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Citation: Egilmezer, E.; Hamilton, S.T.;
Lauw, G.; Follett, J.; Sonntag, E.;
Schütz, M.; Marschall, M.;
Rawlinson, W.D. Human
Cytomegalovirus Dysregulates
Cellular Dual-Specificity Tyrosine
Phosphorylation-Regulated Kinases
and Sonic Hedgehog Pathway
Proteins in Neural Astrocyte and
Placental Models. Viruses 2024,16, 918.
https://doi.org/10.3390/v16060918
Academic Editor: Michael McVoy
Received: 12 January 2024
Revised: 29 May 2024
Accepted: 30 May 2024
Published: 5 June 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
viruses
Article
Human Cytomegalovirus Dysregulates Cellular
Dual-Specificity Tyrosine Phosphorylation-Regulated Kinases
and Sonic Hedgehog Pathway Proteins in Neural Astrocyte and
Placental Models
Ece Egilmezer 1,2 , Stuart T. Hamilton 1,2, Glen Lauw 3, Jasmine Follett 3, Eric Sonntag 4, Martin Schütz 4,
Manfred Marschall 4and William D. Rawlinson 1,2,3,*
1Serology and Virology Division, Microbiology, NSW Health Pathology, Prince of Wales Hospital,
Sydney 2031, Australia; ece.egilmezer1@health.nsw.gov.au (E.E.)
2School of Clinical Medicine, University of New South Wales, Kensington 2052, Australia
3
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney 2033, Australia
4Institute for Clinical and Molecular Virology, Friedrich-Alexander University of Erlangen-Nürnberg,
91054 Erlangen, Germany; manfred.marschall@fau.de (M.M.)
*Correspondence: w.rawlinson@unsw.edu.au; Tel.: +61-293829113; Fax: +61-293984275
Abstract: Human cytomegalovirus (CMV) infection is the leading non-genetic cause of congenital
malformation in developed countries, causing significant fetal injury, and in some cases fetal death.
The pathogenetic mechanisms through which this host-specific virus infects then damages both the
placenta and the fetal brain are currently ill-defined. We investigated the CMV modulation of key
signaling pathway proteins for these organs including dual-specificity tyrosine phosphorylation-
regulated kinases (DYRK) and Sonic Hedgehog (SHH) pathway proteins using human first trimester
placental trophoblast (TEV-1) cells, primary human astrocyte (NHA) brain cells, and CMV-infected
human placental tissue. Immunofluorescence demonstrated the accumulation and re-localization
of SHH proteins in CMV-infected TEV-1 cells with Gli2, Ulk3, and Shh re-localizing to the CMV
cytoplasmic virion assembly complex (VAC). In CMV-infected NHA cells, DYRK1A re-localized
to the VAC and DYRK1B re-localized to the CMV nuclear replication compartments, and the SHH
proteins re-localized with a similar pattern as was observed in TEV-1 cells. Western blot analysis in
CMV-infected TEV-1 cells showed the upregulated expression of Rb, Ulk3, and Shh, but not Gli2.
In CMV-infected NHA cells, there was an upregulation of DYRK1A, DYRK1B, Gli2, Rb, Ulk3, and
Shh. These
in vitro
monoculture findings are consistent with patterns of protein upregulation and re-
localization observed in naturally infected placental tissue and CMV-infected ex vivo placental explant
histocultures. This study reveals CMV-induced changes in proteins critical for fetal development,
and identifies new potential targets for CMV therapeutic development.
Keywords: cytomegalovirus; pathogenesis; developmental pathways; astrocytes; placenta
1. Introduction
Human cytomegalovirus (CMV) is the leading infectious cause of fetal malformation
in developed countries [
1
]. The incidence of CMV births in developed countries ranges
from 0.2 to 2.0%, and ranges from 0.6% to 6.1% in developing countries [
1
,
2
]. Congenital
CMV infection can be asymptomatic, or result in fetal neural malformations including
microcephaly, intracranial calcifications, cerebral palsy, mental disability, sensorineural
hearing loss, seizures, and visual impairment [
3
–
5
]. The transplacental transmission of
CMV from the infected placenta across the materno-fetal interface is the crucial and rate-
limiting step for fetal infection [
6
]. In certain cases, infection may be restricted to the
placenta instead, and there is evidence that placental CMV infection may indirectly result
Viruses 2024,16, 918. https://doi.org/10.3390/v16060918 https://www.mdpi.com/journal/viruses
Viruses 2024,16, 918 2 of 13
in adverse pregnancy outcomes [
7
–
9
]. The pathogenetic mechanisms of CMV-induced fetal
and placental malformation have not yet been fully elucidated.
Cellular protein kinases are dysregulated by CMV infection, and thought to be utilized
by CMV to facilitate viral replication in a range of cells [
10
]. We have previously shown that
dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) play a critical role in
CMV replication in fibroblast cells, placental trophoblasts and placental ex vivo explant
cultures [
11
]. The DYRK family of proteins are key regulators of cell growth, apoptosis,
and differentiation, with DYRK1A and DYRK1B being the best characterized among the
five mammalian DYRKs [
12
]. The DYRK1A protein is active in the nucleus and cytoplasm,
and regulates cellular quiescence, and the proliferation and differentiation of neuronal
progenitor cells, while also modulating signaling pathways including Sonic Hedgehog
(SHH) [
13
–
17
]. DYRK1A is considered a significant gene in the Down syndrome phenotype
and has been implicated in other neurological malformations, some of which are seen
in congenital CMV infection [
18
]. These include intrauterine growth restriction (IUGR),
developmental delay, and microcephaly [
19
–
21
]. DYRK1B, which has in comparison
been less characterized, is associated with cellular differentiation, cell cycle arrest, and
modulation of the SHH pathway [22,23].
One of the key functions of DYRK proteins is to modulate the SHH pathway, which is
necessary for determining cell fate, patterning, proliferation, and differentiation [
12
]. In
fetal brain development, the SHH pathway mediates midbrain and neural tube pattern
formation, synapse formation, promotion of blood–brain barrier integrity, and cerebral
immune quiescence mediated by astrocytes [
24
–
28
]. Recently, the significance of SHH
in the placenta has been explored, showing the SHH pathway is required for placental
development [
29
], and that the dysregulation of the pathway results in adverse pregnancy
outcomes [30].
The effect CMV infection has on dysregulating critical fetal developmental pathways is
emerging as a potential pathogenetic mechanism for fetal injury. Previously, we and others
have shown the virus-supportive impact of DYRK activity on the cultured cell replication
efficiency of CMV [
11
,
31
]. In this study, we utilized different
in vitro
and ex vivo cell
culture systems to model (i) the placenta using first trimester extravillous trophoblast cells
(TEV-1), placental explants, and clinically infected placental tissue to inform mother to child
transmission (MTCT), and (ii) the fetal brain using normal human astrocytes (NHA). We
show that CMV infection dysregulates DYRKs (DYRK1A, DYRK1B) and the SHH pathway
(Shh morphogen, Gli2 transactivator, Ulk3 kinase, and Rb tumor suppressor protein). The
CMV-induced dysregulation of these proteins that are vital for fetal development suggests
an important viral pathogenetic mechanism for fetal neural and placental injury.
2. Methods
2.1. Cell Lines and Preparation of Virus Stocks
Human first-trimester extravillous trophoblast (TEV-1) cells were maintained in Ham’s
F10 Nutrient Mix (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal
bovine serum (FBS; Interpath, Melbourne, VIC, Australia) and 100 U/mL penicillin G,
100 U/mL streptomycin and 29.2
µ
g/mL L-glutamine (1
×
PSG; Gibco, Sydney, NSW,
Australia). Human NHA primary astrocytes (Lonza, Basel, Switzerland) were maintained
in low-glucose Dulbecco’s modified Eagle medium (DMEM; Gibco) supplemented with
10% FBS and 1
×
PSG. Cell lines were mycoplasma-free and maintained at 37
◦
C with
5% CO2.
The CMV strain AD169 (ATCC) was propagated in human MRC-5 cells maintained in
MEM supplemented with 2% FBS and 1
×
PSG. The CMV strain Merlin (UL128+, RL13
−
)
was propagated in RPE-1 cells as previously described [
32
]. Viral titres were determined
using standard plaque assays.
Viruses 2024,16, 918 3 of 13
2.2. Clinical Placentae and Placental Villous Explant Histocultures
Clinical placental tissue was derived from formalin-fixed paraffin embedded (FFPE)
placentae collected from still-born infants as part of a retrospective case series, as previously
described [
9
]. The placenta with confirmed CMV infection was from a 21-week gestational
age pregnancy and uninfected controls were matched for gestational age.
Mock and Merlin-infected placental villous explant histocultures from term placenta
were derived from FFPE explant sections from a previous study (under ethics approval
SESIAHS HREC 09/126) as previously described [31,33].
2.3. Infection of TEV-1 Cells, and NHA Cells with CMV
The TEV-1 and NHA cells were inoculated with CMV AD169 or CMV Merlin for
Western blot analysis (0.5 pfu/cell for NHA and 2 pfu/cell for TEV-1), and inoculated
with CMV AD169, CMV Merlin, or UV-inactivated CMV Merlin for immunofluorescence
analysis (0.1 pfu/cell for NHA and 2 pfu/cell for TEV-1). Different multiplicities of infection
for Western blot analysis were used to give approximately similar levels of infection,
as determined by IFA between the different cell types. Lower MOIs were used for IFA
experiments, to prevent all cells from being infected by 7 dpi, thereby allowing comparisons
between CMV-infected and un-infected cells within the same experiment. Mock-infected
cultures were established concurrently. Plates were centrifuged at 770
×
gfor 30 min
followed by 2 h incubation at 37
◦
C with 5% CO
2
, after which the medium was replaced
with fresh cell culture media. Cells were incubated at 37
◦
C with 5% CO
2
with cell culture
media being replaced with fresh media at 4 dpi.
2.4. Immunofluorescence
The TEV-1 and NHA cells were seeded in 6-well plates with underlying coverslips
and infected with CMV AD169 and Merlin, as described above. Immunofluorescence
staining was performed at days 1, 4, and 7 post-infection as previously described [
34
].
Formalin-fixed paraffin-embedded (FFPE) tissue sections of 4
µ
m were de-paraffinized and
rehydrated followed by antigen retrieval using Tris-EDTA buffer (pH 9.0) for 20 min at 95
◦
C.
These sections were incubated for one hour with primary antibodies for CMV detection
with mouse mAb anti-HCMV immediate early (IE1p72) and early (pUL44) antibody cocktail
(IE/E; clones DDG9 and CCH2; Dako, Sydney, NSW, Australia), mouse mAb-pp28 (Abcam,
Cambridge, UK) and DYRK and Sonic Hedgehog (SHH) proteins with rabbit pAb-DYRK1A
(Abcam), rabbit mAb-DYRK1B (Abcam), rabbit mAb-Sonic Hedgehog (Abcam), rabbit
mAb-ULK3 (Abcam), rabbit mAb-Rb (Abcam) and rabbit pAb-Gli2 (Abcam). In our
methods, we did not utilize an Fc blocking step as we have previously shown no differences
in staining patterns observed with and without Fc blocking [
31
,
34
]. Sections and slides
were incubated with secondary antibodies Alexa Fluor 488 goat anti-mouse and 594 goat
donkey anti-rabbit (Invitrogen, Waltham, MA, USA; 1:1000 dilution) for 30 min. DAPI
(Invitrogen) was added to each slide, mounted with coverslips and imaged as previously
described [31].
As previously described, mock and Merlin-infected TEV-1 cells were stained with the
non-specific control rabbit
β
-galactosidase (Abcam) antibody and PBS, to ensure staining
was not due to non-specific primary and secondary antibody binding, respectively [31].
2.5. Western Blot Analysis
Western blot analysis was performed in duplicate using standard procedures, as de-
scribed previously [
35
]. Immunostaining was performed using mAb anti-CMV immediate
early (IE1p72) and early (pUL44) antibody cocktail (IE/E; clones DDG9 and CCH2; Dako),
mAb-
β
-actin (Ac-15, Sigma, St. Louis, MO, USA), rAb-DYRK1A (Abcam), rAb-DYRK1B
(Abcam), rAb-Shh (Abcam), rAb-ULK3 (Abcam), rAb-Rb (Abcam) and rAb-Gli2 (Abcam).
HRP-conjugated anti-mouse or anti-rabbit secondary antibodies (Pierce) were used as sec-
ondary antibodies. Protein bands were visualized using chemiluminescence. Densitometry
Viruses 2024,16, 918 4 of 13
analysis was performed with ImageJ software (version 1.51j8) using experimental replicates
and bands were normalized to β-actin expression.
3. Results
3.1. CMV Infection of Trophoblast Cells Results in Accumulation and Re-Localization of
SHH Proteins
In a previous study, we showed that the AD169 and Merlin infection of TEV-1 cells
resulted in cellular re-localization and accumulation of DYRK1A and DYRK1B [
31
]. In this
study, immunofluorescence was performed on mock- and Merlin-infected TEV-1 cells at
7dpi for the Sonic Hedgehog signaling (SHH) proteins Gli2, Rb, Ulk3, and Shh (Figure 1).
In mock-infected TEV-1 cells, Gli2, Ulk3, and Shh proteins were diffusely observed predom-
inately in the cell cytoplasm. Merlin infection resulted in the accumulation of Gli2, Ulk3,
and Shh in clusters of punctate staining in the cell cytoplasm. These proteins predominantly
re-localized to the virion assembly complex (VAC), as demonstrated by co-localization with
the VAC-associated CMV protein pp28 (UL99) (Figure 1). The Rb protein was observed in
the nucleus of uninfected TEV-1 cells. Merlin infection induced the accumulation of Rb in
the nucleus and partial re-localization to the VAC with punctate staining.
Viruses2024,16,xFORPEERREVIEW4of13
Dako),mAb‐β‐actin(Ac‐15,Sigma,St.Louis,MO,USA),rAb‐DYRK1A(Abcam),
rAb‐DYRK1B(Abcam),rAb‐Shh(Abcam),rAb‐ULK3(Abcam),rAb‐Rb(Abcam)and
rAb‐Gli2(Abcam).HRP‐conjugatedanti‐mouseoranti‐rabbitsecondaryantibodies
(Pierce)wereusedassecondaryantibodies.Proteinbandswerevisualizedusingchemi‐
luminescence.DensitometryanalysiswasperformedwithImageJsoftware(version
1.51j8)usingexperimentalreplicatesandbandswerenormalizedtoβ‐actinexpression.
3.Results
3.1.CMVInfectionofTrophoblastCellsResultsinAccumulationandRe‐LocalizationofSHH
Proteins
Inapreviousstudy,weshowedthattheAD169andMerlininfectionofTEV‐1cells
resultedincellularre‐localizationandaccumulationofDYRK1AandDYRK1B[31].In
thisstudy,immunofluorescencewasperformedonmock‐ andMerlin‐infectedTEV‐1
cellsat7dpifortheSonicHedgehogsignaling(SHH)proteinsGli2,Rb,Ulk3,andShh
(Figure1).Inmock‐infectedTEV‐1cells,Gli2,Ulk3,andShhproteinswerediffuselyob‐
servedpredominatelyinthecellcytoplasm.Merlininfectionresultedintheaccumulation
ofGli2,Ulk3,andShhinclustersofpunctatestaininginthecellcytoplasm.Thesepro‐
teinspredominantlyre‐localizedtothevirionassemblycomplex(VAC),asdemonstrated
byco‐localizationwiththeVAC ‐associatedCMVproteinpp28(UL99)(Figure1).TheRb
proteinwasobservedinthenucleusofuninfectedTEV‐1cells.Merlininfectioninduced
theaccumulationofRbinthenucleusandpartialre‐localizationtotheVACwithpunc‐
tatestaining.
Figure1.CMVinfectionofplacentaltrophoblastcellsresultsintheaccumulationand
re‐localizationofSHHproteins.TEV‐1trophoblastcellsinfectedwithCMVstrainMerlin(2
pfu/cell)ormock‐infectedandstainedforGli2,Rb,Ulk3orShhproteinandCMVimmediateearly
IE1p72/earlypUL44(IE/E)ortruelatepp28proteinsat7dpi.Thewhitearrowshighlightthelo‐
calizationofpp28staining.Mock‐andMerlin‐infectedculturesincubatedwithnon‐specificrabbit
mAbanti‐β‐galactosidaseorPBSasprimaryantibodyservedasnegativecontrols.
Figure 1. CMV infection of placental trophoblast cells results in the accumulation and re-localization
of SHH proteins. TEV-1 trophoblast cells infected with CMV strain Merlin (2 pfu/cell) or mock-
infected and stained for Gli2, Rb, Ulk3 or Shh protein and CMV immediate early IE1p72/early pUL44
(IE/E) or true late pp28 proteins at 7 dpi. The white arrows highlight the localization of pp28 staining.
Mock- and Merlin-infected cultures incubated with non-specific rabbit mAb anti-
β
-galactosidase or
PBS as primary antibody served as negative controls.
The staining of cells with a non-specific rabbit
β
-galactosidase primary antibody or
incubation with PBS did not result in any staining of the VAC, demonstrating the results
were not due to non-specific CMV-derived Fc receptor binding.
To confirm that the changes observed in protein localization were due to CMV repli-
cation rather than a cellular stress response or virus-cell binding/entry, TEV-1 cells were
Viruses 2024,16, 918 5 of 13
inoculated with UV-inactivated Merlin. No changes were observed in the localization
or accumulation of SHH proteins relative to mock-infected TEV-1 cells (Supplementary
Figure S1).
3.2. CMV-Induced Trophoblast Accumulation and Re-Localization of Rb, Ulk3, and Shh, but Not
Gli2 Is Caused by Upregulation of Expression
Western blot analyses were performed to determine the effects of CMV-infection on
the expression of SHH proteins in TEV-1 cells (Figure 2A). Densitometry analysis using
ImageJ was performed to quantitate protein expression in CMV-infected cells relative to
the uninfected (mock) control cells at days 1, 4, and 7 post-infection, and the bands were
normalized to
β
-actin (Figure 2B). At 1 dpi, no alterations were observed in Gli2, Rb, Ulk3,
or Shh levels in Merlin-infected cells relative to mock. At 4 dpi, Rb, Ulk3, and Shh protein
levels increased, but not Gli2 in Merlin-infected TEV-1 cells relative to mock. At 7dpi,
relative to mock-infected cells, Rb, Ulk3, and Shh protein expressions increased, and Gli2
exhibited a small reduction in protein levels in Merlin-infected cells.
Viruses2024,16,xFORPEERREVIEW5of13
Thestainingofcellswithanon‐specificrabbitβ‐galactosidaseprimaryantibodyor
incubationwithPBSdidnotresultinanystainingoftheVAC ,demonstratingtheresults
werenotduetonon‐specificCMV‐derivedFcreceptorbinding.
ToconfirmthatthechangesobservedinproteinlocalizationwereduetoCMVrep‐
licationratherthanacellularstressresponseorvirus‐cellbinding/entry,TEV‐1cellswere
inoculatedwithUV‐inactivatedMerlin.Nochangeswereobservedinthelocalizationor
accumulationofSHHproteinsrelativetomock‐infectedTEV‐1cells(Supplementary
FigureS1).
3.2.CMV‐InducedTrophoblastAccumulationandRe‐LocalizationofRb,Ulk3,andShh,butNot
Gli2IsCausedbyUpregulationofExpression
WesternblotanalyseswereperformedtodeterminetheeffectsofCMV‐infectionon
theexpressionofSHHproteinsinTEV‐1cells(Figure2A).Densitometryanalysisusing
ImageJwasperformedtoquantitateproteinexpressioninCMV‐infectedcellsrelativeto
theuninfected(mock)controlcellsatdays1,4,and7post‐infection,andthebandswere
normalizedtoβ‐actin(Figure2B).At1dpi,noalterationswereobservedinGli2,Rb,
Ulk3,orShhlevelsinMerlin‐infectedcellsrelativetomock.At4dpi,Rb,Ulk3,andShh
proteinlevelsincreased,butnotGli2inMerlin‐infectedTEV‐1cellsrelativetomock.At
7dpi,relativetomock‐infectedcells,Rb,Ulk3,andShhproteinexpressionsincreased,
andGli2exhibitedasmallreductioninproteinlevelsinMerlin‐infectedcells.
Figure2.CMVinfectionoftrophoblastcellsresultsinincreasedproteinexpressionofkeySHH
proteins.(A)TEV‐1trophoblastcellswereinfectedwithCMVstrainMerlin(2pfu/cell)andcells
harvestedat1,4and7dpiwithcellularGli2,Rb,Ulk3,ShhorCMVimmediateearlyIE1p72protein
expressionmeasuredusingWes ternblot.(B)Gli2,Rb,Ulk3orShhproteinexpressionfoldchange,
normalizedtoβ‐actinlevelsinMerlin‐infectedcellsrelativetomock‐infectedcellsusingdensi‐
tometry.Resultsderivedfromduplicateexperimentsanddensitometryresultspresentedasmean±
SD.
Figure 2. CMV infection of trophoblast cells results in increased protein expression of key SHH
proteins. (A) TEV-1 trophoblast cells were infected with CMV strain Merlin (2 pfu/cell) and cells
harvested at 1, 4 and 7 dpi with cellular Gli2, Rb, Ulk3, Shh or CMV immediate early IE1p72 protein
expression measured using Western blot. (B) Gli2, Rb, Ulk3 or Shh protein expression fold change,
normalized to
β
-actin levels in Merlin-infected cells relative to mock-infected cells using densitometry.
Results derived from duplicate experiments and densitometry results presented as mean ±SD.
3.3. CMV Infection of Normal Human Astrocyte Cells Results in Accumulation and
Re-Localization of DYRK and SHH Proteins
We have previously shown that the CMV infection of placental trophoblasts, ex vivo
placental explants, and naturally infected clinical placentae results in the upregulation and
re-localization of DYRK1A to the cytoplasm, and VAC and DYRK1B to the nucleus [
31
].
Viruses 2024,16, 918 6 of 13
Immunofluorescence was performed at 7 dpi on mock-, AD169-, and Merlin-infected NHA
cells for DYRK and SHH proteins (Figure 3). In mock-infected NHAs, DYRK1A, Gli2, and
Ulk3 were diffusely localized in the nucleus and cytoplasm. The DYRK1B and Rb proteins
were diffusely localized in the nucleus, and Shh in the cytoplasm. Upon AD169 or Merlin
infection, the aberrant re-localization and accumulation of these proteins were detected.
The DYRK1A, Gli2, and Shh proteins accumulated in the cytoplasmic virion assembly
complex. The small punctate accumulation of DYRK1B was observed in infected cell nuclei
corresponding to the CMV nuclear replication compartments. Rb and Ulk3 accumulated in
both the nucleus and cytoplasm of infected cells relative to mock.
Viruses2024,16,xFORPEERREVIEW6of13
3.3.CMVInfectionofNormalHumanAstrocyteCellsResultsinAccumulationand
Re‐LocalizationofDYRKandSHHProteins
WehavepreviouslyshownthattheCMVinfectionofplacentaltrophoblasts,exvivo
placentalexplants,andnaturallyinfectedclinicalplacentaeresultsintheupregulation
andre‐localizationofDYRK1Atothecytoplasm,andVACandDYRK1Btothenucleus
[31].Immunofluorescencewasperformedat7dpionmock‐,AD169‐,andMerlin‐infected
NHAcellsforDYRKandSHHproteins(Figure3).Inmock‐infectedNHAs,DYRK1A,
Gli2,andUlk3werediffuselylocalizedinthenucleusandcytoplasm.TheDYRK1Band
Rbproteinswerediffuselylocalizedinthenucleus,andShhinthecytoplasm.Upon
AD169orMerlininfection,theaberrantre‐localizationandaccumulationofthesepro‐
teinsweredetected.TheDYRK1A,Gli2,andShhproteinsaccumulatedinthecytoplas‐
micvirionassemblycomplex.ThesmallpunctateaccumulationofDYRK1Bwasob‐
servedininfectedcellnucleicorrespondingtotheCMVnuclearreplicationcompart‐
ments.RbandUlk3accumulatedinboththenucleusandcytoplasmofinfectedcellsrel‐
ativetomock.
Figure3.CMVinfectionofhumanbrainastrocytecellsresultsinthesimilaraccumulationand
re‐localizationofSHHproteinsasplacentaltrophoblastcells.NHAastrocytecellsinfectedwith
CMVAD169orMerlinstrain(0.1pfu/cell)ormock‐infectedandstainedforDYRK1A,DYRK1B,
Gli2,Rb,Ulk3orShhproteinandCMVimmediateearlyIE1p72/earlypUL44(IE/E)andtruelate
pp28(UL99)proteinsat7dpi.
Figure 3. CMV infection of human brain astrocyte cells results in the similar accumulation and
re-localization of SHH proteins as placental trophoblast cells. NHA astrocyte cells infected with CMV
AD169 or Merlin strain (0.1 pfu/cell) or mock-infected and stained for DYRK1A, DYRK1B, Gli2, Rb,
Ulk3 or Shh protein and CMV immediate early IE1p72/early pUL44 (IE/E) and true late pp28 (UL99)
proteins at 7 dpi.
No differences in re-localization patterns were observed between AD169- and Merlin-
infected NHA cells. The CMV-induced accumulation and localization patterns for the SHH
proteins were comparable to patterns observed in CMV-infected placental trophoblast cells
(Figure 1).
Merlin-infected NHA cells were additionally co-stained for the DYRK (DYRK1A and
DYRK1B) and SHH (Gli2, Rb, Ulk3, and Shh) proteins with the true late CMV protein pp28.
Similar to TEV-1 cells (Figure 1), these proteins predominantly re-localized to the VAC
(Figure 3). The inoculation of NHA cells with UV-inactivated Merlin showed no changes in
Viruses 2024,16, 918 7 of 13
the localization or accumulation of DYRK or SHH proteins relative to mock-infected NHA
cells (Supplementary Figure S1).
3.4. CMV-Induced Normal Human Astrocyte Accumulation and Re-Localization of DYRK and
SHH Proteins Is Caused by Upregulated Expression
Western blot analyses were performed to determine the effects of CMV-infection on
the expression of DYRK and SHH proteins in NHA cells (Figure 4). Densitometry analysis
using ImageJ was performed to quantitate protein expression in CMV-infected cells relative
to the uninfected (mock) control cells at days 1, 4, and 7 post-infection, and the bands were
normalized to
β
-actin. At 7 dpi, DYRK1A was only upregulated in AD169-infected cells
but not in Merlin-infected cells, which may be a biological consequence of the difference
between CMV strains. Upregulation was detected in DYRK1B, Gli2, Rb, Ulk3, and Shh
in both AD169- and Merlin-infected NHA cells. The changes in protein expression in
CMV-infected NHA cells were time-dependent, and these proteins were dysregulated
during early to late stages of CMV replication, i.e., 4 dpi and 7 dpi.
Viruses2024,16,xFORPEERREVIEW7of13
Nodifferencesinre‐localizationpaernswereobservedbetweenAD169‐andMer‐
lin‐infectedNHAcells.TheCMV‐inducedaccumulationandlocalizationpaernsforthe
SHHproteinswerecomparabletopaernsobservedinCMV‐infectedplacentaltropho‐
blastcells(Figure1).
Merlin‐infectedNHAcellswereadditionallyco‐stainedfortheDYRK(DYRK1A
andDYRK1B)andSHH(Gli2,Rb,Ulk3,andShh)proteinswiththetruelateCMVpro‐
teinpp28.SimilartoTEV‐1cells(Figure1),theseproteinspredominantlyre‐localizedto
theVAC(Figure3).TheinoculationofNHAcellswithUV‐inactivatedMerlinshowedno
changesinthelocalizationoraccumulationofDYRKorSHHproteinsrelativeto
mock‐infectedNHAcells(SupplementaryFigureS1).
3.4.CMV‐InducedNormalHumanAstrocyteAccumulationandRe‐LocalizationofDYRKand
SHHProteinsIsCausedbyUpregulatedExpression
WesternblotanalyseswereperformedtodeterminetheeffectsofCMV‐infectionon
theexpressionofDYRKandSHHproteinsinNHAcells(Figure4).Densitometryanaly‐
sisusingImageJwasperformedtoquantitateproteinexpressioninCMV‐infectedcells
relativetotheuninfected(mock)controlcellsatdays1,4,and7post‐infection,andthe
bandswerenormalizedtoβ‐actin.At7dpi,DYRK1Awasonlyupregulatedin
AD169‐infectedcellsbutnotinMerlin‐infectedcells,whichmaybeabiologicalconse‐
quenceofthedifferencebetweenCMVstrains.UpregulationwasdetectedinDYRK1B,
Gli2,Rb,Ulk3,andShhinbothAD169‐andMerlin‐infectedNHAcells.Thechangesin
proteinexpressioninCMV‐infectedNHAcellsweretime‐dependent,andtheseproteins
weredysregulatedduringearlytolatestagesofCMVreplication,i.e.,4dpiand7dpi.
Figure4.CMVinfectionofhumanbrainastrocytecellsresultsinincreasedproteinexpressionof
keySHHproteins.(A)NHAastrocytecellswereinfectedwithCMVstrainAD169orMerlin(0.5
Figure 4. CMV infection of human brain astrocyte cells results in increased protein expression of key
SHH proteins. (A) NHA astrocyte cells were infected with CMV strain AD169 or Merlin (0.5 pfu/cell)
and cells were harvested at 1, 4 and 7 dpi, with cellular DYRK1A, DYRK1B, Gli2, Rb, Ulk3, Shh
or CMV immediate early IE1p72 protein expression measured using Western blot. (B) DYRK1A,
DYRK1B, Gli2, Rb, Ulk3 or Shh protein expression fold change, normalized to
β
-actin levels in Merlin-
infected cells relative to mock-infected cells using densitometry. Results derived from duplicate
experiments and densitometry results presented as mean ±SD.
Viruses 2024,16, 918 8 of 13
3.5. The CMV Upregulation of SHH Proteins Occurs in In Vivo and Ex Vivo Human
Placental Models
Expressions of the SHH proteins Gli2, Rb, Ulk3, and Shh were investigated in multi-
cellular human placentae infected with CMV both naturally and ex vivo to complement
the evidence in cell culture monolayers, as we reported previously for DYRK1A and
DYRK1B [
31
]. Both Gli2 and Rb were faintly expressed in cells composing the placental villi
in both CMV-infected and uninfected placentae (Figure 5). In CMV-infected placental sec-
tions, cytoplasmic and some nuclear re-localization of Gli2 was observed in CMV-infected
cells. Rb staining in CMV-infected cells showed increased presence in the nucleus compared
to uninfected placental cells, with no signs of punctate staining, as was observed in cell
monocultures (Figure 5). Ulk3 was primarily concentrated in the cytoplasm of cells within
the villi of CMV-infected and uninfected placentae, with a modest increase in cytoplasmic
staining in CMV-infected cells in infected tissue. Shh expression was detected primarily
in the cytoplasm of cells within the villi, again with increased cytoplasmic staining in
CMV-infected cells of the placental tissues.
Viruses2024,16,xFORPEERREVIEW8of13
pfu/cell)andcellswereharvestedat1,4and7dpi,withcellularDYRK1A,DYRK1B,Gli2,Rb,Ulk3,
ShhorCMVimmediateearlyIE1p72proteinexpressionmeasuredusingWesternblot.(B)
DYRK1A,DYRK1B,Gli2,Rb,Ulk3orShhproteinexpressionfoldchange,normalizedtoβ‐actin
levelsinMerlin‐infectedcellsrelativetomock‐infectedcellsusingdensitometry.Resultsderived
fromduplicateexperimentsanddensitometryresultspresentedasmean±SD.
3.5.TheCMVUpregulationofSHHProteinsOccursinInVivoandExVivoHumanPlacental
Models
ExpressionsoftheSHHproteinsGli2,Rb,Ulk3,andShhwereinvestigatedinmul‐
ticellularhumanplacentaeinfectedwithCMVbothnaturallyandexvivotocomplement
theevidenceincellculturemonolayers,aswereportedpreviouslyforDYRK1Aand
DYRK1B[31].BothGli2andRbwerefaintlyexpressedincellscomposingtheplacental
villiinbothCMV‐infectedanduninfectedplacentae(Figure5).InCMV‐infectedplacen‐
talsections,cytoplasmicandsomenuclearre‐localizationofGli2wasobservedin
CMV‐infectedcells.RbstaininginCMV‐infectedcellsshowedincreasedpresenceinthe
nucleuscomparedtouninfectedplacentalcells,withnosignsofpunctatestaining,aswas
observedincellmonocultures(Figure5).Ulk3wasprimarilyconcentratedinthecyto‐
plasmofcellswithinthevilliofCMV‐infectedanduninfectedplacentae,withamodest
increaseincytoplasmicstaininginCMV‐infectedcellsininfectedtissue.Shhexpression
wasdetectedprimarilyinthecytoplasmofcellswithinthevilli,againwithincreased
cytoplasmicstaininginCMV‐infectedcellsoftheplacentaltissues.
Figure5.CMVinfectioninnaturallyinfectedandMerlin‐infectedplacentaltissueresultsinthe
upregulationandre‐localizationofkeySHHproteins.Naturallyinfectedclinicalplacentae,Mer‐
Figure 5. CMV infection in naturally infected and Merlin-infected placental tissue results in the
upregulation and re-localization of key SHH proteins. Naturally infected clinical placentae, Merlin-
infected ex vivo placental explants or uninfected clinical placentae and placental explants stained
for CMV immediate early/early protein (IE/E) and Gli2, Rb, Ulk3 and Shh proteins. Representative
images of clinical placentae derived from 22-week gestational age placental tissue matched for
gestational age control or placental villous explant histocultures from term placentae. The white
arrows indicate infected cells of interest in each panel.
Viruses 2024,16, 918 9 of 13
4. Discussion
Congenital cytomegalovirus pathogenesis likely involves cellular dysregulation in
placental and fetal compartments, both of which likely contribute to fetal damage [
36
]. We
have previously shown DYRKs play a critical role in CMV replication in
in vitro
fibroblasts,
placental trophoblasts, and placental ex vivo explant cultures, as well as the dysregulation
of these proteins in naturally infected clinical placental tissue [
11
,
31
]. Protein kinases play a
critical role in the regulation of cellular functions through signal transduction cascades [
37
],
and as a result, the dysregulation of these kinases represents an interesting pathogenetic
mechanism for fetal neural malformation.
The importance of the DYRK-influenced SHH pathway to the development of pla-
centae is an emerging area of interest. The Shh and Gli2 proteins are required for ap-
propriate placental development and pregnancy maintenance in murine models [
29
]. A
study recently reported the dysregulation of the SHH pathway in preeclampsia placentae
samples [
30
]. In this study, we showed that the Merlin infection of human first trimester
extravillous trophoblast (TEV-1) cells increased Rb, Ulk3, and Shh protein levels relative to
uninfected (mock) controls. Evidence suggests that a reduction in Ulk3 mRNA levels in
Shh-responsive cells increases the cells’ potency to transmit the Shh signal [
38
]. Similarly,
Rb has been shown to exhibit a negative correlation with the SHH pathway [
39
]. Taken
together, the increase in Ulk3 and Rb in CMV-infected TEV-1 cells suggests the suppression
of the SHH pathway in infected placentae. In primary cytotrophoblasts, it has been re-
ported that trophoblast syncytialization is modulated by non-canonical SHH signaling [
30
].
Cytotrophoblasts undergo syncitialization during placental development to form the syncy-
tiotrophoblast layer, which is essential in transporting almost all nutrients from the mother
to the fetus [
40
,
41
]. Similarly, the infection of cytotrophoblasts with CMV has also been
shown to suppress syncytialization [
42
]. Changes in the rates of syncytialization have
been observed in several placentae pathologies, including intrauterine growth restriction
(IUGR) or miscarriage [
43
], both of which are associated sequelae of CMV infection [
44
].
The dysregulation of SHH signaling in trophoblasts during CMV infection may therefore
be a potential mechanism for aberrant syncitialization, resulting in the adverse pregnancy
outcomes observed in cases of congenital CMV infection.
DYRKs are regulators of the SHH pathway, which is critical during embryogenesis and
fetal brain development [
16
,
24
,
25
,
45
]. We have previously shown DYRK proteins play a
critical role in CMV replication in fibroblasts, placental trophoblasts, and three-dimensional
placental ex vivo explant cultures [
11
,
31
]. We have also shown the upregulation of Ulk3,
Gli2, and Rb in CMV-infected HFF cells [11]. Here, we show CMV-induced dysregulation
of the critical placental and neuro-developmental pathways, DYRK (DYRK1A, DYRK1B)
and SHH (Gli2, Rb, Ulk3, and Shh), utilizing NHA cells to model the CMV infection of
human astrocytes. Astrocytes are the most abundant cell type in the brain. They have
critical functions including modulating the cerebral immune response, providing structural
support for neurons, modulating synaptic activity and plasticity, and regulating neuro-
transmitters [
46
–
49
]. Human astrocytes are susceptible to CMV infection [
50
,
51
]. Here, we
show that both AD169 and Merlin strain-infected NHA cells exhibited increased DYRK1A
and DYRK1B protein levels relative to mock. The DYRK1A gene, which is considerably
better characterized compared to DYRK1B, is significantly associated with the Down syn-
drome phenotype [
18
]. The over-expression of DYRK1A is a suggested mechanism for
inducing the neurofibrillary degeneration in Down syndrome individuals through tau
hyperphoshorylation [
52
]. In a study with 14 individuals with de novo heterozygous vari-
ants of DYRK1A, all were reported to have congenital microcephaly, intellectual disability,
developmental delay, and speech impairments, all of which are also sequelae of congenital
CMV infection [
18
]. This suggests a possible link between the CMV-induced dysregulation
of DYRK proteins, and the development of neurological sequelae. These data, combined
with the findings in this study, show that the CMV infection of NHAs dysregulates DYRK
protein levels and localization, suggesting a mechanism of CMV-induced fetal neural injury.
The differential re-localization of DYRK1A and DYRK1B observed in CMV-infected NHA
Viruses 2024,16, 918 10 of 13
cells, with similar re-localization patterns as were previously observed in placental models,
suggests CMV dysregulates the two protein kinases in separate processes during viral
replication [31].
The SHH pathway, which is modulated by DYRKs, is essential for fetal development,
facilitating blood–brain barrier integrity, cerebral immune quiescence mediated by astro-
cytes, and axon pathfinding [
24
–
28
,
53
]. The SHH signaling pathway plays a significant
role in astrocyte function, particularly the crosstalk between astrocytes and neurons [
53
].
In NHA cells, CMV infection induced an increase and re-localization in all SHH proteins
by 7dpi. Recently, it has been reported that astrocyte-specific SHH pathway activation is
necessary for synapse formation [
54
], and the selective disruption of SHH in astrocytes
substantially increases synapse numbers [
27
]. Synapse number and distribution is critically
regulated during brain development, and failure to establish an appropriate organiza-
tion of synapses is the hallmark of a number of neurodevelopmental disorders such as
autism [
27
,
55
]. Future experiments could benefit from the use of confocal microscopy
due to its ability to examine the three-dimensional aspect of culture samples. This would
facilitate further analysis of the CMV-induced co-localization patterns of DYRK and SHH
proteins. The dysregulation of SHH proteins in NHA cells reported in this study suggests a
possible mechanism for the development of neurodevelopmental disease.
This study revealed the dysregulation of cellular kinases (DYRKs) and proteins from
the key developmental pathway, SHH, in CMV-infected placental and brain models. The
CMV-induced dysregulation of protein levels and re-localization in the placental and
neural models is indicative of potential viral pathogenetic mechanisms. These data can
be further utilized to investigate CMV-induced adverse pregnancy outcomes and novel
therapeutic discovery in preventing the dysregulated signaling pathways of the placenta
and fetus [9,35,56].
Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/v16060918/s1.
Author Contributions: Conceptualization, E.E., W.D.R., S.T.H. and M.M.; methodology, E.E., W.D.R.
and S.T.H.; validation, E.E. and S.T.H.; formal analysis, E.E., S.T.H. and G.L.; investigation, E.E., S.T.H.,
G.L. and J.F.; resources, W.D.R., S.T.H. and M.M.; data curation, E.E., S.T.H., G.L. and J.F.; writing—
original draft, E.E.; writing—review and editing, E.E., S.T.H., W.D.R., M.S. and M.M.; visualization,
E.E., S.T.H. and W.D.R.; supervision, W.D.R., S.T.H., E.S. and M.M.; project administration, W.D.R.,
S.T.H. and E.E.; funding acquisition, W.D.R., S.T.H. and M.M. All authors have read and agreed to
the published version of the manuscript.
Funding: This study was supported by grants from the Thelma Pyne Postdoctoral Award (Hamilton—
PS64847) and the Australia–Germany Joint Research Cooperation Scheme (RG192195).
Informed Consent Statement: For the placental villous explant histocultures sections used in this
study, placentae were collected with informed consent from women undergoing elective Caesarean
section delivery who had a healthy pregnancy and were not in labour under the ethics approval
SESIAHS HREC 09/126.
Data Availability Statement: The authors confirm that the data supporting the findings of this study
are available within the article and Supplementary Materials.
Acknowledgments: We thank the University of NSW Sydney, and NSW Health Pathology for their
ongoing support. We would like to acknowledge Irina Voineagu for kindly providing NHA cells.
Conflicts of Interest: The authors declare no conflict of interest.
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