack C, Ogden SC, Wen Z, Qian X, Li Y, Yao B, Shin J, Zhang F, Lee EM, Christian KM, Didier RA, Jin P, Song H, & Ming GL (2016). Zika Virus Infects Human Cortical Neural Progenitors and Attenuates Their Growth. Cell stem cell, 18 (5), 587-90 PMID: 26952870 

Garcez PP, Nascimento JM, de Vasconcelos JM, Madeiro da Costa R, Delvecchio R, Trindade P, Loiola EC, Higa LM, Cassoli JS, Vitória G, Sequeira PC, Sochacki J, Aguiar RS, Fuzii HT, de Filippis AM, da Silva Gonçalves Vianez Júnior JL, Tanuri A, Martins-de-Souza D, & Rehen SK (2017). Zika virus disrupts molecular fingerprinting of human neurospheres. Scientific reports, 7 PMID: 28112162 

Gabriel, E., Ramani, A., Karow, U., Gottardo, M., Natarajan, K., Gooi, L., Goranci-Buzhala, G., Krut, O., Peters, F., Nikolic, M., Kuivanen, S., Korhonen, E., Smura, T., Vapalahti, O., Papantonis, A., Schmidt-Chanasit, J., Riparbelli, M., Callaini, G., Krönke, M., Utermöhlen, O., & Gopalakrishnan, J. (2017). Recent Zika Virus Isolates Induce Premature Differentiation of Neural Progenitors in Human Brain Organoids Cell Stem Cell DOI: 10.1016/j.stem.2016.12.005 

Souza BS, Sampaio GL, Pereira CS, Campos GS, Sardi SI, Freitas LA, Figueira CP, Paredes BD, Nonaka CK, Azevedo CM, Rocha VP, Bandeira AC, Mendez-Otero R, Dos Santos RR, & Soares MB (2016). Zika virus infection induces mitosis abnormalities and apoptotic cell death of human neural progenitor cells. Scientific reports, 6 PMID: 28008958

Ghouzzi VE, Bianchi FT, Molineris I, Mounce BC, Berto GE, Rak M, Lebon S, Aubry L, Tocco C, Gai M, Chiotto AM, Sgrò F, Pallavicini G, Simon-Loriere E, Passemard S, Vignuzzi M, Gressens P, & Di Cunto F (2017). ZIKA virus elicits P53 activation and genotoxic stress in human neural progenitors similar to mutations involved in severe forms of genetic microcephaly and p53. Cell death & disease, 8 (1) PMID: 28102842 

Caldon CE, & Musgrove EA (2010). Distinct and redundant functions of cyclin E1 and cyclin E2 in development and cancer. Cell division, 5 PMID: 20180967 

Gromley A, Jurczyk A, Sillibourne J, Halilovic E, Mogensen M, Groisman I, Blomberg M, & Doxsey S (2003). A novel human protein of the maternal centriole is required for the final stages of cytokinesis and entry into S phase. The Journal of cell biology, 161 (3), 535-45 PMID: 12732615

Shimada M, Matsuzaki F, Kato A, Kobayashi J, Matsumoto T, & Komatsu K (2016). Induction of Excess Centrosomes in Neural Progenitor Cells during the Development of Radiation-Induced Microcephaly. PloS one, 11 (7) PMID: 27367050 

Meitinger F, Anzola JV, Kaulich M, Richardson A, Stender JD, Benner C, Glass CK, Dowdy SF, Desai A, Shiau AK, & Oegema K (2016). 53BP1 and USP28 mediate p53 activation and G1 arrest after centrosome loss or extended mitotic duration. The Journal of cell biology, 214 (2), 155-66 PMID: 27432897 

Liu H, Gong M, French BA, Liao G, Li J, Tillman B, & French SW (2015). Aberrant modulation of the BRCA1 and G1/S cell cycle pathways in alcoholic hepatitis patients with Mallory Denk Bodies revealed by RNA sequencing. Oncotarget, 6 (40), 42491-503 PMID: 26623723 

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

Li H, Saucedo-Cuevas L, Shresta S, & Gleeson JG (2016). The Neurobiology of Zika Virus. Neuron, 92 (5), 949-958 PMID: 27930910

Tabata T, Petitt M, Puerta-Guardo H, Michlmayr D, Wang C, Fang-Hoover J, Harris E, & Pereira L (2016). Zika Virus Targets Different Primary Human Placental Cells, Suggesting Two Routes for Vertical Transmission. Cell host & microbe, 20 (2), 155-66 PMID: 27443522

Retallack H, Di Lullo E, Arias C, Knopp KA, Laurie MT, Sandoval-Espinosa C, Mancia Leon WR, Krencik R, Ullian EM, Spatazza J, Pollen AA, Mandel-Brehm C, Nowakowski TJ, Kriegstein AR, & DeRisi JL (2016). Zika virus cell tropism in the developing human brain and inhibition by azithromycin. Proceedings of the National Academy of Sciences of the United States of America, 113 (50), 14408-14413 PMID: 27911847 

Brault JB, Khou C, Basset J, Coquand L, Fraisier V, Frenkiel MP, Goud B, Manuguerra JC, Pardigon N, & Baffet AD (2016). Comparative Analysis Between Flaviviruses Reveals Specific Neural Stem Cell Tropism for Zika Virus in the Mouse Developing Neocortex. EBioMedicine, 10, 71-6 PMID: 27453325 

El Costa H, Gouilly J, Mansuy JM, Chen Q, Levy C, Cartron G, Veas F, Al-Daccak R, Izopet J, & Jabrane-Ferrat N (2016). ZIKA virus reveals broad tissue and cell tropism during the first trimester of pregnancy. Scientific reports, 6 PMID: 27759009 

Quicke KM, Bowen JR, Johnson EL, McDonald CE, Ma H, O'Neal JT, Rajakumar A, Wrammert J, Rimawi BH, Pulendran B, Schinazi RF, Chakraborty R, & Suthar MS (2016). Zika Virus Infects Human Placental Macrophages. Cell host & microbe, 20 (1), 83-90 PMID: 27247001 

Delvecchio R, Higa LM, Pezzuto P, Valadão AL, Garcez PP, Monteiro FL, Loiola EC, Dias AA, Silva FJ, Aliota MT, Caine EA, Osorio JE, Bellio M, O'Connor DH, Rehen S, de Aguiar RS, Savarino A, Campanati L, & Tanuri A (2016). Chloroquine, an Endocytosis Blocking Agent, Inhibits Zika Virus Infection in Different Cell Models. Viruses, 8 (12) PMID: 27916837

Wells MF, Salick MR, Wiskow O, Ho DJ, Worringer KA, Ihry RJ, Kommineni S, Bilican B, Klim JR, Hill EJ, Kane LT, Ye C, Kaykas A, & Eggan K (2016). Genetic Ablation of AXL Does Not Protect Human Neural Progenitor Cells and Cerebral Organoids from Zika Virus Infection. Cell stem cell, 19 (6), 703-708 PMID: 27912091 

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