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.
Lorenz, I. C., et al. (2002). "Folding and dimerization of tick-borne encephalitis virus envelope proteins prM and E in the endoplasmic reticulum." J Virol 76(11): 5480-5491.
Ma, H., et al. (2015). "A CRISPR-Based Screen Identifies Genes Essential for West-Nile-Virus-Induced Cell Death." Cell Rep 12(4): 673-683.
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.
p
Pena, J. and E. Harris (2011). "Dengue virus modulates the unfolded protein response in a time-dependent manner." J Biol Chem 286(16): 14226-14236.
Pena, J. and E. Harris (2012). "Early dengue virus protein synthesis induces extensive rearrangement of the endoplasmic reticulum independent of the UPR and SREBP-2 pathway." PLoS One 7(6): e38202.
T
Perreira, J. M., et al. (2016). "Functional Genomic Strategies for Elucidating Human-Virus Interactions: Will CRISPR Knockout RNAi and Haploid Cells?" Adv Virus Res 94: 1-51.
Reggiori, F., et al. (2010). "Coronaviruses Hijack the LC3-I-positive EDEMosomes, ER-derived vesicles exporting short-lived ERAD regulators, for replication." Cell Host Microbe 7(6): 500-508.
Schrader, M., et al. (2015). "The different facets of organelle interplay-an overview of organelle interactions." Front Cell Dev Biol 3: 56.
Wideman, J. G. (2015). "The ubiquitous and ancient ER membrane protein complex (EMC): tether or not?" F1000Res 4: 624.
