Virology tidbits

Virology tidbits

Wednesday, 14 September 2016

ZIKV: antivirals and the cell cycle, TBK-1 relocalisation and immune signalling

Zika Virus (ZIKV) is an emerging flavivirus that was first isolated in 1947 from a sentinel
monkey in Uganda as part of study that aimed to identify novel pathogens and despite sporadic local outbreaks in countries such as Gabon, Nigeria, Cambodia, Malaysia and Indonesia followed by the first major outbreak in Yap/Federal States of Micronesia 2007  only caused mild disease in humans with up to 80% of asymptomatic cases.

The emergence of ZIKV combined with severe pathogenicity following the outbreak in French Polynesia 2013/2014 with an excess of 30000 patients and particular the introduction of ZIKV to Brazil  as early as 2013 as suggested by molecular clock analysis however raised questions about the molecular evolution of ZIKV since ZIKV was previously only associated with arthralgia and a mild febrile illness but not neuropathological disorders including abnormal foetal brain development and Guillain-Barre Syndrome (GBS) that were first identified in Pernambuco/Brazil and in a retroactive study of the 2013 outbreak in French Polynesia.

ZIKV is a flavivirus closely related to Dengue Virus (DENV), Japanese Encephalitis Virus (JEV) and Yellow Fever Virus (YFV) with a single stranded positive stranded RNA genome of approximately 10800 bp. Similar to DENV, JEV and YFV, the ZIKV RNA encodes for a single polyprotein that it is cleaved into the structural (Capsid (C), pre-membrane (prM), and envelope (E)) and non-structural (NS1, NS2A, NS2B, NS3, NS4A, 2K, NS4B, and NS5) proteins with the replication taking place in the cytoplasm of infected although at least the C and NS5 proteins localise to the nucleolus and to nuclear speckles respectively, suggesting that the nuclear localisation of these proteins might be required for efficient replication of JEV probably due to the interaction of the JEV core protein with B23, thus relocalising B23 to the nuclear periphery. In contrast to JEV core protein however, the DENV core protein does not co-localize with B23. In the case of NS5, the expression of DENV NS5 interacts with components of the cellular spliceosome –in particular with components of the U5 small nuclear ribonucleoprotein particle- and thus disrupts the maturation of cellular pre-mRNA by decreasing the efficiency of pre-mRNA processing, thus contributing to the downregulation of cellular gene expression. Presently it is not known to which extent ZIKV derived proteins interfere with these processes.

Both DENV and ZIKV NS5 have been shown to inhibit the nuclear translocation of STAT2 and thus antiviral signaling, suggesting that ZIKV and DENV NS5 exhibit similar if not identical properties and similar to DENV, so called “viral factories” or viral replication centers are formed in the cytoplasm of ZIKV infected cells which contain both viral (progeny) RNA as well as viral proteins. Since ZIKV RC are similar to the viral replication centers of other positive strand RNA viruses and are positive for LC3, it has been proposed that these are formed by utilizing the autophagic machinery although in A549 cells infected with the South Pacific ZIKV PF-25013-18 no LC3-B positive structures have been identified (in contrast with ZIKV 766 infected human keratinocytes or ZIKV SPH 2015 infected human astrocytes) and chloroquine inhibits ZIKV replication in infected U87 glioblstoma cells.

In any case, as mentioned above, both the ZIKV outbreak in French Polynesia and the current outbreak in the Americas are are associated with neurological abnormalities, namely foetal microcephaly/micrencephaly, lissencephaly, hydrocephaly, cortical/periventricular calcifications, hypoplasia of the brain stem and spinal cord, necrosis  and other congenital abnormalities such as focal pigment mottling of the retina, optic nerve abnormalities and chorioretinal atrophy in foetuses and newborns of previously infected women as well as uveitis, conjunctivitis and GBS in adults. These observations suggest that ZIKV is neurotrophic, a finding which was first reported in mice following the isolation of ZIKV from the sentinel monkey (for further details see previous discussion here). Subsequent studies demonstrated that ZIKV enters neuronal and non-neuronal cells via different receptors, the phosphatidylserine TAM receptor Axl that is enriched on the surface of human glial cells and the main receptor, and with DC-SIGN, TIM-1 and Tyro-3 as minor receptors.
Consequently, recently published studies which have been discussed in extensio before, suggest that ZIKV can infect human neural progenitor cells (hNPC) derived from induced pluripotent stem cells, brain organoids and neurospheres that are derived from embryonic stem cells or induced pluripotent stem cells as well as two foetal cell lines. These studies showed that ZIKV induces caspase-3 dependent apoptosis which may be preceded by mitochondrial depolarization and subsequent activation of caspase-3 via the release of cytochrome-c although the mechanism leading to mitochondrial depolarisation has not been elucidated (see here for discussion). ZIKV infected foetal neural tissue samples derived 13 -16 weeks post conception exhibits high levels of infection in the ventricular and subventricular zone which are positive for radial glia cells  with only a small number of mature neurons being infected and later ( 18 weeks pcw), suggesting that postmitotic neurons are not susceptible to ZIKV which is confirmed by the absence of Axl in mature neurons.

More recent studies also identified the vaginal mucosa and lacrimal glands of mice as being susceptible for ZIKV thus providing a model of sexual transmission and viral persistence respectively. Interestingly, ZIKV infection of the adult neurosensory retina induces apoptosis as measured by TUNEL staining yet does not induce significant pan-retinal abnormalities.

Antiviral drugs: targeting caspase-3

Recently, a drug repurposing screen identified several small molecule inhibitors that inhibit ZIKV induced caspase-3 dependent apoptosis in ZIKV FSS 13025 (Cambodia 2010) or ZIKV MR766 (Uganda 1947) infected SNB-19 glioblastoma, human astrocytes and hNPC. In this assay, 194 compounds were tested using two high-throughput assays with one measuring both cell viability at 72 hrs p.i. and caspase-3/-7 activity at 6 hrs p.i. and the other measuring the caspase-3/-7 activity in a primary screen followed secondary screen measuring both cell viability and caspase-3/-7 activity which is then followed by tertiary screen that involves a ZIKV replication assay, 2D & 3D neural cell models (such as hNPC and brain organoids) and in addition measuring the effect of drug combinations on ZIKV replication and cell viability. Despite causing apoptosis in all cell types tested, ZIKV MR766 induced apoptosis can only be prevented by 35 compounds tested in all cell types, with 54 inhibiting apoptosis in human astrocytes, 57 in SNB-19 glioblastoma cells and 48 in hNPC, whereas only 1 compound –a pan-caspase inhibitor (Emericasan)- inhibiting both caspase-3/-7 activity and increasing the viability of ZIKV MR766, ZIKV FSS 13025 and ZIKV PRVABC59 infected hNPC/SNB-19 cells and brain organoids. In contrast to Emericasan, the vast majority of screened not only inhibited apoptosis but also had a negative impact on cell proliferation even in the absence of viral infection.

Figure: Effect of tested compounds on cell viability of ZIKV infected cells (Astrocytes, SNB-19 glioblastoma cells
and hNPC) Negative cell viablity=toxic effect even in absence of ZIKV 

Figure: Effect of tested compounds on ZIKV induced caspase-3 activity 

Besides preventing ZIKV induced apoptosis, Emericasan also reduced viral replication as measured by determining viral titres and measuring the expression levels of the viral NS1 protein, suggesting that the inhibition of cellular caspases also inhibits viral replication. One possibility is that ZIKV induced activation of caspase-3/-7 and/or other caspases inactivates Beclin-1 induced autophagy by cleaving Beclin-1 at AA 133 and AA149 (TDVD133 and DQLD149 respectively) thus not only preventing autophagy but also localising the resulting Beclin-1 C terminal fragment to the mitochondria, inducing the release of Cytochrome-c in addition to cleaving Phosphatidylinositol-3-Kinase (PI3KC3)/vacuolar protein sorting complex-34 (Vps-34). Restoring Beclin-1 dependent autophagy in Emericasan treated and ZIKV infected cells therefore might contribute to the inhibition of viral replication.

Additionally, the replication of ZIKV FSS13025 and ZIKV PRVABC59 in SNB-19 cells and human astrocytes can be efficiently inhibited by Cyclin dependent kinase inhibitors (Cdki) such as PHA-690509, Niclosamide  and Seliciclib that inhibit the progression of the cell cycle, indicating that cellular Cdk might phosphorylate the viral NS5 and/or NS5a protein similar to the DENV-2, TBE  and YFV NS5 or BVDV NS5a or that the progression in particular from the G1 phase of the cell cycle to S phase might be required for efficient ZIKV replication similar to Mouse Hepatitis Virus (MHV), Infectious Bronchitis Virus (IBV), SARS-CoV and Coxsackievirus B1. Further experiments are however needed to determine the role of Cdk’s in ZIKV replication which might involve using Cdk -/- MEF and/or siRNA targeting specific Cdk. The disadvantage of using Cdki however is that the proliferation of ZIKV PRVABC59 infected/Cdki treated cells as measured by EdU incorporation is significantly decreased compared to non-infected hNPC at 72 hrs p.i. thus limiting the use in utero. Cdki however might be useful in treating adults, preventing sexual transmission and/or prolonged shedding of ZIKV in urine, saliva and tears.

Figure: (A) Niclosamide: targeting viral entry (B) Cdki: targeting multiple cdk

ZIKV and the cell cycle: G2 and mitotic arrest

As discussed in a previous post, the in utero infection of (mouse) foetal brains with ZIKV SZ01 decreases the expression of proteins that previously have been linked to the development of microcephaly, in particular those that are involved in the separation of chromosomes during metaphase and anaphase, suggesting that ZIKV infected embryonic and/or foetal cells might exhibit incomplete cytokinesis and subsequent apoptosis, a notion that is supported by previous observations that  hNPC infected with ZIKV MR766 also exhibit a decrease in the expression of the very same genes confirming that the downregulation of genes regulating mitotic progression might arrest infected cells in G2/M phase of the cell cycle which is confirmed by flow cytometry analysis of infected hNPC. In addition to hNPC, more recent data indicate that the infection of neuroepithelial cells derived from the Neocortex (NCX-NES) derived from human specimens ranging from 5 to 8 weeks postconception with ZIKV FSS 13025 not only support viral replication as evidenced by the expression of the viral NS1 protein but also undergo caspase-3 dependent apoptosis including nuclear fragmentation and pyknosis as well exhibiting decreased cell proliferation as indicated by the absence of the proliferation marker Ki-67 starting at day 3.5 p.i. and continuing until day 6.5 p.i. . In contrast to NCX-NES cells, mature neurons do not support viral replication as measured by the presence of NS1 probably due to the absence of the entry receptor, Axl, and do not show a significant increase in apoptosis. Similar to the ZIKV SZ01 isolate, ZIKV FSS13025 and the ZIKV BE 243 strain (derived from the current outbreak in Brazil) also infect radial glial cells (RGC) of the ventricular zone (VZ), subventricular zone (SVZ) and the intermediate zone (IZ/SP), all of which contain PCNA positive proliferating cells and VIM positive RGC cells, of ex vivo foetal brain slices with viral replication being detected as early as day 3.5 p.i. , similar to NCX-NES cells. Most interestingly however, only ZIKV infected and ZIKV NS1 positive cells exhibit an aberrant cell morphology which indicates that primary proliferating neuronal cells infected with an ZIKV replication competent strain (but not with an UV inactivated strain) induce a cell cycle arrest.
In addition to downregulating the expression of genes related to mitotic progression such as Aurora Kinase-B, activation of the innate immune response can induce apoptosis, i.e. via IRF-3 mediated activation of Bax by Sendai Virus (SeV). In this case, the dsRNA intermediate activates IPS-1 which in turn recruits TANK-binding Kinase-1 (TBK-1) which in turn phosphorylates and activates IRF-3, the latter binding Bax and translocating to the mitochondrion.
In the case of ZIKV infected NCX-NES cells or foetal brain slices neither ZIKV FSS13025 nor ZIKV PE243 increases the expression of TBK-1 but rather relocalises TBK-1 from the centrosome to mitochondria thus potentially preventing the phosphorylation of the centrosomal protein CEP170 and the mitotic apparatus protein NuMA as well as g-tubulin, leading to mitotic defects such as aberrant cytokinesis characterised by the presence of a cleavage furrow in G1 phase of the cell cycle and/or subsequent apoptosis due to mitotic catastrophe. The presence of cells with a cleavage furrow might also explain the presence of a small percentage of ZIKV positive (postmitotic) neurons.

In addition, inhibiting the progress of mitosis, TBK-1 also might recruit IRF-3 and Bax to the mitochondria thus providing an alternative pathway culminating in apoptosis independent of mitotic arrest. Further experiments are needed to distinguish both pathways. Paradoxically the inhibition of TBK-1 with inhibitors such as BX795 or Amlexanox exacerbates ZIKV induced apoptosis. One reason might be that the relocalisation of TBK-1 in ZIKV infected cells also induces mitophagy by recruiting p62/SQSTM-1 and/or optineurin so that treating infected cells with TBK-1 also decreases mitophagy and thus the clearance of damaged mitochondria.
Besides the induction of apoptosis, mitochondrial localisation of TBK-1 may also interfere with immune signalling by disrupting STING mediated phosphorylation of TBK-1 at perinuclear granulae and thus the translocation of IRF-3 to the nucleus, thus subsequently inhibiting the Interferon response. In this context, results from both WNV and DENV-1, -2 and -4 infected primary endothelial cells and HEK 293T cells indicate that the viral encoded NS2A and NS4B inhibit the phosphorylation of both TBK-1 and IRF-3 and subsequent induction of Interferon-b, with DENV-1/-2/-4 NS4A uniquely inhibiting TBK-1 and IKKε-directed signalling. In a similar way, PEDV has been shown to inhibit TBK-1 signalling as well. Additionally, the expression of ZIKV NS4B protein might –similar to to DENV NS4B- induce the elongation of mitochondria in infected cells and disrupting the ER-Mitochondiria contact (MAM) which is critical for immune signalling and thus abrogating the celluar RIG-1 dependent interferon response.

Figure: ZIKV and DENV-1/-2/-4 NS4A and TBK-1 mediated signalling:
targeting phosphorylation of IRF-3

Apart from inhibiting mitotic progression, interfering with TBK-1 dependent immune signalling  and inducing apoptosis, mitochondrial TBK-1 might also induce lipophagy and thus promote viral replication by inducing mitophagy via recruitment of p62/SQSTM-1, NDP52 and/or Optineurin. Again further studies are warranted.

In conclusion, mitotic arrest of ZIKV infected primary neuronal cells might be caused by multiple reasons. As discussed before, based on gene expression analysis of ZIKV MR766 infected hNPC ZIKV might induce DNA replication stress that ultimately induces cell cycle arrest and aberrant mitosis; if this cell cycle delay is preceded by a prolonged S phase similar to IBV has not been determined. Mitotic progression of ZIKV infected hNPC might also be delayed by downregulating components of the mitotic machinery such as Aurora-A/B, components required for chromosome segregation or the Anaphase Promoting Complex/Cyclosome (APC/C). ZIKV induced activation of autophagy might also inhibit the onset of mitosis and thus arrest cells in G2 phase of the cell cycle in addition to inhibiting the progression of S to G2 and/or G2 progression. Again, further studies using EdU or BrdU incorporation to determine cell cycle progression in ZIKV infected cells are needed (both synchronised and non-synchronised cells).

Table: Genes related to mitotic progression that are up- or downregulated in ZIKV MR766 infected hNPC

The inhibition of viral replication by Cdki indicates that Cdk’s are essential for viral replication and further studies using siRNA and/or specific inhibitors should clarify the contribution of individual Cdk such as Cdk-4/-6, Cdk-2, Cdk-3, Cdk-1 and -since ZIKV infects neuronal cells- also Cdk-5. The latter is of particular interest since the inhibition of Cdk-5 has been shown to confer protection against neuronal apoptosis of cerebral granule neurons and prevent aberrant S-phase entry of postmitotic neurons.  
In addition to (potentially) phosphorylating viral proteins, Cdk might facilitate ZIKV replication indirectly by creating favourable conditions for viral entry. In the case of DENV-2 and DENV-3, HepG2 cells have been demonstrated to be more permissive for both infection and viral replication in G2 phase of the cell cycle compared to G1 or S phase which might explain why the combination of Niclosamide and a Cdki increases the cell viability of ZIKV infected human astrocytes and hNPC as well as decreasing viral replication.

Inhibiting caspase-3 dependent apoptosis also might prevent the cleavage of Beclin-1 and thus promote autophagy and increase cell viability in addition in preventing mitotic entry. It is crucial therefore to determine if caspase-3 inhibition has a negative effect on cell proliferation. Based on the results obtained from treating ZIKV infected astrocytes with Cdki, it may be possible that despite limiting ZIKV replication and increasing cell viability pan-caspase inhibitors might not allow the proliferation of primary neuronal cells infected with ZIKV.
Finally, similar to Porcine Respiratory Syndrome Virus (PRRSV) induced apoptosis, following the initial activation of caspase-3, the cleavage of Beclin-1 in ZIKV infected cells might enhance mitochondrial depolarisation by releasing the pro-apoptotic BH3 only protein Bad, followed by the localisation of Bad to the mitochondria where it forms a dimer with the anti-apoptotic Bcl-XL thus inactivating Bcl-XL.  Since so far cleavage of Beclin-1 in ZIKV infected cells has not been demonstrated, it remains to be seen if this is the case or not.

Figure: Hypothetical network of ZIKV induced changes to the cell cycle in infected cells

Further reading

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Friday, 24 June 2016

Flavivirus host factors: importance of the ER in viral replication

With the emergence of ZIKV in the Americas there has been a renewed interest in flaviviruses, in particular those that are transmitted by insects which historically only generated limited interest in the research community due to their inability to infect vertebrates or only causing relative mild illness.
In recent years however, the increase in infections caused by a number of flavivirus’ including West Nile Virus (WNV), Yellow Fever Virus (YFV), Dengue Virus (DENV) and Zika Virus (ZIKV) transmitted by insects such as Aedes sp. and Culex sp. in the Pacific islands, the Americas and more recently in Africa renewed an interest in these viruses and the advent of new technologies allows to study the virus-host interactions and assists in the identification of potential therapeutic targets.

Flavivirus infection and the EMC: pro-apoptotic during WNV infection whilst supporting replication of DENV, ZIKV and YFV?

The Endoplasmic Reticulum (ER) membrane complex (EMC) was originally discovered as part of a complex allowing the tethering Mitochondria to the ER and thus facilitating the exchange of lipids between the ER and the outer mitochondrial membrane (OMM) but later also being required for the assembly of multipass ER membrane proteins as well as the ER associated degradation (ERAD) pathway. Whilst it has been demonstrated that all EMC proteins interact with the mitochondrial translocase of the OMM (TOM) protein 5 (TOM-5), the role of the EMC in the assembly of proteins is less well characterised.
Genomic screens using RNAi and a CRISPR/Cas9a assay targeting 19052 genes, the replication of both ZIKV and DENV has been shown to depend on the presence of at least four components of the EMC, namely EMC-1, -3, -4 and -5, suggesting that the ability of maintaining tethering mitochondria to the ER and/or to process multipass ER membrane proteins is a significant factor for ZIKV and DENV replication in HeLa cells and 293T cells.
Loss of EMC decreases the level of both intracellular E protein and viral RNA as early as 40 min following the infection of HeLa cells with either DENV-2/NGC and ZIKV MR766, similar to Axl depleted cells, suggesting that EMC is a significant factor for viral binding and/or viral entry, with viral entry being suggested be the limiting factor as opposed to binding of viral particles. One mechanism might be that the loss EMC might prevent decapsidation of the viral genome by targeting endosomes to the lysosome and thus induce the degradation of viral RNA (see below). 

The dependence of ZIKV and DENV on the integrity of the EMC therefore might extend beyond facilitating viral entry to the formation of the viral replication centre as well as the release of viral particles via COPII independent pathways similar to Mouse Hepatitis Virus. Further studies using the DENV and ZIKV replicon systems are however needed to characterize the role of EMC in viral replication and release as well in initiating the ERAD response. In the case of WNV, the expression of seven genes –including EMC-2 and -3- has been shown to be crucial to WNV induced cell death in HeLa and 293FT cells via the ERAD pathway whereas in DENV-NGC1 and various ZIKV strains (MR766, PR 2015 and Cambodia) infected HeLa cells the knockout of EMC-1, -2, -4, or-5 reduces viral replication, suggesting that the EMC supports viral replication as determined by intracellular staining for the viral E protein at 48 hrs p.i. . Closer examination suggests that in addition to a potential role of EMC in the formation of viral RC and/or transport of viral particles to the cell surface, EMC has also a role in viral entry, specifically after viral binding but prior to viral endocytosis. Although the role of the EMC is only poorly characterized, it might be possible that the knockout of EMC might promote the degradation of viral particles and/or the RC by targeting viral RC (and late endosomes containing viral particles following viral entry) to lysosomes and thus promote the degradation rather than release of mature viral particles. This hypothesis is supported by findings that the position and timing of endosome fission is dependent on the ER contact site. In Cos-7 cells expressing mCherry-Rab7 (a marker for late endosomes) and GFP- Sec61β (a marker for the ER), a small cargo containing Rab7+ compartment buds from a larger vacuolar Rab7+ compartment with an ER tubule localised perpendicular to the fission site that “cups” the bud just prior fission. Closer examination of these sites revealed that prior fission components of the retromer complex including FAM21 (which is involved in endosomal sorting) co-localise to the site of fission, indicating that the localization of proteins to the fission site determines the sorting of cargo. Future work however is needed to determine the role of the EMC in the sorting of endosomes and the role of EMC during the formation of viral RC; thus any role of EMC in the role of the development of ZIKV and DENV RC is hypothetical.

An additional role for EMC in the replication of both ZIKV and DENV might the recruitment of mitochondria and thus the facilitation of lipophagy. The transfer of phospholipids from the ER to Mitochondria is believed to be non-vesicular and to occur at sites of close contact between the ER and Mitochondria. In S. cerevisiae, deletion of EMC components leads to a decreased transfer of phosphatidylserine (PS) from the ER to Mitochondria which as a consequence contain decreased levels of both PS and Phosphatidyletholamine (PE), thus leading to decreased cell growth.  Since both PS and PE are also involved in the formation of lipid droplets (LD), EMC deficiency might also impact viral replication by decreased formation of LD and thus decreased lipophagy. Again, more research is needed to verify the involvement of EMC in LD synthesis during ZIKV and DENV infection.
In the case of WNV, the deletion of EMC-2 partially prevents WNV induced apoptosis, indicating that EMC-2 is a factor for viral induced apoptosis. Besides the potential involvement of ERAD in WNV induced apoptosis not much is known and indeed speculative. One possible mechanism is that viral proteins associate with EMC-2 and thus increase ER stress, inducing ERAD and apoptosis due to lipid depletion. On the other hand, deletion of EMC-2 mitigates WNV induced apoptosis. It might be necessary therefore to monitor the ER stress response in WNV infected EMC-2-/-   cells by artificially inducing the accumulation of unfolded proteins in the ER.

The importance for the ER for ZIKV replication is further strengthened by observations that in cells infected with various members of the Flaviviridae –including but not limited to WNV, DENV, YFV, and Japanese Encephalitis Virus (JEV)- viral proteins are localised to the ER and indeed viral proteins are processed at the ER prior viral assembly. As has been discussed in prior posts, the localisation of viral proteins from JEV at the ER induces the ER stress response concomitant with the formation of autophagosomes and ZIKV infection of primary human fibroblasts has been associated with an increase in autophagosomes.
The role of the ER in the replication of Flavivirus’ has been further supported by recent findings that the expression of sgRNAs related to the ERAD response (including EMC-4 and -6), ER translocation machinery or the Oligosaccharyl transferase complex (OST) decreases the replication of WNV (Kunjin), ZIKV H/PF/2013, YFV (12D vaccine strain), JEV and DENV-2 in 293T cells as well as in a Drosophila cell line, DL-1. Two of the genes tested encode for two of the five components of the cellular Signal Peptidase Complex (SPC), namely SPCS-1 and SPCS-3. Indeed, WNV, JEV, DENV and ZIKV do not replicate in SPCS-1-/-  293T and SPCS-1-/-  Huh 7.5 cells and both WNV and DENV-2 are not replicating in U2OS cells transfected with either siRNA targeting SPCS-1 or SPCS-3, suggesting that viral polyprotein consisting of the structural and non-structural proteins requires to be processed at the ER by SPCS-1 and/or SPCS-3.

Figure: Prototype Flavivirus genome
This notion is supported by results showing that in SPCS-1-/-  293T cells infected with WNV the levels of both the viral prM and E protein are reduced at 12 hrs p.i. and non-cleaved prM, E proteins are detectable at 24 hrs p.i., whereas cleavage of the viral C protein in SPCS-1-/-  293T cells is not affected with similar finding in cells transfected with a prM-E-C plasmid, indicating therefore that the cellular Signal Peptidase complex is required for the cleavage of prM and E (but not C). Further experiments revealed that the leader sequence preceding the viral E protein however is not cleaved by SPCS (in contrast to the leader sequence preceding prM), suggesting that the cleavage of prM is necessary for the processing of E in a sequential manner. In a similar way the processing of the viral NS1 protein depends on the previous processing of prM but itself is not dependent processed by SPCS-1.

In line with these results, the loss of EMC might induce the accumulation of misfolded viral proteins and/or decreased incorporation of viral proteins into the ER and thus decrease viral replication.

Figure: Localisation of the signal sequence of prM in the context of structural and non-structural protein
localisation in the ER 

In conclusion, the ER -in particular the EMC and Signal Peptidase Complex- plays a pivotal role in the replication of ZIKV and DENV. Whilst the connection between the EMC and viral replication is still obscure, the role of ER localised cellular signal peptidases is better characterized (at least for WNV) although questions remain, in particular if the cleavage of prM leader sequence induces a structural change that increases the stability of E which would be consistent with a chaperone-like role for prM in the folding of E in cells infected with Tick Borne Encephalitis Virus and evidenced by lower expression levels of both prM and E in SPCS-1-/-  293T cells expressing prM and E derived from WNV
compared to wt cells.
In addition to EMC and SPCS-1/-3, the genome wide CRISPR/Cas9a assay based screen identified other ER resident proteins that are required for viral replication, including components of OST and the ER translocation machinery such as OST-C and Sec61β whose contribution to viral replication is still undetermined. 

It might be also of interest to explore the question if prM co-localises and/or interact directly with SPSC-1 or other components of Signal Peptidase complex? As mentioned above, the absence of the EMC might induce the relocalisation of endosomes to lysosomes and thus affect sorting. One of the questions to be answered therefore is if the absence of the EMC -or components of the EMC- induces the localisation of viral RC to lysosomes and thus prevents the release of viral particles. Further studies are needed to address these and other questions.

Further reading

Savidis G., et al.(2016) “Identification of Zika Virus and Dengue Virus Dependency Factors using Functional Genomics” Cell Reports 16, 1–15

Zhang, et al. (2016) “A CRISPR screen defines a signal peptide processing pathway required by flaviviruses” 
Nature doi:10.1038/nature18625

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