Download Host protein Snapin interacts with human cytomegalovirus pUL130

Host protein Snapin interacts with human cytomegalovirus pUL130
and affects viral DNA replication
GUILI WANG1,2 , GAOWEI REN1 , XIN CUI1 , ZHITAO LU1 , YANPIN MA1 , YING QI1 , YUJING HUANG1 ,
ZHONGYANG LIU1 , ZHENGRONG SUN1,* and QIANG RUAN1,*
1
Virus Laboratory, The Affiliated Shengjing Hospital, China Medical University, Shenyang 110004, China
2
Department of Pediatrics, The Third Affiliated Hospital of Liaoning Medical College,
Jinzhou 121001 Liaoning, China
*Corresponding author (Email, ZS – [email protected]; QR – [email protected])
The interplay between the host and Human cytomegalovirus (HCMV) plays a pivotal role in the outcome of an infection.
HCMV growth in endothelial and epithelial cells requires expression of viral proteins UL128, UL130, and UL131
proteins (UL128-131), of which UL130 is the largest gene and the only one that is not interrupted by introns. Mutation of
the C terminus of the UL130 protein causes reduced tropism of endothelial cells (EC). However, very few host factors
have been identified that interact with the UL130 protein. In this study, HCMV UL130 protein was shown to directly
interact with the human protein Snapin in human embryonic kidney HEK293 cells by Yeast two-hybrid screening, in vitro
glutathione S-transferase (GST) pull-down, and co-immunoprecipitation. Additionally, heterologous expression of
protein UL130 revealed co-localization with Snapin in the cell membrane and cytoplasm of HEK293 cells using
fluorescence confocal microscopy. Furthermore, decreasing the level of Snapin via specific small interfering RNAs
decreased the number of viral DNA copies and titer in HCMV-infected U373-S cells. Taken together, these results suggest
that Snapin, the pUL130 interacting protein, has a role in modulating HCMV DNA synthesis.
[Wang G, Ren G, Cui X, Lu Z, Ma Y, Qi Y, Huang Y, Liu Z, Sun Z and Ruan Q 2016 Host protein Snapin interacts with human cytomegalovirus
pUL130 and affects viral DNA replication. J. Biosci. 41 173–182] DOI 10.1007/s12038-016-9604-2
1.
Introduction
Human cytomegalovirus (HCMV) is ubiquitous throughout
populations worldwide and represents a significant public
health challenge in both developed and developing countries
(Mocarski et al. 2007). Severe forms of the disease are most
commonly observed when immune systems have been compromised by infection (e.g. HIV/AIDS) or immunosuppressive therapy (e.g. in transplant recipients). HCMV is also the
leading viral cause of congenital abnormalities and mental
retardation in newborns.
Keywords.
HCMV can infect a wide range of cell types in vivo
(Sinzger et al. 1996; Kahl et al. 2000; Riegler et al. 2000;
Bissinger et al. 2002) and establish lytic, persistent, and
latent infections in many different cells (Wills et al. 2005;
Britt 2008). HCMV utilizes two different pathways for host
cell entry. Infection of fibroblasts occurs by direct fusion of
the virion envelope with the plasma membrane, and this
requires glycoproteins gB and gH/gL. In contrast, in epithelial and endothelial cells (EC), membrane fusion takes place
in vesicles following internalization by endocytosis or
micropinocytosis and requires an additional complex
DNA replication; HCMV; Snapin; UL130
Abbreviations used: Co-IP, Co-immunoprecipitation; HCMV, Human cytomegalovirus; ORF, open reading frame; PCR, polymerase
chain reaction; siRNAs, small interfering RNAs
http://www.ias.ac.in/jbiosci
Published online: 7 April 2016
J. Biosci. 41(2), June 2016, 173–182 * Indian Academy of Sciences
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composed of gH, gL, and UL128, UL130, and UL131
(UL128-131) (Ryckman et al. 2006; Sinzger 2008; Haspot
et al. 2012; Wussow et al. 2014). Genes encoding UL128131 are transcribed with the 3′ coterminal at late kinetics
(Akter et al. 2003; Hahn et al. 2004), although their mRNA
initiation points may be different (Sun et al. 2010). Their
proteins are produced when HCMV replication is at the stage
of virion assembly and release (Akter et al. 2003; Hahn et al.
2004). The pUL128-131 is required for efficient infection
and cell-to-cell spread in several cell types, including monocytes, epithelial, endothelial, and myeloid cells (Revello and
Gerna 2010; Straschewski et al. 2011; Schuessler et al.
2012; Nogalski et al. 2013), either by binding to cell surface
receptors or promoting nuclear translocation of virions
(Sinzger et al. 2000; Ryckman et al. 2008a; Sinzger 2008).
However, pUL128-131 restricts either cell-to-cell transmission or virus production in fibroblast cultures (Stanton et al.
2010). Further investigation into the interactions between
virus proteins and host cell proteins will yield important
information regarding the process of viral entry and replication, which is critical for developing new strategies and
novel compounds for the treatment and prevention of
HCMV infection.
Of the genes in the pUL128-131, the UL130 is centrally
located, is the largest gene, and is the only one that is not
interrupted by introns (Akter et al. 2003; Hahn et al. 2004).
pUL130 is a luminal glycoprotein that is inefficiently secreted from infected cells but is incorporated into the virion
envelope in a Golgi-matured form (Patrone et al. 2005).
The C terminus of pUL130 serves an important function
for infection of endothelial cells by HCMV, and mutation
of the C-terminal of pUL130 causes a reduction of EC
tropism (Schuessler et al. 2010). However, few host factors
that interact with pUL130 have been identified to date.
In this study, the human protein Snapin was identified as
directly interacting with HCMV pUL130. In addition, the
viral DNA level was found to increase in HCMV-infected
U373 cells over-expressing Snapin and decrease in cells
treated with Snapin specific siRNA. These results suggest
that Snapin, the pUL130 interacting protein, has a role in
modulating HCMV DNA synthesis.
2.
Material and methods
2.1
Cells and viruses
Human embryonic lung fibroblast MRC-5 cells, astrocytoma
U373MG cells, and human embryonic kidney HEK293 cells
were maintained in Dulbecco’s modified Eagle medium
(DMEM) supplemented with 10% fetal bovine serum at
37°C in 5% CO2. HCMV clinical strain Han was at ages of
<5 months and isolated from urine samples of neonates
J. Biosci. 41(2), June 2016
admitted to the Pediatrics Department, Affiliated Shengjing
Hospital of China Medical University. The virus was inoculated into MRC-5 cells maintained in minimal essential
medium (MEM) supplemented with 2% fetal calf serum
(HyClone, Logan UT, USA) and penicillin-streptomycin in
an incubator at 37°C, 5% CO2.
2.2
Yeast two-hybrid screening
The coding sequence of HCMV UL130 was amplified by
polymerase chain reaction (PCR) using HCMV Han strain
DNA (Genbank No: GQ981646) as a template with primers
5′-CCGGAATTCTTGTCGACCCTGCGGCTTCTGC
TTCGTCAC-3′ and 5′-CGCGGATCCGGTACCTCAAA
CGATGAGATTGGGATG -3′. After digestion, the UL130
sequence was cloned into the pGBKT7 vector (Clontech,
Mountain View, CA, USA) between the EcoRI and BamHI
sites downstream of the c-Myc coding sequence, resulting in
pGBKT7-UL130. The inserted sequence of the pGBKT7UL130 was confirmed by sequencing (Invitrogen
Biotechnology Co., Ltd., Shanghai, China).
Yeast two-hybrid experiments were performed according
to the manufacturer's instructions (Matchmaker GAL4 TwoHybrid System 3, Clontech, USA). Briefly, Saccharomyces
cerevisiae strain AH109 was co-transformed with pGBKT7UL130 and a human fetus brain cDNA library, which was
cloned into pACT2 (pACT2-cDNA; Clontech Laboratories,
Inc.). Positive colonies were selected on synthetic dropout
(SD) medium lacking tryptophan, leucine, adenine, and histidine (SD-minus Trp/Leu/Ade/His (Quadruple dropouts,
QDO)) and tested for β-galactosidase activity by a colonylift filter assay as described previously (To et al. 2011).
Plasmids containing sequences encoding the interaction partners of UL130 (designated as pACT2-cDNA) were extracted
from the positive colonies and rescued by electrotransformation into competent Escherichia coli DH5α.
Each pACT2-cDNA plasmid rescued from E. coli was cotransformed into yeast together with the pGBKT7-UL130 or
the pGBKT7 empty vector. The transformed cells were
plated on QDO plates and subjected to a β-galactosidase
activity test. Human gene sequences in pACT2-cDNA from
blue yeast colonies were identified using the sequencing
primer 5′-AATACCACTACAATGGAT-3′, and analysed
by the BLAST network server at the National Center for
Biotechnology Information (http://www.ncbi.nlm.gov/blast).
2.3
Glutathione S-transferase (GST) pull-down assay
The coding sequence of one of the HCMV UL130 candidate
interacting proteins, Snapin, was obtained from a selected
cDNA clone (pACT2-Snapin) and GST-tagged by cloning
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into the pGEX-4T-2 vector between the EcoRI and SamI
sites, yielding pGEX-4T2-Snapin.
A GST-pull-down experiment was performed according
to the manufacturer's instructions (MagneGST™ Pull-Down
System, Promega, Madison, WI, USA). Briefly, c-Myclabelled UL130 protein (c-Myc-UL130) was expressed from
the pGBKT7-UL130 in a TNT (Quick coupled transcription/
translation reaction) T7 Quick Reaction (McMahon and
Anders 2002). GST-labelled Snapin protein (GST-Snapin)
was expressed in BL21 (DE3) cells transfected with the
pGEX-4T2-Snapin and induced with isopropyl β-D-1-thiogalactopyranoside (IPTG). As a bait protein, 20 μL of GSTSnapin was incubated with 80 μL of c-Myc-UL130 at room
temperature for 1.5 h on a rotating platform. Then, the
reaction products were incubated with MagneGST particles
for 30 min. After washing three times with buffer, the bound
proteins on the MagneGST particles were eluted with elution
buffer and solubilized in 2× SDS sample buffer. The recovered proteins were subjected to electrophoresis on a 12%
SDS-polyacrylamide gel, and the separated proteins were
transferred electrically onto nitrocellulose membranes
(Sigma–Aldrich, St. Louis, MO, USA). Western blot analyses were performed using mouse anti-c-Myc or goat antiGST monoclonal antibodies (Pierce, Rockford, IL, USA)
and corresponding peroxidase-conjugated secondary antibodies. The membranes were then incubated with chemiluminescent substrate provided in the Western
Chemiluminescent Substrate kit (Thermo Pierce, Rockford,
IL, USA). Signals were revealed using a Molecular Imager
ChemiDoc XRS System (Bio-Rad, Inc., Hercules, CA,
USA).
2.4
Co-immunoprecipitation (co-IP) analysis
The coding sequence of UL130 in the pGBKT7-UL130 was
obtained and cloned into the pCMV-HA vector between SfiI
and NotI sites, designated as pCMV-HA-UL130. The coding
sequence of Snapin in the pGEX-4T2-Snapin was also
obtained and cloned into SfiI and XhoI sites of pCMVmyc, designated as pCMV-myc-Snapin. All constructs were
confirmed by DNA sequencing (Invitrogen Biotechnology
Co., Ltd, Shanghai, China). HEK293 cells were cotransfected with pCMV-myc-Snapin and pCMV-HAUL130 using Lipofectamine 2000 (Invitrogen, Carlsbad,
CA, USA). The cell lysates were harvested at 48 h post
transfection. Co-immunoprecipitation experiments were performed using the ProFound Mammalian HA Tag IP/Co-IP
and c-Myc Tag IP/Co-IP kits following the manufacturer’s
protocols (Pierce). The Myc-Snapin and HA-UL130 were
detected by Western blot using mouse anti-Myc or rabbit
anti-HA antibodies (Clontech, USA), respectively, and
corresponding peroxidase-conjugated secondary antibodies.
2.5
Cellular localization assay
To express UL130 fusion protein labelled with an enhanced
green fluorescent protein (EGFP) tag, the UL130 coding
region was amplified from the HCMV Han strain using
primers 5′-CGGAATTCCCTGCGGCTTCTGCTTCGT-3′
and 5′-CGCGGATCCTCAAACGATGAGATTGGG-3′.
Then, the product was cloned into the pEGFP-C1 vector
(Clontech, USA) via the EcoRI and BamHI sites, yielding
pEGFP-C1-UL130. Similarly, the coding sequence of
Snapin in the Snapin-containing cDNA clone was amplified
by PCR with primers 5′-CGGAATTCCATGGCGG
G GG C T G GT T C C - 3′ and 5 ′ - C G CG GA T C C T T AT
TTGCCTGGGGAGCC-3′. The products were cloned into
the pDsRed-C1 vector (Clontech, USA) via the EcoRI and
BamHI sites, yielding pDsRed-C1-Snapin. All constructs
were confirmed by DNA sequencing (Invitrogen
Biotechnology Co., Ltd., Shanghai, China).
HEK293 cells (5×106) were seeded in 6-cm dishes 24 h
before transfection. At 75% confluence, the cells were transfected with 4 μg pEGFP-C1-UL130, 4 μg pDsRed-C1Snapin, or a mixture of 2 μg of pEGFP-C1-UL130 and
2 μg of pDsRed-C1-Snapin using Lipofectamine 2000
(Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. At 48 h post-transfection, the cells were
subjected to DAPI staining (Invitrogen, Carlsbad, CA, USA)
and expression of the fluorescently labelled EGFP-UL130
and DsRed-Snapin was analysed using a TCS SP2 Leica
laser scanning confocal microscope (Nikon Eclipse C1
Plus, Tokyo, Japan) at 488 nm or 543 nm, respectively.
2.6 Stable expression of Snapin and transfection of
Snapin-specific small interfering RNA (siRNA) into cells
To generate a U373MG cell line that stably expressed
Snapin (U373-S) and a control cell line, pDsRed-C1Snapin and the empty vector pDsRed-C1 were transfected
into U373 cells, respectively. At 48 h post-transfection,
neomycin was added to the culture medium at a final concentration of 600 μg/mL. Neomycin-resistant cells were
subsequently selected in the presence of neomycin for 2
weeks. Total protein was extracted from the cells and equal
amounts of the protein were subjected independently to
Western blot analysis. The expression level of Snapin in the
selected cell clones was determined by Western blot using a
mouse anti-Snapin antibody (Abcam, Hong Kong, China) and
goat anti-mouse IgG antibody conjugated with horseradish
peroxidase (Vector Laboratories, Burlingame, CA, USA) as
following. As an internal control, the expression level of
cellular glyceraldehyde -3-phosphate dehydrogenase
(GAPDH) in the cells was detected using a mouse antiGAPDH antibody and goat anti-mouse antibody conjugated
with horseradish peroxidase (Vector Laboratories).
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Guili Wang et al.
U373-S cells (1×105) were seeded in 12-well plates and
transfected with Silencer® Select Pre-Designed Snapin
siRNAs (Ambion, Grand Island, NY, USA) or control
siRNA (C-siRNA) molecules (Santa Cruz Biotechnology,
Santa Cruz, CA, USA). The antisense sequences of the
Snapin specific siRNAs were: 5′-UUAGCCGUCUCAGU
CGUUCct-3′ (S-siRNA-1) and 5′-UACGUGAGAGUC
GAGCUGCtg-3′ (S-siRNA-2). For each well, 0.5 μl of 10
nM siRNA and 3 μL of Lipofectamine (Invitrogen,
Carlsbad, CA, USA) were diluted separately in 100 μL of
Opti-MEM (Invitrogen, Carlsbad, CA, USA). After 5 min of
incubation at room temperature, both solutions were mixed
together. Twenty min later, the mixture was added to the
cells. At 10 h post-transfection, siRNA-containing medium
was removed. The cells were washed and incubated with
complete DMEM supplemented with 10% fetal bovine serum. At 10 h after the first round of transfection, the
transfection was repeated. The transfected cells, together
with U373MG (U373), U373-C (U373-C), and U373-S
(U373-S) cells that were cultured in parallel, were infected
with the HCMV Han strain at a multiplicity of infection
(MOI) of 1 at 24 h after the second round of transfection.
Total protein was extracted from the infected cells at 72 h
post infection. The level of Snapin in the infected cells was
determined by Western blot as described above. The experiments were repeated three times.
2.7
Viral growth and quantitative PCR analysis of
viral infection
To assay viral growth, we infected cells (n=1×105) with
HCMV at an MOI of 1. We collected the cells and medium
at 5 days postinfection and prepared viral stocks. We determined the titers of the viral stocks by infecting 1×105 human
foreskin fibroblasts and counting the number of plaques 10–
14 days after infection. The values obtained were averages
from triplicate experiments. Quantitative PCR (qPCR) analysis of viral DNA was conducted as follows. Briefly, DNA
was isolated from the HCMV-infected cells using the
TIANamp Genomic DNA Kit (Tiangen Biotech Co. Ltd.,
Beijing, China) according to the manufacturer’s instructions.
Viral DNA was amplified and quantified using HCMV
UL83-specific primers (5′-GTCAGCGTTCGTGTTTCCCA
-3′ and 5′-GGGACACAACACCGTAA AGC-3′) and the
SYBR Green PCR Master Mix kit (Applied Biosystems,
USA) on an ABI Prism 7300 Sequence Detection System
(Applied Biosystems, USA). The number of viral DNA
copies was normalized to that of β-actin detected in the same
sample using primers 5′-CGGAACCGCTCATTGCC-3′ and
5′-ACCCACACTGTG CCCATCTA-3′. The amplification
conditions were 95°C for 10 min and 40 cycles of 95°C for
15 s and 60°C for 1 min.
J. Biosci. 41(2), June 2016
The relative quantity of viral DNA copies was calculated
using a modified comparative CT method (2−ΔΔCT), in
which CT was defined as the cycle number at which fluorescence reached a set threshold value. The difference in the
CT value between the target gene and the corresponding
internal control β-actin gene, ΔCT (CTgene – CTβ-Actin),
was calculated. Then, the change in ΔCT of the treated
group relative to the control group, ΔΔCT (ΔCTtreated –
ΔCTcontrol), was computed. The relative number of HCMV
DNA copies was described using the equation 2−ΔΔCT. The
experiments were repeated three times.
2.8
Statistics
Data are shown as mean±SD. Statistical significance was
determined by analysis of variance (ANOVA). A p-value
<0.05 was considered to be statistically significant.
3.
3.1
Results
Snapin was selected as a candidate binding protein of
HCMV UL130 by a yeast two-hybrid screen
To identify cellular factors that potentially interact with
HCMV pUL130, a yeast two-hybrid screen was carried out
by transforming S. cerevisiae AH109 with the pGBKT7UL130 and a cDNA library derived from human fetal brain.
Two of the identified constructs, Homo sapiens SNAPassociated protein (pACT2-Snapin), which contain the full
length coding sequence of Snapin, were consistently found
to interact with pUL130. The nucleotide sequence of
pACT2-Snapin was 99% identical to that of the human
Snapin sequence in NCBI (Genbank No: NM_012437.5)
(table 1).
3.2
Interaction between HCMV pUL130 and host Snapin
was identified by GST pull-down assay
To detect the binding ability between HCMV pUL130 and
host Snapin in vitro, GST-tagged Snapin was used as a bait
protein and c-Myc-tagged pUL130 was used as the prey
protein in GST pull-down experiments. The recovered products were detected with a mouse anti-c-Myc monoclonal
antibody and a goat anti-GST monoclonal antibody, respectively. As shown in figure 1, an approximate 50 kDa c-Myc
labelled UL130 protein was captured by an approximate
41 kDa GST-tagged Snapin protein stabilized on the
MagneGST particles. These results reveal that UL130 protein has the ability to interact with Snapin protein in vitro.
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Snapin interacts with HCMV pUL130
Table 1. Homologous genes interacted with HCMV UL130 protein were analysed by comparing the gene sequences with human genome
from Genebank
Homologous genes
Identical clone
Homology (%)
Homo sapiens DnaJ (Hsp40) homolog
Homo sapiens forkhead box G1(FOXG1)
Homo sapiens glycoprotein, synaptic2
Homo sapiens SNAP-associated protein
Homo sapiens chromosome 9 open reading
Homo sapiens chromosome 12 open reading
Homo sapiens similar to putative DNA dependent
ATPase and helicase
2
1
1
2
1
1
1
1
96
100
98
99
100
99
99
96
Note: The sequencing results of positive clones, which were screened by Yeast two-hybrid experiment between HCMV UL130 and a
human fetus brain cDNA library, were analysed by the BLAST network server at the National Center for Biotechnology Information (http://
www.ncbi.nlm.gov/blast). Seven homologous genes were obtained by comparing with human genome of Genbank.
3.3 Interaction between HCMV pUL130 and
host Snapin protein was identified by co-IP
To further determine whether the interaction between
pUL130 and Snapin occurs in vitro, a co-IP assay was
performed using proteins expressed from pCMV-MycSnapin and pCMV-HA-UL130 in co-transfected HEK293
cells. As shown in figure 2, both the c-Myc-tagged Snapin
and HA-tagged UL130 proteins were detected in the recovered products immunoprecipitated with either anti-c-Myc or
anti-HA antibodies. These results confirm the specific
Figure 1. Interaction between host Snapin and HCMV pUL130
protein was analysed by GST pull-down. GST-tagged Snapin was
used as the bait protein and c-Myc-tagged UL130 was used as the
prey protein. The recovered products were detected with either
mouse anti-c-Myc monoclonal antibody or goat anti-GST monoclonal antibody. The ‘GST pull-down’ shows the c-Myc-tagged
pUL130 and only GST control (lane 1) or GST-snapin (lane 2)
captured by the MagneGST particles; The ‘input’ shows the c-Myclabelled protein that was expressed from pGBKT7-UL130 in vitro
(lane 3) or the protein lysates of BL21(DE)3 transfected with GSTSnapin expressing plasmid pGEX-4T2-Snapin (lane 4).
interaction between the HCMV pUL130 and host Snapin
protein in human cells.
3.4
Co-localization of pUL130 and Snapin proteins
in human cells was identified by fluorescence
confocal microscopy
To determine whether HCMV pUL130 and Snapin proteins
localize within the same cellular compartment, HEK293
cells were transfected with pEGFP-UL130, pDsRedSnapin, or both plasmids. The fusion proteins of GFPUL130 (figure 3A) and DsRed-Snapin (figure 3B) were
mainly localized in the membrane and cytoplasm of
Figure 2. Interaction between c-Myc-tagged Snapin and HAtagged pUL130 was identified by co-immunoprecipitation.
HEK293 cells were co-transfected with plasmids pCMV-c-mycSnapin and pCMV-HA-UL130. Cell lysates were prepared at 48 h
post transfection. The input protein samples (lane 1) and samples
immunoprecipitated with either anti-c-myc (lane 2) or anti-HA
antibodies (lane 3) were separated on SDS-containing polyacrylamide gels and analysed with anti-c-myc and anti-HA antibodies,
respectively. Negative control was analysed simultaneously (lane
4). Both the c-Myc-tagged Snapin and HA-tagged UL130 protein
were detected in the recovered products.
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Guili Wang et al.
Figure 3. Localization of pUL130 and Snapin expressed in HEK293 cells. HEK293 cells were transiently transfected with pEGFP-UL130
(A), pDsRed-Snapin (B), or both (C). At 48 h post transfection, nuclear DNA was stained with DAPI (blue image). Localization of the
expressed proteins was observed by fluorescent confocal microscopy. ‘Merge’ represents the overlapping image of fluorescence from
expressed proteins and nuclear staining in the same field of view.
HEK293 cells and were expressed simultaneously
(figure 3C).
3.5
Effect of Snapin protein on HCMV DNA synthesis and
lytic infection
To study the effect of Snapin on HCMV DNA replication, a
stable Snapin-expressing cell line, U373-S, was constructed.
U373-S cells were then transfected with Snapin-specific
siRNA. U373-S cells and Snapin-specific siRNA-transfected
U373-S cells showed no obvious change in the cell growth
during compared to U373MG cells (data not shown), suggesting that changes in Snapin expression did not result in
J. Biosci. 41(2), June 2016
significant cytotoxicity. Western blot results showed that the
expression level of Snapin in U373-S cells was five-fold
higher than in the parental U373MG cells and cells transfected with the empty vector (U373-C). The levels of the
over-expressed and endogenous Snapin proteins in the SsiRNA-1 and S-siRNA-2 transfected cells collected at 72 h
post-HCMV infection were significantly reduced (75.59%
and 92.45%, respectively) compared with cells transfected
with control siRNA (figure 4A).
To determine whether changes in Snapin expression alter
HCMV DNA synthesis and lytic infection, the number of
viral DNA copies and titer was detected. At 72 h postinfection, an approximate 5-fold increase in the number of
viral DNA copies (UL83/β-Actin) was observed in the
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Snapin interacts with HCMV pUL130
Figure 4. Effects of Snapin on HCMV DNA synthesis were detected by real-time PCR. U373-S cells were transfected with Snapinspecific siRNAs (S-siRNA-2 or S-siRNA-1) or control siRNA (C-siRNA). The cells, together with U373MG (U373), U373-C (U373-C),
and U373-S (U373-S) cells were infected with HCMV Han strain at MOI of 1. (A) At 72 h post-infection, the level of Snapin protein was
detected by Western blot analysis using a mouse anti-Snapin antibody (Abcam). The expression of cellular glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) was used as the internal control. (B) The values of the relative levels of HCMV DNA and titers (mean±SD from
triplicate experiments). The relative level of HCMV DNA and titers are shown compared to the number of viral DNA copies in the parental
U373MG cells (U373).
Snapin-expressing U373-S cells compared with U373-C
cells (p=0.018) and the parental U373MG cells (p=0.033).
Moreover, the number of viral DNA copies was significantly
reduced in cells treated with anti-Snapin S-siRNA-1 molecules (84%, p=0.020) and anti-Snapin S-siRNA-2 molecules
(89%, p=0.028) compared with U373-C and parental
U373MG cells (figure 4B). At the same time, viral progeny
production in these cells was determined. Virus stocks were
prepared from the infected cultures (cells and culture medium together), and their titers were determined as similar with
viral DNA copies. The two Snapin specific siRNA molecules (S-siRNA-1 and S-siRNA-2) yielded similar effects on
Snapin expression and HCMV DNA replication and their
titer, indicating that these results were not due to off-targets
effects of the siRNAs. These findings suggest that the increase of Snapin expression in HCMV-infected cells benefits
HCMV DNA synthesis and vice verse.
4.
Discussion
Similar to other viral infections, HCMV is known to perturb
a number of host cell functions. The majority of these perturbations presumably optimize host cell functions and
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Guili Wang et al.
conditions to support viral persistence and productive viral
replication (Kalejta and Shenk 2002; Landolfo et al. 2003).
Despite the clear importance of UL128-131 in virion cell
tropism, the mechanisms that regulate their relative abundance during HCMV infection have for the most part
remained elusive. Our results have revealed that host protein
Snapin interacts with pUL130, as determined by two yeasthybrid screening, GST pull-down assay, and co-immunoprecipitation. Co-localization of these two proteins in both the
cytosol and the plasma membrane was confirmed by confocal microscopic analysis. These observations suggest that the
host factor Snapin may play an important role at the time of
HCMV entry into cells.
A few host factors have been identified that interact
with HCMV proteins and are involved in all stages of
the virus life cycle (Scholz et al. 2003). Host factors
may have important roles in viral entry, replication, budding, release, pathogenesis, and restricting cross-species
transmission. Snapin is small protein of 15 kDa and only
136 amino acids. Snapin is a ubiquitous cellular protein,
and it is located in both the cytosol and the plasma membrane in neuronal and non-neuronal cells (Ilardi et al.
1999; Vites et al. 2004; Ruder et al. 2005). In neuronal
cells, Snapin is a component of the SNARE (soluble Nethylmaleimide-sensitive fusion protein attachment protein
receptor) complex that is required for synaptic vesicle
docking and fusion (Buxton et al. 2003). Despite this fact,
Snapin interacts with a number of molecules to regulate
their function (Wang et al. 2009). In other types of cells,
Snapin interacts with components of the biogenesis of
lysosome-related organelles complex 1 (BLOC-1), which
is a ubiquitously expressed multi-subunit protein complex
required for normal biogenesis of specialized organelles of
the endosomal-lysosomal system, such as melanosomes
and platelet-dense granules (Starcevic and Dell'Angelica
2004; Ruder et al. 2005). HCMV transcriptionally regulates expression of some cellular soluble Nethylmaleimide-sensitive fusion protein attachment protein
receptors (SNAREs) genes whose products are involved in
membrane fusion events (Hertel and Mocarski 2004).
Given that SNARE proteins drive many membrane fusion
events within eukaryotic cells, it is likely that in infected
cells, HCMV exploits/manipulates these cellular proteins
to produce new virions. The export of gH/gL complexes
from the endoplasmic reticulum (ER) to the Golgi apparatus and cell surface has previously been shown to dramatically increase when UL128, UL130, and UL131 were coexpressed with gH/gL (Ryckman et al. 2008b). Snapin
also plays a role in cell vesicle transport and fusion and
has been implicated in the regulation of exocytosis and
endocytosis (Hertel and Mocarski 2004). Taken together,
this suggests that the interaction between Snapin and
HCMV UL130 may impact the transport and fusion of
J. Biosci. 41(2), June 2016
HCMV from the endoplasmic reticulum to the Golgi or
from the Golgi to the cell membrane.
In our study, the number of HCMV DNA copies and
synthesis in an HCMV-infected Snapin-expressing U373
cell line was significantly higher than that in normal
U373MG cells. In addition, decreased Snapin expression in
the Snapin-expressing U373 cell line via siRNA resulted in a
significant decrease in the number of viral DNA copies in
HCMV-infected Snapin-expressing U373 cells. These
results suggest that the increase of Snapin expression in
HCMV-infected cells benefits viral DNA replication synthesis and vice verse.
In contrast, Snapin over-expression in U373 cells has
previously been shown to down-modulate HCMV viral
DNA synthesis and lytic infection by interaction with viral
primase UL70 (Shen et al. 2011). This opposing observation
maybe because of the differences of using strains. The
HCMV Towne strain was used in the study (Shen et al.
2011), however, the HCMV clinical strain was used in our
study. According to our previous published article (Sun et al.
2009), the coding region of UL130 gene in clinical strains
is 645 bp in length, whereas that in the Towne strain is
690 bp. UL130 gene in Towne strain has a frameshift near
the 3′ end of the gene since double T insertion. It was
observed that a cysteine codon, which was conserved in
clinical strains, was substituted by serine codon at the 3′
end of UL130 in Towne strain. In addition, UL130 protein in
Towne strain contains an additional casein kinase II phosphorylation site and an N-myristoylation site, which were
absent in clinical strains and Merlin strain. The UL130
putative protein of clinical strains contains a putative chemokine domain from amino acids 46 to 120, although it lacks
two of the four cysteines that are strictly conserved in a
chemokine of this type.
U373MG is an epithelia-like cell line derived from neuronal tissues. It is highly permissive to HCMV replication
and has been widely used in HCMV infection studies
(Mocarski et al. 2007). In our study, Snapin was identified
as interacting with pUL130 and modulating HCMV
DNA replication in U373MG cells. Because of the necessary
roles of HCMV pUL128-131 in epithelial cell infection, it
could be speculated that Snapin may be involved in the
HCMV infection by interacting with pUL130, except for
the known mechanism (Patrone et al. 2005; Schuessler
et al. 2010). It is interesting to understand whether the
influence of pUL130, together with the other proteins of
pUL128-131, on HCMV infection or the regulation of
Snapin on HCMV DNA synthesis is mediated by their
interaction.
In conclusion, the present study demonstrated the interaction between HCMV pUL130 and Snapin and confirmed
the modulating role of Snapin on HCMV DNA replication.
Future studies are needed to elucidate the mechanism of the
Snapin interacts with HCMV pUL130
pUL130–Snapin interaction in regulating HCMV DNA
replication.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (81171580 and 81171581) and the
Outstanding Scientific Fund of Shengjing Hospital.
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MS received 01 August 2015; accepted 03 March 2016
Corresponding editor: SHAHID JAMEEL
J. Biosci. 41(2), June 2016