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Title: Dysregulation of RNA editing may help explain pathogenicity mechanisms of 1
congenital Zika syndrome and Guillain-Barre syndrome 2
3
Helen Piontkivska 1, 2, *, Noel-Marie Plonski 2, Michael M. Miyamoto 3, and Marta L. 4
Wayne 3, 4 5
6
1 Department of Biological Sciences and 2 School of Biomedical Sciences, Kent State University, 7
Kent, OH 44242, USA 8
3 Department of Biology, University of Florida, Gainesville, FL 32611, USA 9
4 Emerging Pathogens Institute, University of Florida, Gainesville, FL 32611, USA 10
11
* Corresponding Author 12
Helen Piontkivska 13
Department of Biological Sciences 14
Kent State University 15
Kent, OH 44242, USA 16
Telephone: (330) 672-3620 17
Fax: (330) 672-3713 18
E-mail: [email protected] 19
20
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27401v1 | CC BY 4.0 Open Access | rec: 3 Dec 2018, publ: 3 Dec 2018
2
Abbreviations: 21
ADAR, Adenosine Deaminases Acting on RNA 22
AGS6, Aicardi-Goutieres syndrome 6 23
ALS, amyotrophic lateral sclerosis 24
CDC, Centers for Disease Control and Prevention 25
CDI, inhibitory Ca2+-feedback 26
CMV, cytomegalovirus 27
CZS, Congenital Zika syndrome 28
GBS, Guillain-Barre syndrome 29
IFN, interferon 30
ISGs, interferon-stimulated genes 31
ISREs, interferon-sensitive response elements 32
WNV, West Nile virus 33
Zika virus (ZIKV) 34
35
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27401v1 | CC BY 4.0 Open Access | rec: 3 Dec 2018, publ: 3 Dec 2018
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Abstract: 36
Many Zika virus (ZIKV) pathogenesis-related studies have focused primarily on virus-driven 37
pathology and neurotoxicity, instead of considering the possibility of pathogenesis as an 38
(unintended) consequence of host innate immunity: specifically, as the side-effect of an 39
otherwise well-functioning machine. The hypothesis presented here suggests a new way of 40
thinking about the role of host immune mechanisms in disease pathogenesis, focusing on 41
dysregulation of post-transcriptional RNA editing as a candidate driver of a broad range of 42
observed neurodevelopmental defects and neurodegenerative clinical symptoms in both infants 43
and adults linked with ZIKV infections. We collect and synthesize existing evidence of ZIKV-44
mediated changes in expression of adenosine deaminases that act on RNA (ADARs), known 45
links between abnormal RNA editing and pathogenesis, as well as ideas for potential 46
translational applications, including genomic profile-based molecular diagnostic tools and/or 47
treatment strategies. 48
49
50
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27401v1 | CC BY 4.0 Open Access | rec: 3 Dec 2018, publ: 3 Dec 2018
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Introduction: ZIKV as a causative agent for an emergent public health crisis 51
Following discovery of a link between Zika Virus (ZIKV, a flavivirus) infection in pregnancy 52
and microcephaly in infants in 2015 [1], this public health crisis continues to present a significant 53
hazard to human health. The Global Disease Burden study estimated that ~7·60 million ZIKV 54
infections occurred in 2016, most of which were in Latin America and the Caribbean [2]. While 55
the vast majority of these cases were asymptomatic infections, with only a small portion 56
resulting in Zika-attributable Guillain-Barré syndrome (GBS) and congenital Zika syndrome 57
(CZS) births, infants born with the CZS will face life-long profound disability [3] and their 58
families face extreme economic burdens as well. Already over 6900 pregnancies with ZIKV 59
infected mothers have been recorded in the US and the US territories as of July 17, 2018 (CDC), 60
from which over 300 infants were born with CZS. Nor are adults immune: known consequences 61
of ZIKV infection in adults include GBS [4, 5], a debilitating peripheral neuropathy that is 62
potentially life-threatening and costly to treat [6]. However, there is currently no available vaccine 63
for ZIKV, nor antiviral treatments. The efficacy of prevention strategies, such as protecting 64
oneself from mosquito bites or sexual abstinence during confirmed infection, is compromised by 65
the frequently asymptomatic nature of ZIKV infection [7] as well as the wiliness of mosquitoes. 66
In the absence of effective prevention, insights into the mechanisms behind ZIKV-associated 67
pathogenesis are critical to the development of treatments that can ameliorate potential ZIKV 68
sequelae. 69
70
What is congenital Zika syndrome (CZS)? 71
The link between ZIKV infection in pregnancy and congenital Zika syndrome (CZS) is a 72
particularly pressing concern, in part because we do not yet fully understand the extent of 73
neurodevelopmental defects – particularly those not obvious at birth - associated with it [8]. CZS 74
includes a broad range of neurodevelopmental defects beyond microcephaly [9, 10], including 75
ocular [11] and auditory defects [12]. The overall risk of ZIKV-linked birth defects has been 76
estimated at up to ~15% for infections in the 1st trimester [13], potentially impacting thousands of 77
infants in the US and US territories alone. However, the risk extends into infections that occur 78
later in pregnancy [14], and central nervous system defects may be present without microcephaly 79
[15, 16]. A recent CDC report showed an increase in prevalence of birth defects potentially 80
consistent with CZS in US areas with documented local ZIKV transmission [8]. Flash flooding 81
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accompanying major hurricanes (such as those associated with 2017 hurricanes Harvey, Irma 82
and Maria in TX, FL and PR, respectively) and the diverse set of ZIKV transmission routes, 83
including non-vector-borne transmission via sexual contact and bodily fluids, such as sweat, 84
saliva or tears [17], likely expand the portion of ZIKV-exposed US population, similar to the 85
observed expansion of West Nile virus (WNV) infections following hurricane Katrina in 2005 86
[18]. 87
88
ZIKV-mediated pathogenesis? 89
Despite major efforts focused on ZIKV research over the last three years, our understanding of 90
ZIKV-mediated pathogenesis remains limited, in part because the symptoms and sequelae of 91
ZIKV infection span from asymptomatic to major birth defects in infants, as well as severe 92
adverse outcomes in some adults. Below we highlight a broad variety of clinical symptoms and 93
outcomes associated with ZIKV infection, with the explicit purpose of identifying connectedness 94
of ZIKV sequelae across different life stages. For in-depth comprehensive surveys focused on 95
fetal or adult outcomes, we refer the reader to the following studies [9, 19, 20]. 96
97
What do we know so far? On the one hand, most ZIKV infections in adults are benign and self-98
limiting [21], with as many as 80% having asymptomatic infections [7]. It is worth noting that 99
these asymptomatic carriers are still capable of infecting others [19]. Moreover, the risk of 100
neurodevelopmental fetal defects appears to be similar between symptomatic and asymptomatic 101
pregnant women [19]. While relatively few pediatric-focused studies of ZIKV infections are 102
available, the available evidence suggests that the majority of acute ZIKV infections in children 103
are similarly mild in nature to adult ones [22], although recent reports have begun to associate 104
ZIKV with other clinical issues, such as acute abdominal symptoms [23] or post-infection 105
vasculitis resulting in pediatric stroke [24]. 106
107
During pregnancy, ZIKV poses a substantial risk of fetal neurodevelopmental defects, resulting 108
in profound long-term disability and immense costs of future care, influencing individuals and 109
communities [21, 25]. The full spectrum of CZS is still being elucidated, coupled with concerns 110
that ZIKV-related outcomes are underreported, particularly when ZIKV infections result in later 111
onset of developmental delays and learning disabilities without visible microcephaly [26], or 112
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includes other systemic abnormalities that manifest postnatally, such as dysphagia [27], seizures 113
[28], and hearing loss [29]. For example, recent evidence suggests that ZIKV infection in 114
pregnancy may have been linked with microcephaly and other neurodevelopmental defects in the 115
US (Hawaii) as early as 2007, with several (likely travel-related) births in 2009 and 2011 being 116
definitively linked to maternal ZIKV-positive antibodies [30]. In part due to lack of long term, 117
longitudinal studies, the long-term consequences of an in utero ZIKV infection are poorly 118
understood, particularly in infants lacking visible symptoms of CZS and/or from asymptomatic 119
infections; however, there are reported cases of CZS being diagnosed post-birth because the 120
infants showed neurodevelopmental delays and developed postnatal microcephaly [31, 32]. Recent 121
abnormal imaging from normocephalic infants from ZIKV-positive pregnancies [33] and pregnant 122
pigtail macaques [16] further underscores challenges associated with detection of ZIKV-mediated 123
brain injury that may appear subtle, yet could have profound neurocognitive and psychiatric 124
consequences later. 125
126
In addition to causing severe neurodevelopmental defects in prenatal infections, ZIKV infections 127
have also been linked to severe neurotoxicity in some adults, including Guillain-Barre Syndrome 128
(GBS) [4, 5, 34]. GBS is an acute paralytic neuropathy, an inflammatory disorder of the peripheral 129
nervous system, and is one of the leading causes of acute flaccid paralysis worldwide [35, 36]. 130
Despite recent progress in immunotherapy [37], GBS remains a life-threatening condition, often 131
requiring complex and expensive care [38]. Up to 20% of GBS cases result in severe disability, or 132
even death [36], while many other patients experience ongoing motor or sensory issues, fatigue 133
and pain [39]. Compared to other morbidities, ZIKV-triggered GBS represents a very small 134
fraction of the global disease burden [3]. Nonetheless, GBS is associated with substantial risks 135
(including death) as well as financial costs for affected individuals [21]. Moreover, there are 136
challenges with early GBS diagnosis, particular in atypical presentations and/or pediatric patients 137
[38]. For example, cranial nerve enhancement - thought to be a part of GBS in pediatric patients 138
[40] - was recently described from an imaging study of seemingly normal infant born after ZIKV-139
positive pregnancy. However, not all infants from ZIKV-positive pregnancies receive the 140
recommended postnatal imaging [13, 41]. Moreover, the molecular mechanisms behind GBS 141
remain poorly understood [5, 42]. In addition to its association with GBS, the list of neurological 142
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and other consequences of ZIKV infection in adults and children – including 143
meningoencephalitis, paresis, and vasculitis - keeps growing [11, 24, 43]. 144
145
In other words, ZIKV infection appears to be associated with a broad and disparate range of 146
clinical and subclinical symptoms and outcomes. We would like to propose a novel hypothesis 147
that can help explain the multitude of diverse ZIKV infection outcomes both pre- and post-148
natally. While it requires experimental validation, this hypothesis is firmly grounded in evidence 149
from multiple areas of study, and allows us to make connections between previously un-150
connectable observations. It also enables us to generate new, testable hypotheses that may enable 151
development of effective treatment and prevention strategies. 152
153
Hypothesis: Connecting the dots 154
We now build the case that ZIKV-mediated pathogenicity – both fetal and adult - is due to 155
dysregulation of the activity of the RNA-editing enzymes, ADARs (Adenosine Deaminases 156
Acting on RNA), which in turn leads to inappropriate levels of post-transcriptional editing of 157
key proteins involved in metabolism of calcium and other ions, and homeostasis in neural 158
cells, thereby triggering a cascade of diverse neurological effects (Figure 1). We propose that 159
viral-triggered dysregulation of ADAR editing results in a handful of amino acid changes in key 160
neural proteins involved in ion transport and neurotransmitter metabolism. Either an excess or a 161
paucity of developmentally-appropriate edits lead to changes in ion permeability and excitability, 162
ultimately resulting in excitotoxicity and neuronal demise [44-46]. 163
164
What are ADARs? 165
ADARs (adenosine deaminases that act on RNA) are RNA editing enzymes that deaminate 166
adenosines (A) to produce inosines (I) within double-stranded RNAs [47]. These A-to-I changes 167
are translated as A-to-G substitutions in mRNA transcripts, thereby modulating proteome 168
diversity and expression by introducing nonsynonymous (amino acid changing) substitutions or 169
splice site changes, and serving as a mechanism of dynamic and nuanced post-transcriptional 170
regulation of gene expression [48]. There are three mammalian ADAR loci - ADAR, ADARB1 171
and ADARB2 [49]. Only ADAR and ADARB1 have proven deaminase activity [50], while 172
ADARB2 is thought to play a regulatory role through competition with other ADARs for 173
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substrate [51]. It should be noted that expression and editing activities of one family member can 174
be influenced by expression and editing activities of the other ADARs [52, 53]. ADARs play a 175
particularly prominent role in the nervous system as the majority of ADAR editing target genes 176
are expressed in the nervous system, including brain [49]. 177
178
ADAR expression is elevated in ZIKV-infected cells that exhibit cytopathic effects 179
This piece of evidence comes from a recent study that examined consequences of ZIKV infection 180
in three cell lines of primary human neural stem cells originally derived from three individual 181
fetal brains [54]. Two lines (K048 and K054) exhibited signs of reduced neuronal differentiation 182
(which is consistent with other ZIKV infected brain studies, including in vivo), while such signs 183
were not noticed in the third line, G010 [54]. Differential gene expression analysis showed global 184
similarities in transcriptome changes in K048 and K054, which were not shared with G010, 185
particularly among genes involved in innate immune pathways [54]. Their supplementary list of 186
differentially expressed genes reported that ADAR gene expression increased significantly in 187
K048 and K054 cells in response to ZIKV infection, while remained the same in G010 cells 188
(with 1.63 and 1.1-fold change, with the false discovery rate P values < 0.05 in K048 and K054, 189
but not in G010, respectively). In other words, only the cells with the observable cytopathic 190
effects exhibited an increase in ADAR expression (of course, the relationship between 191
expression and editing may not necessarily be linear [55]). While no significant change in 192
expression of ADARB1 and ADARB2 was reported, it should be noted that changes in the 193
ADAR gene expression can interfere with specific editing activity of the other ADARB1 enzyme 194
[52, 56]. 195
196
Our re-analysis of the McGrath et al. (2017) transcriptome data [54] shows that ADAR editing 197
activity per se, not merely expression of ADAR, appears to be higher in the cells with more 198
prominent cytopathic effects. Using the SnpEff variant annotation tool implemented in GATK, 199
we tallied nucleotide changes between mock and ZIKV-infected cells and found that ZIKV-200
infected samples harbor significantly higher numbers of A-to-G-changes (i.e., changes that can 201
be attributed to ADAR activity) in K048 cells compared to mock-infected cells (Mood’s median 202
test, P = 0.014). However, no significant differences were observed in K054 cells, where 203
cytopathic effects were not as prominent as in K048, and in G010 cells (P > 0.05). An alternative 204
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variant calling was performed using GATK Haplotype caller, and the distribution of missense A-205
to-G variants was examined in K048 cells, which had the highest read depth. Of the genes with 206
variants not shared between mock and ZIKV-infected cells (in other words, those less likely to 207
be genetic polymorphisms), there was a significantly higher fraction of genes with variants in at 208
least 2 samples in ZIKV-infected cells compared to mock-infected cells (10 versus 70 in mock 209
cell, and 59 versus 127 in ZIKV-infected cells), including well-known editing target GRIA3. 210
However, these preliminary analyses have significant limitations, one of which is the significant 211
difference in sequencing depth between samples (K048 had twice as many reads as the other two 212
cell lines) Thus, these preliminary findings offer only an initial glimpse into the potential effects 213
of ADAR editing in ZIKV infection, and require further experimental confirmation, including 214
careful validation of editing changes (if any) of specific key neural genes and/or whether 215
potential differences of normal editing patterns among individuals may be contributing to 216
pathogenesis mechanisms. Nonetheless, these preliminary findings are consistent with the 217
proposed hypothesis about the role of dysregulated editing changes in ZIKV infections; although 218
it remains to be determined whether these changes result from the redirection of ADAR p150 219
editing activity away from the host editome to ZIKV, or through changes in normal host editing 220
patterns because of increased ADAR p150 editing activity and/or interference between different 221
ADARs. 222
223
Appropriate ADAR editing is required for proper neuronal development 224
Severe CNS phenotypes are observed in ADAR knockouts and ADAR deficiency or loss of 225
function studies (e.g., [57]), including embryonic lethality [58], epilepsy, motor neuron death [59], 226
microcephaly and neural tube defects [60], as well as shortened life-span in worms [61]. Likewise, 227
decreased ADARB1 editing activity plays a role in the pathogenesis of a common motor neuron 228
disease, sporadic amyotrophic lateral sclerosis (ALS) [62], and various brain and neurological 229
cancers [52, 63]. 230
231
Mutations and polymorphisms in ADAR loci, such as the ADARB2 locus polymorphisms, have 232
been implicated in complex neurological phenotypes such as migraines [64], autism [65], and 233
genetic epilepsy [66]. Likewise, mutations in the ADAR gene that affect efficacy of RNA binding 234
and/or of A-to-I editing [67] have been associated with a rare human Mendelian disease, Aicardi-235
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Goutieres syndrome (AGS6) [68]. In its typical form, the damage caused by AGS resembles 236
neurodevelopmental pathology due to in utero infection [69], and is associated with an increased 237
level of interferon and upregulation of interferon-stimulated genes [70]. 238
239
ADAR editing is enriched in the nervous system [71]. One prominent example of ADAR editing 240
in the brain is the Q/R site of the glutamate receptor subunit GRIA2 (also known as GluA2), 241
where virtually complete (~100%) editing [72] of genomic glutamine codon 607 (Q607) to 242
arginine (R) results in multiple changes of the receptor properties, including cellular permeability 243
to calcium (Ca2+) [73]. Underediting of Q607R GRIA2 site (e.g., in the case of ADARB1 244
deficiency) results in increased influx of calcium, and neuronal death [74]. GRIA2 has an 245
additional ADAR editing site, R764G, which is edited mostly by ADAR, and results in different 246
electrophysiological properties. Editing at R764G gradually increases during neural 247
development, with the edited isoform fraction in adult brains ranging from 50% to 85% [75]. 248
249
Another well-documented ADAR editing event (via ADARB1) results in I-to-M, Q-to-R, and Y-250
to-C amino acid changes in the mammalian calcium voltage-gated channel subunit alpha1 D 251
channel CACNA1D (also known as CaV1.3) within the IQ domain, a calmodulin-binding site 252
responsible for regulating sensitivity to inhibitory Ca2+-feedback (CDI) [76]. Edited CACNA1D 253
proteins are expressed in both brain tissue and the surface membranes of primary neurons; 254
neurons with edited CACNA1D proteins show decreased CDI, as part of likely selective fine-255
tuning of Ca2+ feedback for low-voltage activated Ca2+ influx [76]. 256
257
GRIA2 and CACNA1D are not the only ADAR editing targets critical for neural function. 258
Functionally relevant editing targets include other glutamate receptors, such as glutamate 259
receptor subunit GRIA3 (also known as GluA3) [77], receptors for other neurotransmitters, 260
including excitatory and inhibitory neurotransmitters, such as serotonin 2C receptor 5HT2CR [78] 261
and the alpha3 subunit of gamma-aminobutyric acid (GABA) receptor (Gabra3) [79], respectively, 262
as well as ion channels (e.g., voltage-dependent potassium channel KCNA1 [80]), and 263
microRNAs [81]. Disruption of editing of these targets is known to lead to changes in excitability, 264
kinetics and other crucial functions of these receptors and ion channels, ultimately resulting in 265
neurodevelopmental defects, decreased proliferation and neuronal death [45]. In other words, 266
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seemingly minor amino acid changes in these proteins have the potential to significantly change 267
electrophysiology, calcium permeability and metabolism, among other properties, in neural (and 268
likely glial) cells, thus initiating a broad array of downstream neurophysiological changes such 269
as those observed in CZS and GBS. Indeed, because of glutamate’s fundamental role as a major 270
excitatory neurotransmitter in as many as 80 to 90% of brain neurons, most neurons and many 271
glial cells have glutamate receptors in their membranes [82]. Glutamatergic signaling also plays a 272
critical role in regulation of neural development, including neurotoxic effects of increased 273
calcium permeability due to, for example, inappropriate editing of GRIA2 [83]. 274
275
These examples offer a potential common explanation to the breadth of the observed clinical 276
symptoms and defects in both fetal and adult ZIKV infections, where variation in specific 277
manifestations across developmental stage at the time of infection can be attributed to the 278
nuanced spatio-temporal regulation of ADAR editing – and/or its infection-mediated 279
dysregulation, particularly in neural cells [84]. 280
281
ADAR is part of the antiviral response 282
Expression of ADARs, specifically, the ADAR1 p150 isoform, is regulated by interferon as part 283
of cell-autonomous immunity [85], with ADAR editing playing a role in an anti-viral activity, as 284
part of the innate immune response [86]. Recent experimental ZIKV studies have shown that the 285
interferon (IFN) type I response plays a role in ZIKV infection, that is that in human ZIKV 286
infections, expression of IFN cytokines increases, followed by increased expression of IFN-287
regulated genes, including ADARs [54]. Because the interactions between ZIKV and IFN pathway 288
activation are complex (e.g., may include mechanisms that suppress IFN signaling, such as 289
through action of viral NS5 protein [87], or by targeting STAT1 [88] and STAT2 [89]), and may 290
vary between ZIKV strains and/or specific variants of human genes or combination of the two 291
phenomena, that may help explain why not all ZIKV-positive pregnancies – or even fetuses from 292
the same pregnancy – show neurodevelopmental defects [90]. 293
294
Our recent work showed that ADAR editing plays a role in the molecular evolution of RNA 295
viruses [91], including ZIKV [92]. These results indicate that ADAR editing is active during ZIKV 296
infection. While it remains to be determined whether observed ADAR editing footprints were 297
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due to ADAR action in the mosquito or mammalian host (or both) [92], studies of the mammalian 298
immune response support the hypothesis that ADAR (specifically, the IFN-induced ADARp150 299
isoform) is activated in ZIKV infections. 300
Dysregulation of ADAR editing could potentially contribute to ZIKV pathogenesis via decreased 301
host editing (i.e., if ADAR editing activity is re-directed from the host editome to ZIKV) or via 302
increased host editing due to elevated expression of ADAR p150. ADAR p150 might directly 303
alter editing patterns, or do so indirectly if a stoichiometric change between ADAR p150 and 304
other ADARs modifies editing patterns. As noted previously, in mice both ADAR and ADARB1 305
knockouts can be fatal, e.g., due to underediting of key transcripts such as GRIA2 [58, 74, 93]. There 306
is mounting evidence that off-target editing by ADAR is also deleterious in humans [94]. 307
308
Thus, one of the potential explanations for mechanisms of congenital Zika syndrome may be 309
attributed to the disruption of editing and regulatory function of ADAR genes due to ZIKV-310
mediated IFN pathway activation (Figure 1). Specifically, physiological consequences of ADAR 311
editing dysregulation may be particularly critical for key synaptic signaling proteins, which in 312
turn result in shifts in calcium metabolism and homeostasis in the developing brain. For example, 313
changes in ADAR editing patterns can be linked to changes in (i) calcium permeability, and thus, 314
channel conductivity of GRIA2 and GRIA3 [95], and (ii) changes in how multiple isoforms of 5-315
HT2C – that have different binding affinities and functional potencies of agonists [96] – are 316
distributed within the brain. Appropriate ADAR editing thus could influence synaptic 317
transmission, as well as regulation of cell proliferation and neuronal differentiation [97], in 318
proliferative zones of the developing brain [98]. The changes of 5-HT2C editing have been linked 319
to mental disorders and suicide [99], although the full extent of editing consequences remains to 320
be elucidated [100]. Dysregulation of ADAR activity can also be potentially connected to the 321
neurodegeneration observed in GBS, where IFN-triggered elevated expression of ADAR results 322
in abnormal editing of proteins involved in synaptic signaling, thereby changing the ion 323
homeostasis of neurons, and hence, triggering the subsequent cascade of effects, similar to what 324
is observed in CZS. 325
326
327
328
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Conclusions and prospects 329
We posit that the experimental evidence from both the CZS and the GBS cases available so far is 330
consistent with the expectations of the hypothesis presented here. However, a causal link 331
between ADAR editing and subsequent neurodevelopmental or neurodegenerative sequelae in 332
CZS or GBS remains to be demonstrated. A critical first step would be to document ADAR 333
expression and ADAR editing levels in ZIKV infection in pregnancy and GBS, including self-334
regulation aspects of ADAR loci themselves [101], as well as expression and activity of calcium 335
homeostasis and metabolism proteins. Simultaneously, we need to elucidate the patterns and 336
regulatory processes involved in nuanced spatio-temporal changes in ADAR editing in brain 337
development during normal as well as ZIKV-positive pregnancies, and how these changes are 338
linked to observed clinical symptoms, because we still have only a limited understanding of the 339
complex regulation of ADAR editing across cells, tissues, developmental stages and individuals. 340
The observation of differential editing activity and its ramp-up during brain development [102] 341
must be supplemented by deeper understanding of how editing is regulated during different 342
stages of brain development and/or different locations in the CNS. This can be greatly aided by 343
the recently developed single-cell transcriptomics approaches [103]. 344
Understanding how ADAR editing influences brain development and function may lead to 345
development of treatment strategies that can be applied to newly infected ZIKV-positive 346
pregnant women, as well as to infants born or later diagnosed with CZS. It is likely that in utero 347
treatment strategies would vary depending on the stage of pregnancy (stemming from the 348
changing role of ADAR-edited proteins) at different stages of prenatal development. Treatment 349
strategies might range from (tailored) applications of existing immunotherapies to regulate 350
expression of interferon and/or other players of the cell-autonomous immunity pathway that are 351
upstream regulators of ADAR expression [85], to development of biomarkers of editing as well as 352
compounds targeting the editing process itself [104]. Similarly, we need to better understand the 353
role of ADAR editing and the functional implications of it (including quantitative aspects of how 354
much gets edited during any given time) on its respective targets during neurodegeneration of 355
GBS. 356
357
There might be an opportunity to intervene in the regulation of ADAR editing during/shortly 358
after ZIKV infection with the aim of preventing/diminishing the spectrum of ZIKV 359
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pathogenic/neurodevelopmental sequelae. For example, during pregnancy, within two-three 360
weeks of diagnosed ZIKV infection (i.e., within two-three weeks after the initial rash appears 361
[105]) a short course of drugs leading to the suppression of the type I interferon pathway can be 362
expected to result in down-regulation (or prevention of activation) of fetal ADAR activity. This 363
can be achieved by administering treatments currently used, e.g., to treat lupus [106]. Decisions to 364
use such treatments will be complicated by the fact that not all pregnant women exhibit clinical 365
signs of ZIKV infection [19]. However, the timing of asymptomatic infections can be inferred if 366
there is a finite window of exposure, by definition problematic in ZIKV-endemic areas. A similar 367
course of treatment can be applied if the early GBS symptoms were to appear a few weeks post-368
ZIKV infection (similar to that in HCV infections, [107]). Furthermore, the role of the recently 369
discovered member of the type III interferon pathway in the immune response needs to be 370
considered too [108]. Additionally, development of treatments specifically aimed at the reversal 371
of deleterious editing effects can also be pursued, e.g., via a combination of short-term 372
stimulation of expression of ADAR editing targets, administration of antagonists to the edited 373
protein versions and/or suppression of ADAR editing. 374
375
The same molecular mechanism postulated here to explain ZIKV pathogenesis may underlie 376
other conditions. For example, the increased editing of the calcium channel CaV1.3 has been 377
observed in dopamine-containing neurons affected in Parkinson’s disease, a second most 378
frequent neurodegenerative disease in the world [109], in which degeneration and death of these 379
(‘dopaminergic’) neurons is associated with motor symptoms of the disease [110]. Currently we 380
have neither a cure for it, nor a firm understanding of its underlying mechanisms [110, 111], 381
although it appears that (some form of) calcium dysregulation likely plays a prominent role [112]. 382
This dysregulation would result in oxidative and proteolytic stress and neuroinflammation that 383
may lead to mitochondrial dysfunction or vice versa [112, 113]. Evidence suggests that even in early 384
stages of Parkinson’s the calcium homeostasis is disturbed, in part attributable to what appears to 385
be a shift in cells reliance on (editable) CaV1.3 in lieu of CaV1.2 [114]. It is unclear whether the 386
changes in channel kinetics are due to CaV1.3 editing, and/or differential expression of 387
alternatively spliced isoforms CaV1.3 [115], as the study lacked the needed antibody resolution 388
[114]. In either case, this scenario offers an explanation of yet another neurodegenerative condition 389
(in addition to GBS) with unclear aetiology that could potentially be linked to possible changes 390
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in ADAR editing patterns induced by prior infections (e.g., [116]), likely of minor viral variety, 391
which have mild to subclinical symptoms that nonetheless have the potential to (over)activate 392
ADARs as part of cell-autonomous immunity. Could changes in edited calcium channels also 393
explain the brain calcification observed following prenatal CMV (a herpesvirus) infection [117]? 394
Interestingly, brain calcification is also a common finding in CZS infants, born with or without 395
microcephaly [32], again suggesting a role for ADAR editing in ZIKV pathogenesis. 396
Ultimately, we would like to call for systematic, concerted and broad efforts to understand the 397
nuances of spatio-temporal distribution of ADARs and associated editing activity throughout the 398
brain and primary neurons, both in pregnancy and postnatally, as well the influence of viral 399
infections (including ZIKV) on such activity and subsequent clinical consequences. 400
401
Funding 402
This work was partially supported Kent State University Research Council Seed Award, and 403
Brain Health Institute Pilot Award to HP. MLW was supported by the National Institutes of 404
Health grant number GM083192. 405
406
407
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Figure 1.
A
B
408
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409
Figure 1. Simplified (A) and detailed (B) summaries of our new hypothesis linking ZIKV 410
infection to congenital Zika syndrome and Guillain-Barre syndrome. (B) Overview of ZIKV 411
infection and the proposed mechanistic model of ZIKV-mediated pathogenesis. Upon infection, 412
ZIKV activates Toll-like-Receptor 3 (TLR3), which in turn activates the interferon (IFN) 413
pathway [118, 119]. Interferon-sensitive response elements (ISREs) upregulate interferon-stimulated 414
genes (ISGs), including the ADAR p150 isoform [85]. Thus, in addition to ADAR editing of viral 415
RNA as part of cell-autonomous immunity, ZIKV-driven activation of ADARp150 may also 416
lead to dysregulation of ADAR editing within host cells. These changes in levels of ADAR 417
editing may be due to increased abundance of ADAR p150 enzyme copies and/or due to 418
interference between ADAR p150 and other ADARs, e.g., through substrate competition with 419
ADARB1 [52, 101]. In turn, ZIKV-induced changes in editing patterns of host transcripts – such as 420
neurotransmitter receptors and transporters - can result in physiological changes in host proteins, 421
such as changes in ion permeability and excitability as illustrated by GRIA2 and GRIA3 [73, 77], in 422
turn leading to excitotoxicity and neuronal demise [45, 46]. Artistic drawing is by Peggy Muddles, 423
@vexedmuddler. 424
425
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