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Multiple Apoptotic/Necrotic Pathways May Be Involved In Zika Virus Neurotropic Brain Injury

  • Hale O'mana'o Biomedical Research
Multiple Apoptotic/Necrotic Pathways May Be Involved In
Zika Virus Neurotropic Brain Injury
Robert Ricketson, MD
Institute of Pure and Applied Knowledge
February 16, 2016
Zika virus (ZIKV) is a member of the Flaviviridae family, which includes West Nile Virus, St.
Louis encephalitis virus, Kunjin virus, yellow fever virus, Dengue virus, and Japanese
encephalitis virus. Cellular apoptosis (cell death) following infection appears to be dependent
upon several factors, such as viral load, host factors, and specific viral protein induced
apoptosis/necrosis pathways, many of which have yet to be fully defined.
P53-BAX-mt Apoptosis and Necrosis
West Nile Virus (WNV) infections cause severe clinical manifestations including chorioretinitis,
acute flaccid paralysis syndrome and fatal meningoencephalitis. Both necrosis and apoptosis is
observed morphologically. In West Nile Virus (WNV) infections, viral load at with a high
infectious dose (multiplicity of infection (m.o.i) > 10, necrosis was the predominant form of cell
death. Apoptosis was observed when the infectious dose was at a low m.o.i. of <1 [1]. That
observation may suggest separate, distinct pathways.
The neuroinvasive WNV capsid protein has been identified to result in apoptosis via HDM2
sequestration to the nucleolus. Specifically, HDM2 binds p53 and targets it for degradation at
the proteasome. Inhibition of the HDM2-p53 complex leads to stabilization of p53 and apoptosis
through the Bax-mt pathway. Stressed conditions prevent HDM2-mediated p53 degradation and
result in p53 activation, inducing apoptosis [2].
The C-terminus of WNV-Cp has been shown to mediate cytotoxic effects on cells [3] through
HDM2, an ubiquitin protein ligase that suppresses the transcriptional activity of the tumor
suppressor p53 and promotes its degradation. P53 is a major activator in the BAX-mt apoptotic
pathway. The mutant (deletion of 106-123) proved unable to induce the translocalization of
HDM2 into the nucleolus. This mutant proved consistently unable to bind to HDM2, and also
proved incapable of inducing p53 and BAX, which suggests that the C-terminus is responsible
for WNV-Cp’s cytotoxic effects. The WNV-Cp has been shown to block the binding of HDM2 to
p53 via phosphorylation by protein kinase C (PKC) at amino acid residues residing near Ser-
83 or within Ser-99 to Ser/Thr-100 and subsequent nucleolar sequestration of HDM2,
preventing its interaction with p53. P53 is then free to activate BAX, leading to mitochondrial
permeabilization and apoptosis, releasing cytochrome c into the cytosol. West Nile virus capsid
protein interaction with importin and HDM2 protein is regulated by protein kinase C-mediated
phosphorylation. [4]. The N-terminal 170 residues of Rubella Virus (RV) capsid protein domain,
known to cause microcephaly, have also been demonstrated to induce BAX-mediated
apoptosis via upregulation of p53 [5, 6].
Apoptosis regulator BAX protein is reported to interact with, and increase the opening of, the
mitochondrial voltage-dependent anion channel (VDAC), which leads to the loss in membrane
potential and the release of cytochrome c. BAX mediated apoptosis and necrosis occurs by
binding to, and antagonizing the apoptosis repressor BCL2. BAX also is known to effect
cerebral cortex development (GO0021987) and its related term Microcephalin (IPR022047) [7].
Microcephalin injury by viral protein or molecular mimicry has yet to be reported but needs
further consideration.
The apoptotic mechanism of apoptosis necrosis along with observed histopathologic changes is
worthy of consideration. The expression of BAX is regulated by the tumor suppressor p53. The
majority of BAX is found in the cytosol, but upon initiation of the apoptotic signaling, BAX
undergoes a conformational shift and becomes mitochondrial membrane associated. Organelle
disruption, therefore, is a characteristic of BAX-mt pathway apoptosis (Figure 1).
Figure 1. BAX mediated mitochondrial permeabilization with release of cytochrome c. Note the
organelle disruption (red arrow) [Reference image from Whelan et al [8] ].
This result in the release of cytochrome c and other pro-apoptotic factors from the mitochondria
is often referred to as mitochondrial outer membrane permeabilization. Through necrosis and
organelle disruption, the subsequent release of cytochrome c from the disrupted mitochondria
leads to activation of caspases. This defines a direct role for BAX in mitochondrial outer
membrane permeabilization and necrosis with organelle disruption and necrosis [8].
Caspase-9 Apoptosis
Apoptosis can occur through distinct caspase-independent and -dependent pathways [9]. Yang
et al in 2002 also reported that the WNV capsid protein (WNV-Cp) drives apoptosis in vitro
through the mitochondrial/caspase-9 pathway and subsequent caspace-3 activation in the
brain [10]. Samuel et al in 2007, reported their findings that subsequent to infection, WNV
induced Caspase-3 activation and apoptosis in the brain [11]. Inhibition of caspases in WNV
infection has been shown to limit apoptosis [12].
Caspase-9 is activated by the mitochondrial release of cytochrome c into the cytosol (Zou et al.,
1999) and initiates the induction of Caspace-3. Caspase-3 induces cells to undergo
characteristic morphological changes in caspace-independent apoptosis (Table 1, Figure 2):
1. Formation of apoptotic bodies consisting of cytoplasm with tightly packed organelles with
or without a nuclear fragment
2. Organelle integrity is still maintained and remain enclosed within an intact plasma
Figure 2. Caspace apoptosis. The apoptotic body is indicated by the arrow enclosed by
cytoplasm. The mitochondria organelle are intact in contrast to necrosis through BAX induction
Caspase-8 does not appear to be involved in the WNV-Cp induced apoptosis. Deletion of the C-
terminal residues of WNV-Cp reduced the induction of caspase-9 and subsequent apoptosis
and thereby defining the caspases-9 induction domain as residing between residues 67-122 in
the capsid protein domain. In contrast to the BAX pathway, the caspace-9 pathway does not
appear to destabilize the mitochondrial membrane and subsequent mitochondrial outer
membrane permeabilization as observed through the p53-BAX pathway.
Viral NS3 and Caspace Apoptosis
Ramanathan et al demonstrated that the viral protein NS3 alone was sufficient to induce
caspace-8 apoptosis. Two primary domains within NS3 were identified. Expressions of the
protease and helicase domains were sufficient to trigger apoptosis in WNV infections [14].
Chu et al in 2003 [1] found that after cytochrome c was released during initial apoptosis,
caspases-9 was activated increasing caspace-3 leading to further apoptosis. This would
suggest the p53-BAX pathway leads to later caspace-initiated apoptosis via release of
cytochrome c from the mitochondria. This is required for the formation of the apoptosome,
which, in turn, is necessary for activation of pro-caspase-9. This would imply both the p53-BAX
pathway via the viral capsid protein and caspace-9 activation pathways via the NS3 Peptidase 7
and Helicase domains are involved in the necrosis and apoptosis.
Figure 3. Eukaryotic Apoptosis/Necrosis Pathways
Proposed Mechanisms Of Apoptosis/ Necrosis In Zika Virus Infection
The histomorphologic changes in the brain have recently been reported [15]. The authors
reported on a case of an expectant mother who had a febrile illness with rash at the end of the
first trimester of pregnancy while she was living in Brazil.
Numerous histomorphologic findings of both apoptosis and necrosis were identified. Gross
findings included intracranial cortical and subcortical calcifications, lissencephly, and autolysis.
Electron microscopy identified ruptured and lysed neuronal cells in association with numerous
icosahedral virus- like particles. Zika virus was identified by PCR. The large number of viral
particles associated with the necrosis and apoptosis is similar to the previously described
association of primary necrosis at high m.o.i >10 and apoptosis when the multiplicity of infection
is low (m.o.i. <1). Known infectious causes of intracranial calcifications include toxoplasmosis,
rubella, Cytomegalovirus (CMV) and Herpes simplex virus (HSV).
Figure 4. Electron microscopy from ZIKV confirmed microcephaly [15]
The ZIKV capsid domain (1-122; Figure 5 and 6) was first investigated for homology with
neuroinvasive WNV (WNVnv) and Dengue virus representative sequences (DENV1, DENV2,
The N-terminal residues demonstrated the greatest homology, particularly from Gly-40-Pro-61.
The C-terminal residues previously demonstrated to be involved in Bax mediated apoptosis
from WNV-Cp (67-100) were found to have 42% identity-Figure 5). The p53-HDM2 binding
residues which were reported to be phosphorylated and flank the p53-HDM2 binding domain
(Ser-83 and Ser-100) were not found in ZIKV or DENV. In ZIKV and DENV, these serine
residues were replaced by Lys-83 in ZIKV and Gly-83 in DENV1, DENV2, DENV3, and DENV4.
The C-terminal WNVnv Ser-100 residue was replaced by Lys-100 in both ZIKV and The Dengue
virus sequences. However, the serine residues (Ser-83 and Ser-100) were flanked by highly
conserved, positively charged residues (FKK) immediately from positions 84-86 and from 97-
100. These electrostatic interactions may also play a role in BAX mediated injury.
Figure 5. HDM2 Binding Domain in the Flavivirus Capsid Protein (1-122)
The HDM2 critical binding domain is identified by the red bar (residues 83-100). Note the Serine
residues at position 83 and 100 from WNVnv are flanked by conserved, positively charged
Arginine and Lysine residues in all sequences. The amino acid alignment performed with the
CLUSTAL Omega algorithm and visualized with Jalview.
Figure 6. Multiple Sequence Alignment of the Flavivirus Capsid Domain
Figure 7. Hydrophobicity of the capsid domain of ZIKV, WNV, and DENV.
A significant difference is that ZIKV has a greater hydrophobicity profile within the C-terminus of
the capsid protein domain (Figure 7). Those residues are flanked by positively charged Lysine
and Arginine residues that may contribute to interactions.
The Peptidase and Helicase domains were evaluated for homology as these domains
were previously discussed as being sufficient for caspace induced apoptosis. Between
position 1620-1670 of the Peptidase 7 conserved domain in NS3 (Figure 8), the overall
homology was found to have 56% identity, but also 78% positives suggesting the C-
terminus may be the region of shared interaction with caspases.
The Helicase domain from position 1867-1976 (Figure 9) demonstrated 76% homology
and 81% positives within the C-terminus. This may suggest a greater role of the
Helicase domain in caspase induction and apoptosis.
Figure 8. Peptidase 7 Caspace Induction Domain within NS3
Figure 9. Helicase Domain within NS3
1. The viral capsid protein homology indicates the region 83-100 may be responsible for
the p53-BAX mediated necrosis/apoptosis.
2. The histomorphology seen in Zika virus encephalopathy is indicative of both a BAX-mt
and Caspace pathway of necrosis and apoptosis.
3. Caspase-induced apoptosis via the Peptidase 7 and Helicase domain of NS3 has been
previously described with West Nile Virus and may also be involved in ZIKA Virus
4. Significant homology is identified between West Nile Virus, Dengue Virus, and Zika Virus
within the C-terminus of Peptidase 7 and Helicase domains of the NS3 protein, further
giving evidence to cytochrome c-> caspace initiated apoptosis in Zika Virus infection.
5. Both p53-BAX mediated necrosis/apoptosis and Caspace-mediated apoptosis appear to
be active pathways in Zika virus infection as identified by previously reported
histomorphologic features.
6. Causes of selective neurotropism resulting in microcephaly (e.g., Microcephalin injury)
remain to be identified
In summary, it would appear that the C-terminus of the ZIKV Capsid Protein domain may very
well be responsible for the BAX apoptosis/necrosis pathway particularly when the
histomorphologic finding are considered. However, without the phosphorylated serine residues
at the 3’ and 5’ ends, ZIKV and DENV may not be able to bind HDM2 and may use another
mechanism to induce p53, block anti-apoptotic Bcl-2, or bypass BAX and preferentially use
caspase induced apoptosis. The histomorphologic evidence of both apoptotic bodies and
necrosis in intracranial ZIKV infection would implicate both pathways in ZIKV induced
apoptosis/necrosis. Additionally, with the significant homology that exists with the NS3 domain,
specifically the C-terminus of both the Peptidase 7 and Helicase domains, evidence suggests a
caspace induced apoptosis as a result of cytochrome c release following BAX mitochondrial
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