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

Blazevic, J., et al. (2016). "The membrane anchors of the structural flavivirus proteins and their role in virus assembly." J Virol. 90 (14) 6365-6378

Blazquez, A. B., et al. (2014). "Stress responses in flavivirus-infected cells: activation of unfolded protein response and autophagy." Front Microbiol 5: 266.

Blitvich, B. J. and A. E. Firth (2015). "Insect-specific flaviviruses: a systematic review of their discovery, host range, mode of transmission, superinfection exclusion potential and genomic organization." Viruses 7(4): 1927-1959.

Bolling, B. G., et al. (2011). "Insect-specific flaviviruses from Culex mosquitoes in Colorado, with evidence of vertical transmission." Am J Trop Med Hyg 85(1): 169-177.

Calzolari, M., et al. (2016). "Insect-specific flaviviruses, a worldwide widespread group of viruses only detected in insects." Infect Genet Evol 40: 381-388.

Kenney, J. L., et al. (2014). "Characterization of a novel insect-specific flavivirus from Brazil: potential for inhibition of infection of arthropod cells with medically important flaviviruses." J Gen Virol 95(Pt 12): 2796-2808.

Lahiri, S., et al. (2014). "A conserved endoplasmic reticulum membrane protein complex (EMC) facilitates phospholipid transfer from the ER to mitochondria." PLoS Biol 12(10): e1001969.

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Mukhopadhyay, S., et al. (2005). "A structural perspective of the flavivirus life cycle." Nat Rev Microbiol 3(1): 13-22.

Papa, A., et al. (2016). "Insect-specific flaviviruses in Aedes mosquitoes in Greece." Arch Virol.

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