Autophagy in CSFV and ZIKV infected cells: persistence versus neurodegenerative disease

Zika Virus (ZIKV) was first isolated in 1947 from a sentinel monkey in Uganda and associated with human infection in 1954 when neutralizing antibodies were detected in the sera of residents in India, with antibodies also being found in residents from various African countries. ZIKV is mosquitoe-borne Flavivirus that is predominantly transmitted via Aedes Agypti, although sexual transmission (female to male, male to female, male to male) and transmission via blood transfusion has also been reported.

Clinically most cases are asymptomatic and symptomatic cases only present themselves with relative benign symptoms in adults, the exception being Guillain Barre Syndrome. During in the current outbreak in the Americas and –albeit only retroactively identified- in the 2013/2014 outbreak in French Polynesia, ZIKV has been identified to a causative agent of abnormal foetal brain development, leading to congenital defects, namely microcephaly, anomalies of the CNS, miscarriages and in rare cases foetal and/or neonatal death. Studies in both wt and immunodeficient mice confirmed that the infection of pregnant mice with various strains of ZIKV including strains from Asia (SZ01, FSS1305), Oceania (H/PF/2013), Brazil (Paraiba 2015), Puerto Rico (PRVABC59) and Mexico (MEX_1_7) indeed can cause abnormal development of the (foetal) brain by apoptosis of neural progenitor cells similar to representatives of the African strain, ZIKV MR766 (Uganda, 1947) and ZIKV IbH30656 (Nigeria, 1968), tested. In addition, the infection of brain organoids, neurospheres, human (foetal) Neural Progenitor Cells (h (f) NPC) and human Neural Stem Cells (NSC) with various ZIKV strains including a primary isolate from Africa, ZIKV ArB41644, demonstrated that ZIKV can indeed induce apoptosis of proliferating but probably not of mature neurons. Recently the infection of cranial neural crest cells (CNCC) with either ZIKV MR766 or ZIKV H/PF/2013 has been reported to induce high levels of cytokines that are detrimental for neurogenesis, causing bystander apoptosis of uninfected neuronal cells. Infected CNCC do not undergo apoptosis at 24 hrs p.i. but apoptosis is increased at 72 hrs p.i. confirming that both ZIKV MR766 and ZIKV H/PF/2013 can induce apoptosis of CNCC although only approx. 8% of ZIKV infected CNCC undergo apoptosis compared to 4% of mock or DENV infected CNCC, indicating that CNCC support viral infection whilst being protected against apoptosis probably by VEGF as indicated by increased secretion of VEGF which has been shown to inhibit apoptosis of human microvascular endothelial cells (HUMEC).  

In brains of wt C57BL/6 mice intracranial infected with ZIKV MR766 day 7 postnatal and analysed day 4 p.i. brain mass is reduced compared to mock infected mice by 25%, as well as showing an increase of NeuN⁺  and CTIP⁺ cells that also positive for active Caspase-3, indicating that ZIKV MR766 does not or rarely induces apoptosis in corticospinal cells, neuronal progenitor cells and astrocytes but in mature neurons in postnatal mice.  These results are in contrast with previously discussed results that indicate that in foetal mice, mature neurons are ZIKV negative and do not undergo apoptosis upon ZIKV infection. In contrast to mice infected at day 7 postnatal, mice infected at three weeks postnatal do show less severe apoptosis as measured by the presence of active Caspase-3 although similar to day 7 postnatal infected mice, widespread apoptosis of NeuN⁺ cells is detected by the presence active Caspase-3. When interpreting these experiments however, one has to bear in mind that ZIKV MR766 is a neurotrophic strain adapted to mice brain due to repeated intracranial passage.

Autophagy and viral infection

Autophagy is a cellular degradation pathway that involves the sequestration of cytoplasmic component such as organelles, protein aggregates or pathogens with (transient) double membrane most commonly derived from the ER, forming a phagosome that ultimately matures into the autophagosome and is degraded by lysosomal enzymes such as acidic hydrolases upon fusion with the lysosome. In general, basal autophagy is part of the turnover of RNA, proteins and organelles but can be induced in cells undergoing various forms of stress, including starvation. Both genome wide screens and large scale proteomic basic screens identified a substantial number of autophagy related regulators, including but not limited to autophagy related genes (ATG) that initiate the formation and the maturation of autophagosomes, aided by improved imaging, as well as degrading substrates such as RNA in lysosomes.

In the case of viral infections, the interactions between positive sense RNA viruses is of particular interest since autophagosomes might contribute to the decay of (viral) RNA in lysosomes by RNase T2, RNASE1 and RNASE6/RNase K6. Although it has not been determined experimentally, viral RNA might be targeted to lysosomes directly (similar to LINE 1 via processing bodies/stress granules that interact with NDP52/CALCOCO2 or p62/SQSTM-1) or as part of a complex with viral and/or cellular proteins that form a RNA-Protein Binding complex (RBP).

Figure: General outline of the autophagy pathway

More importantly however, RNA viruses such as Chikungunya Virus (CHIKV), Coronaviruses’ (CoV), Polio Virus, Measles Virus, DENV, Hepatitis C Virus (HCV) or Classical Swine Fever Virus (CSFV) can utilize autophagosomes and/or double membrane vesicles (DMV) to increase viral replication. As discussed in previous posts, in the case of CHIKV or CoV, the formation of viral replication centers (RC) is dependent on the autophagy machinery, including ATG5 and Beclin-1, whilst in others –such as HCV- autophagy is also required for exit of the mature virions as exosomes. To prevent degradation of assembled viral particles, viral proteins that inhibit the fusion of the autophagosome with the lysosome by binding Beclin-1 for instance are expressed late in the replication cycle.

Figure: Regulation of the autophagy pathway by CoV proteins as an example for the regulation of
autophagy by viral proteins

Autophagy itself can be activated via the inhibition of the Phosphatidylinositol-3 Kinase (PI3-K)-Akt-mTOR pathway, thereby inducing the phosphorylation of the ULK-1 complex and subsequently inducing the formation of the phagosome and autophagosome. This pathway is activated by the binding of growth factors such as Insulin to their respective cell surface receptor followed by the conversion of membrane bound Phosphatidylinositol-4’,5’-bisphosphate (PIP₂) to Phosphatidylinositol-3’,4’,5’-trisphosphate (PIP₃) thus recruiting Akt to the plasma membrane and phosphorylating Akt. Phosphorylated Akt activates the TSC1/2 complex, the GTPase activating protein (GAP) of Rheb. Activated Rheb binds and activates the mTOR-Raptor complex, inhibiting the formation of autophagosomes and thus autophagy.

Indeed, preventing the phosphorylation of Akt both at Thr 308 and Ser473 reduces the phosphorylation of mTOR at Ser2448 thus promoting autophagy whereas the ectopic expression of a constitutively active myristoylated mutant of Akt (Akt3 E17K) inhibits autophagy.

Figure: Akt inhibits the formation of autophagosomes via mTORC-1

The inhibition of autophagy in neuronal cells can induce neurodegeneration and autophagy defects –including lysosomal defects- have been implicated in Huntington Disease and ALS as well as in microcephaly and megalencephaly-polydactyly-polymicrogyria-hydrocephalus.  

CSFV and Autophagy: where autophagy meets viral persistence

Before discussing recent results concerning ZIKV and autophagy, it is worth examining the role of autophagy in cells persistently infected with CSFV and how autophagy contributes to the survival of viral infected cells.

Classical Swine Fever Virus (CSFV) is positive strand RNA virus which is classified as a Pestivirus within the Flaviviridae, causing highly virulent disease in swine characterized by a high fever, leukopenia and hemorrhages with a mortality. In infected PK-15 and 3D4/2 cells, CSFV inhibits the type I Interferon response, leading to persistent infection in the absence of apoptosis. In a recently published study, CSFV has been shown to induce the formation of LC3-II positive (mature) autophagosomes as early as 48 hrs p.i. and both the viral E2 and NS5A proteins have been shown to localise in LC3 positive vesicles as early as 24 hrs p.i. which can be inhibited by 3-Methyladenine (3-MA) as well as by downregulating the expression of either Beclin-1 or LC3-B suggesting that CSFV does induce the formation of autophagosomes as well as autophagic flux as evidenced by decreased levels of p62/SQSTM-1 in CSFV infected PK-5 and 3D4/2 cells.

Autophagy induction by CSFV however not only increases viral replication –presumably by forming viral RC- but also decreases viral induced apoptosis and decreasing mRNA levels of genes related to the type I Interferon response and IFN stimulated genes (ISG). 

CSFV induces apoptosis via activation of the intrinsic apoptotic pathway, i.e. via activation of Caspase-9 and Caspase-3, by inducing the accumulation of mitochondrial reactive oxygen species (ROS) if autophagy is inhibited in infected PK-5 and 3D4/2 cells by shBeclin1 or shLC3 which can be inhibited by either Z-VAD (a pan caspase inhibitor) or Necrostatin-1 (Nec-1/ROS scavenger). In addition to the intrinsic pathway, the extrinsic –Caspase-8 dependent- is also triggered in CSFV infected autophagy deficient cells.

CSFV induction of mitochondrial ROS not only induces the activation of apoptosis but also induces autophagy and thus promotes the clearance of damaged mitochondria via mitophagy, which is indirectly indicated by the increase of copy numbers of mitochondrial DNA in autophagy deficient PK-5 and 3D4/2 CSFV infected cells (flow cytometry using JC-1, NAO or TMRE was not performed) and/or promotes autophagy in a Nrf-2 dependent manner.

The accumulation of ROS in CFSV infected cells also induces antiviral signalling via RIG-1 and MDA-5, including increased expression of TNF/TNF-α, IFN-β/IFNB1 and FAS/TNFRSF6 in autophagy deficient CSFV infected cells, thus inducing the extrinsic pathway in an autocrine manner, which is inhibited in shDDX58, shIFIH1 and shMAVS CSFV infected autophagy deficient cells. The induction of autophagy by CSFV therefore might not only promote the clearance of damaged mitochondria but also the decreasing transcripts of ISG via RNautophagy. Similar to other positive strand RNA viruses, CSFV might also induce autophagy via the ER stress response induced by the viral NS2 and NS5A proteins which might play a role in establishing a persistent infection. In addition, the induction of NFκ-B by oxidative stress can also autophagy. If any of those pathways however contributes to viral persistence has not been investigated yet.

Figure: CSFV inhibitors apoptosis and induces autophagy via multiple pathways

Also, so far it has not been demonstrated if CSFV induced formation of ROS also induces the induction of TLR-9 by mitochondrial DNA. In this scenario, a complex of mtDNA and TLR-9 might be degraded via autophagy and thus abrogate antiviral signalling.

ZIKV and autophagy: impairment of autophagic flux linked to ZIKV induced apoptosis ?

In the case of ZIKV, the formation of autophagosome like vesicles has been observed in ZIKV MR766 infected human primary fibroblasts and keratinocytes and in the cytoplasm of C6/36 cells infected with a ZIKV isolate from a patient in Brazil although in A549 cells infected with ZIKV H/PF/2013 no LC3 positive structures have been detected.

Following the infection human foetal neural stem cells (fNSC) infected with either ZIKV MR766, ZIKV H/PF/2013 or ZIKV IbH30656 the formation of neurospheres is significantly impaired at day 7 p.i., as indicated by fewer neurospheres present which are also smaller in size, due to the induction of apoptosis as measured by TUNEL staining. Ina accordance with previously published results, ZIKV infected fNSC derived neurospheres also exhibit a reduction of cell proliferation measured by both BrdU incorporation, suggesting that ZIKV indeed does affect the proliferation of foetal neuronal precursor cells.

Similar to ZIKV MR766 infected human keratinocytes, the infection of fNSC with ZIKV MR766 or ZIKV IbH30656 induces the formation of LC3-II positive autophagosomes, without affecting autophagic flux, indicating that ZIKV infection of fNSC induces the formation of autophagosomes without affecting the formation of autolysosomes as measured by the degradation of p62/SQSTM-1. It should be noted that the infection of HeLa cells transiently transfected with a GFP/RFP-LC3 tandem plasmid with ZIKV MR766 increases GFP+/RFP+ punctae, indicating that the formation of the autolysosome might be inhibited. The discrepancy can either be due to the use of HeLa cells or it might be possible that ZIKV infection leads to the proteasomal degradation of p62/SQSTM-1 in fNSC similar to DENV-2 infected Huh-7 cells. 

In any case, treatment of ZIKV infected fNSC with Chloroquine decreases viral RNA/viral titres whilst Rapamycin treatment increases levels of both viral RNA and titers as early as 10 hrs p.i. suggesting that autophagic flux is necessary for viral replication. Based on the results described for CSFV above, autophagic flux might be necessary to prevent viral induced apoptosis and/or antiviral signalling; therefore, treatment with Chloroquine might prevent the degradation of viral RNA-TLR complexes and thus induce antiviral signalling whereas in untreated cells residual autophagy might be sufficient for inducing a partial antiviral response.  Treatment of fNSC with 3-MA inhibits viral replication, suggesting that autophagosome formation is required for viral replication, probably for the formation of viral RC. 

The ability to initiate the formation of the autophagosome is further highlighted by results that the replication of ZIKV MR766 in Atg3 -/- MEF and Atg5 -/-/MEF as well as in MEF transfected with siATG13 or siATG3 is lower when compared to wt MEF.

In DENV and HCV infected cells, the localisation of viral proteins induces the formation of viral replication complexes by utilizing the autophagy machinery; in a similar way, CoV nsp-3 and -4 have been shown to induce the formation of LC3-II positive vesicles that are derived from the ER.

In the case of ZIKV, the expression of NS4A and NS4B induces the formation of GFP-LC3 positive punctae in HeLa-GFP-LC3 cells which is more pronounced if both NS4A and NS4B are co-expressed. Similar to ZIKV MR766 infected cells, the co-expression of NS4A and NS4B increases the percentage of GFP+/RFP+ positive punctae, indicating that autophagic flux might be inhibited, although p62/SQSTM-1 levels are decreased which is probably due to proteasomal degradation of p62/SQSTM-1 (as described above). 

Based on immunofluorescence data and on data obtained from DENV NS4A and DENV NS4B, both ZIKV NS4A and NS4B are localised at the ER, suggesting that autophagy might be induced by the ER stress response.

Figure: Topology of DENV NS4A and NS4B 

As described before, the ER stress response can initiate autophagy by negatively regulating the PI-3-K/Akt pathway. Indeed, ZIKV NS4A and NS4B reduce the phosphorylation of Akt at both Thr308 and Ser473 as well as of mTOR at Ser2448 which can be reversed by the expression of a constitutively active form of Akt, Myr-HA-Akt3 E17K, in ZIKV MR766 infected fNSC or HeLa cells, suggesting that NS4A and NS4B induce the ER stress response, although it has not proven experimentally yet. 

Figure:ZIKV NS4A and NS4B inhibits PI3-K and therefore activates the formation of autophagosomes

In addition, the expression of ZIKV NS4A and NS4B decreases the proliferation of fNSC as measured by Ki67, which is more pronounced if both NS4A and NS4B are co-expressed. Since prolonged ER stress induces not only autophagy but also apoptosis, the (co-) expression of NS4A and NS4B might induce both autophagy and apoptosis although the percentage of apoptotic cells following the transfection of fNSC with ZIKV NS4A/B has not been determined.

Figure: ZIKV NS4A and NS4B decrease the expression of Ki-67 and the proliferation of fNSC

In conclusion, the infection of fNSC with ZIKV MR766, ZIKV IbH30656 and ZIKV H/PF/2013 induces apoptosis, decreased cell proliferation, smaller neurospheres and the formation of autophagosomes with impaired autophagic flux, the latter probably induced by both NS4A and NS4B protein. The notion that ZIKV MR766 inhibits the fusion of the autophagosome with the lysosome -or lysosomal maturation- and thus induces neurodegeneration by impairing the degradation of autophagic substrates is supported by findings in ZIKV MR766 infected hNPC, the expression of LAMP-2 is downregulated.

Figure: ZIKV and autophagy

If the impairment of autophagic flux however is linked to the induction of apoptosis in ZIKV infected primary neuronal cells has not been demonstrated. It might be possible that similar to CSFV infected cells, autophagy is required for abrogating antiviral signalling.
It should be noted that the infection of hNPC with ZIKV MR766 downregulates the expression of key proteins involved in the onset and progression of stress induced autophagy. Therefore, ZIKV infection of neuronal and non-neuronal cells might inhibit stress induced and selective autophagy whilst promoting the formation of viral RC. It is therefore important to analyse if the formation of viral RC is totally or only partially dependent on the ability to induce the formation of autophagosomes since viral proteins might be able to induce some of the processes required for the formation of RC independent of the autophagy machinery. 

Table: Genes that are up- or downregulated in ZIKV MR766 infected hNPC

A screen of FDA approved revealed that both Bortezomib and Ivermectin inhibit the replication of ZIKV Mex_1_7 in infected Huh-7, HeLa and/or Human Amnion Epithelial Cells. Both Bortezomib and Ivermectin induce apoptosis of cancer cells by inhibiting autophagy, indicating that in ZIKV infected (neuronal) cells autophagy might not only support viral replication by forming viral RC but also by inhibiting apoptosis at least in the early stages of viral replication. If the induction of autophagy has also consequences for the establishment of persistently infected cells, however remains to be seen.

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