JGV Papers in Press. Published December 11, 2014 as doi:10.1099/jgv.0.000015
Journal of General Virology Heat shock cognate protein 70 isoform D is required for clathrin-dependent endocytosis of Japanese encephalitis virus in C6/36cells --Manuscript Draft-Manuscript Number:
JGV-D-14-00014R1
Full Title:
Heat shock cognate protein 70 isoform D is required for clathrin-dependent endocytosis of Japanese encephalitis virus in C6/36cells
Short Title:
Hsc70D in clathrin-dependent endocytosis of JEV
Article Type:
Standard
Section/Category:
Animal - Positive-strand RNA Viruses
Corresponding Author:
Wei-June Chen, Ph.D. Chang Gung University Kwei-San, Tao-Yuan, TAIWAN
First Author:
Ching-Kai Chuang, Ph.D.
Order of Authors:
Ching-Kai Chuang, Ph.D. Tsong-Han Yang, MS Tien-Huang Chen, Ph.D. Chao-Fu Yang, MS Wei-June Chen, Ph.D.
Abstract:
Japanese encephalitis virus (JEV), one of encephalitic flaviviruses, is naturally transmitted by mosquitoes. During the infection, JEV generally enters host cells via receptor-mediated clathrin-dependent endocytosis that requires the involvement of the 70 kDa heat shock protein (Hsp70). Heat shock cognate protein 70 (Hsc70) is one member of the Hsp70 family and is mainly constitutive; thus, it may be expressed at physiological condition. In C6/36 cells, Hsc70 is up-regulated in response to JEV infection. Since Hsc70 shows no relationship with viruses attaching to the cell surface, it probably does not serve as the receptor according to our results in the present study. In contrast, Hsc70 is evidently associated with virus penetration into the cell and resultant acidification of intracellular vesicles. It suggests that Hsc70 is highly involved in clathrin-mediated endocytosis, particularly at a late stage of viral entry into host cells. Furthermore, we found that Hsc70 is composed of at least three isoforms, including B, C, and D. Of them, the isoform D is the one that helps JEV to penetrate C6/36 cells via clathrin-mediated endocytosis. This study provides relevant evidence that sheds light on the regulatory mechanisms of JEV infection in host cells, especially on the process of clathrin-mediated endocytosis.
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Manuscript Including References (Word document) Click here to download Manuscript Including References (Word document): JGV 14 MS.doc
1 2
Short title: Hsc70D in clathrin-dependent endocytosis of JEV
3
Heat shock cognate protein 70 isoform D is required for clathrin-dependent endocytosis of Japanese encephalitis virus in C6/36cells
4 5 6 7 8 9 10 11 12 13 14 15 16
Ching-Kai Chuang1,#, ¶, Tsong-Han Yang1,#, Tien-Huang Chen1,§, Chao-Fu Yang1, and Wei-June Chen1,2,* 1
Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan 33332, Taiwan
2
Department of Public Health and Parasitology, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan 33332, Taiwan
17 18
# These authors contribute equally to this work.
19
¶ Current address: Department of Plant Pathology, University of Kentucky,
20 21 22 23 24 25 26 27 28 29 30 31 32 33
Lexington, KY 40546-0312, USA. § Current address: Research and Diagnostic Center, Centers for Disease Control, Ministry of Health and Welfare, Taipei 10050, Taiwan. * To whom correspondence should be addressed: [email protected].
Based on the sequences of Hsc70 reported from Aedes albopictus (C6/36 cells), three isoforms (B, C, and D) were identified in this study. Their nucleotide sequences has been deposited to the database in NCBI with accession numbers KJ701753, KJ701754, and KJ701755 for isoforms B, C, and D, respectively.
1
1
Abstract
2
Japanese encephalitis virus (JEV), one of encephalitic flaviviruses, is naturally
3
transmitted by mosquitoes. During the infection, JEV generally enters host cells via
4
receptor-mediated clathrin-dependent endocytosis that requires the involvement of the
5
70 kDa heat shock protein (Hsp70). Heat shock cognate protein 70 (Hsc70) is one
6
member of the Hsp70 family and is mainly constitutive; thus, it may be expressed at
7
physiological condition. In C6/36 cells, Hsc70 is up-regulated in response to JEV
8
infection. Since Hsc70 shows no relationship with viruses attaching to the cell surface,
9
it probably does not serve as the receptor according to our results in the present study.
10
In contrast, Hsc70 is evidently associated with virus penetration into the cell and
11
resultant acidification of intracellular vesicles. It suggests that Hsc70 is highly
12
involved in clathrin-mediated endocytosis, particularly at a late stage of viral entry
13
into host cells. Furthermore, we found that Hsc70 is composed of at least three
14
isoforms, including B, C, and D. Of them, the isoform D is the one that helps JEV to
15
penetrate C6/36 cells via clathrin-mediated endocytosis. This study provides relevant
16
evidence that sheds light on the regulatory mechanisms of JEV infection in host cells,
17
especially on the process of clathrin-mediated endocytosis.
18 19
2
1
INTRODUCTION
2
Endocytosis is a physiological process by which molecules translocate from outside
3
into the intracellular environment of eukaryotic cells (Conner &Schmid, 2003). In
4
most cases, the formation of clathrin-coated vesicles (CCVs) is necessary for
5
endocytosis, specifically clathrin-mediated endocytosis, serving as the endocytic
6
portal into cells (McMahon & Boucrot, 2011). Generally, molecules are packaged into
7
vesicles with the aid of a clathrin coat, followed by a sequence of ligand attachment,
8
invagination, bud formation, and fission (Ungewickell & Hinrichsen, 2007). The
9
efficiency of this process is increased when receptors for extracellular molecules to be
10
internalized are located at the clathrin-coated pit (CCP), which has been implicated as
11
the major site of internalization for receptor-bound ligands (Anderson, 1989) from
12
which free CCVs are released into the cytosol (Rappoport et al., 2004). In turn, the
13
receptor-mediated endocytosis of extracellular molecules requires the involvement of
14
clathrin that is highly dynamic and can be recycled after each round of endocytosis
15
(McMahon & Boucrot, 2011; Merrifield et al., 2005).
16
The infection of host cells via clathrin-mediated endocytosis has been extensively
17
utilized by a variety of viruses including vesicular stomatitis virus (VSV) (Sun et al.,
18
2005), hepatitis C virus (HCV) (Blanchare et al., 2006), hepatitis B virus (HBV)
19
(Cooper & Shaul, 2006), respiratory syncytial virus (RSV) (Gutiérrez-Ortega et al.,
20
2008), human immunodeficiency virus (HIV) (Daecke et al., 2005), and Ebola virus
21
(Bhattacharyya et al., 2010). Orthobunyavirus, one of the encephalitic arboviruses, is
22
also reported to infect mammalian host cells via clathrin-mediated endocytosis as its
23
primary mechanism (Hollidge et al., 2012). This mechanism for infecting host cells is
24
also implemented by various mosquito-borne flaviviruses (Acosta et al., 2008; Chu &
25
Ng, 2004). Among them, Japanese encephalitis virus (JEV) which infects host (either
26
mammalian or mosquito) cells via receptor-mediated endocytosis (Andoh et al., 1998; 3
1
Nawa, 1998) has also been confirmed to be clathrin-dependent (Chai et al., 2013;
2
Yang et al., 2013), except for certain unusual cases (Kalia et al., 2013).
3
Although the mechanism is sophisticated, clathrin-mediated endocytosis requires
4
the involvement of the 70 kDa heat shock protein (Hsp70) (Chang et al., 2002). The
5
Hsp70 family includes Hsp70 and Hsc70, which are molecular chaperones with
6
critical roles in protein folding and trafficking in all eukaryotic cells (Takayama et al.,
7
1999). They may also help to protect cells from a wide array of stresses like elevated
8
temperature and infection (Morano, 2007). Within the Hsp70 family, Hsp70 genes are
9
usually inducible whereas Hsc70 genes are mainly constitutive and usually expressed
10
in the absence of stress (Yamashida et al., 2004). Hsc70s are highly conserved in
11
structure from prokaryotes to eukaryotes (Boorstein et al., 1994). In general, Hsc70
12
can be involved in folding/unfolding and assembly/disassembly of macromolecular
13
structures (Elefant and Palter, 1999; Jiang et al., 2005) as well as anti-apoptotic
14
activity (Powers et al., 2008). Thus, Hsc70 expression is usually beneficial for cells
15
by resisting injury under conditions of minimal stress (Sens et al., 1997).
16
Among viruses using clathrin-dependent endocytosis as the entry mechanism for
17
infecting host cells, the influenza virus M1 protein is known to bind Hsc70 and is
18
required for the efficient production of the virus in infected cells (Watanabe et al.,
19
2006). Hsc70 also plays a role in outer capsid disassembly during cell entry by
20
reoviruses (Ivanovic et al., 2007), further indicating that HSc70 helps a virus infect
21
host cells and successfully replicate via interaction(s) with viral protein(s). In
22
rotaviruses, the Hsc70 protein interacts with the viral VP5 at a post-attachment step
23
during viral entry (Zárate et al., 2003). Even in mosquito cells, a 74 kDa protein likely
24
to be Hsc70 that binds JEV was identified from uninfected C6/36 cells based on an
25
analysis with a co-immunoprecipitation (Co-IP) assay and mass spectrometry (Re et
26
al., 2007). Meanwhile, Hsc70 is also one of the proteins interacting with DENV-2 in 4
1
the membrane fraction, suggesting that it may function to concentrate viral particles
2
and allow efficient interactions between primary receptors and the virus on the cell
3
surface (Paingankar et al., 2010). Accordingly, it was proposed that Hsc70 may play a
4
role as a penetration receptor that mediates JEV entry into C6/36 cells (Re et al.,
5
2007). Nonetheless, the function of Hsc70 is still ambiguous and remains to be
6
clarified for the detail.
7
Based on an EST library previously established in our lab (Chen et al., 2011), a
8
significantly higher number of Hsc70 gene copies were found in DENV-2-infected
9
C6/36 cells. In addition, Hsc70 was further confirmed to be responsive to JEV
10
infection in C6/36 cells according to a follow-up preliminary study. Therefore, the
11
purpose of this study was to investigate in detail how significant this protein is in JEV
12
infection of C6/36 cells. More importantly, we further identified specific Hsc70
13
isoforms in this study and elucidated their different roles in the process of JEV
14
infection to mosquito cells.
15
5
1
RESULTS
2
Expression of Hsc70 is required for JEV infection of C6/36 cells
3
Using the semi-quantitative RT-PCR Hsc70 was repeatedly shown to be upregulated
4
at 12 hpi at the mRNA level in C6/36 cells infected by JEV (MOI = 5) (Fig. 1a).
5
Meanwhile, quantitative RT-PCR revealed there was an increase of some 1.6 folds
6
(Fig. 1b). According to the result of Western blot, expression of Hsc70 was also
7
slightly increased at protein level (Fig. 1c). Through RNA interference (RNAi)
8
derived from a microRNA-based system, ca. 21% of the Hsc expression was revealed
9
by qRT-PCR compared with that detected from cells without knockdown of HSC70
10
(Fig. 2a). The lower level of the Hsc70 was also shown in Hsc-knockdown C6/36
11
cells according to the result from semi-quantitative RT-PCR (Fig. 2b). In the
12
meantime, decreased expression of the Hsc70 protein can clearly be seen in the result
13
from the Western blot analysis in Hsc70-knockdown cells compared to controls (Fig.
14
2c). Upon gene knockdown in C6/36 cells, intracellular viral RNA assessed by
15
RT-PCR was clearly detected as early as 6 hpi in cells without Hsc70 knockdown but
16
not until 12 hpi in cells with Hsc70 knockdown (Fig. 3a). Likely, the virus entered the
17
cells earlier when Hsc70 was not knocked down, implying this chaperone may be
18
essential for JEV entry into mosquito cells. The profile of viral infection in such cells
19
was further monitored by IFA with antibodies against the viral NS3 protein during the
20
infection, showing a large proportion of cells were eventually infected by JEV at 6 hpi
21
in C6/36 cells without the knockdown of Hsc70 (C6/36 or miN). However, the viral
22
NS3 protein was evidently reduced in Hsc70 knockdown cells (Fig. 3b), indicating the
23
necessity of Hsc70 for virus entry. Collectively, Hsc70 could be a crucial advantage
24
allowing JEV propagation in C6/36 cells.
25 26
Hsc70 affects JEV infection in C6/36 cells at the stage of entry 6
1
In C6/36 cells infected by JEV, virus titers in cells with Hsc70 knockdown were
2
evidently lower than in controls (C6/36 and miN) at 24, 48, and 72 hpi, respectively
3
(Fig. 4a). It revealed that Hsc70 plays a role in determining viral growth in C6/36
4
cells although other factors may also be involved as limited reduction of the virus titer
5
was shown in the present result. To investigate the role of Hsc70 on specific stages
6
during JEV infection, C6/36 cells with (miHsc70) or without (C6/36 or miN)
7
knockdown of Hsc70 were inoculated with the virus at MOI = 50 and incubated for 1
8
h at 4 ℃ or 28 ℃, respectively. Using IFA to detect the viral E protein,
9
approximately equal amounts of the viral antigen were observed to bind on the
10
surfaces of cells either with or without Hsc knockdown at 4 ℃ (Fig. 4b). In contrast,
11
less viral E antigen appeared in cell cytoplasm with Hsc70 knockdown when those
12
cells were incubated at 28 ℃ (Fig. 4b) compared to controls. Measurement by
13
qRT-PCR revealed that the amount of intracellular viral RNA was significantly
14
decreased to a level of ~60% in Hsc70-knockdown cells when incubated at 28 ℃ (p
15
< 0.05, Student’s t-test), however, it did not change evidently at 4 ℃ (Fig. 4c). It
16
suggests that JEV penetration into, but not binding onto, C6/36 cells is highly
17
regulated by Hsc70.
18 19
Hsc70 is involved in the endocytosis of JEV by C6/36 cells
20
Texas red conjugated with avidin was used as an endocytosis tracer to confirm the
21
role of Hsc70 in the endocytosis of C6/36 cells either knockdown of Hsc70 or not
22
when incubated at 28 ℃. Results show that tracer-containing vesicles evidently
23
appeared in the cells of controls (C6/36 and miN groups) after exposure to the tracer
24
for 1 min compared to Hsc70-knockdown cells (Fig. 5a). This suggests that tracer
25
internalization during the entry stage was closely associated with the involvement of
26
Hsc70. In C6/36 cells inoculated with JEV to replace the treatment with the tracer, 7
1
vesicles formed in response to viral inoculation were easily detected by the assay for
2
cellular uptake of the endocytic tracer at the initial step, i.e., 0 min post-inoculation, in
3
all groups. However, vesicles were not shown in control group cells but remained as
4
vesicle-like structures in cells with Hsc70 knockdown at 30 min post-inoculation (Fig.
5
5b). This implies that Hsc70 is important for viral particles up-taken by the cell to
6
form and particularly to escape the virus-containing vesicles or endosomes that are
7
usually formed in response to infection by the virus.
8 9
Formation of acidified endosomes in C6/36 cells is dependent on Hsc70
10
The acidification of virus-containing endosomes is an essential step in virus
11
un-coating for releasing the viral genome into the cytoplasm for replication. This can
12
be demonstrated by staining using acridine orange (AO), a fluorescent dye, that shows
13
orange in color for the newly-formed acidified vesicles or endosomes after
14
endocytosis. The results showed that acidified vesicles were prevalent in control
15
group cells (C6/36 and miN), whereas they were dramatically reduced to a relatively
16
low level in cells with Hsc70 knockdown (Fig. 6), suggesting that efficient
17
endocytosis of JEV in C6/36 cells is highly regulated by Hsc70.
18 19
Hsc70 is associated with clathrin-mediated endocytosis of JEV in C6/36
20
cells
21
Clathrin-coated vesicles (CCVs) are usually formed during the process of
22
clathrin-mediated endocytosis, which actually occurs in the JEV infection of C6/36
23
cells. To further prove the role of Hsc70 in this phenomenon, the clathrin light chain
24
(a component of the clathrin triskeleton) fused with RFP (CLC-RFP) was
25
overexpressed in C6/36 cells for monitoring the formation and localization of CCVs.
26
When C6/36 cells were inoculated with JEV (MOI = 50) and incubated at 28 ℃ for 8
1
5 and 30 min, respectively. The results show that CCVs tagged with RFP (CCVs-RFP)
2
were evidently decreased in control group cells at 30 min post-infection compared to
3
those cells infected for 5 min whereas the change was insignificant in
4
Hsc70-knockdown cells (Fig. 7). It indicates that clathrin is normally dissociated from
5
CCVs for recycling, likely back to the plasma membrane in C6/36 cells infected by
6
JEV. It creates an advantage for viral RNA to release from the formed CCVs. In
7
contrast, CCVs may be retained and gradually accumulated in the cytoplasm,
8
blocking the formation of more endosomes containing ingested viral particles due to
9
the knockdown of Hsc70 in C6/36 cells.
10 11
JEV infection to C6/36 cells is decided by the specific Hsc70 isoform
12
Based on the sequences of Hsc70 from Aedes albopictus (C6/36 cells), three isoforms
13
(B, C, and D) were identified in this study. Their nucleotide sequences were retrieved
14
from the database in NCBI by accession numbers KJ701753, KJ701754, and
15
KJ701755 for isoforms B, C, and D, respectively. As shown in the results of RT-PCR,
16
all Hsc70 isoforms, particularly D, were elevated in C6/36 cells infected by JEV
17
infection at MOI = 5 for 12 h (Fig. 8). Using dsRNA to knock down individual Hsc70
18
isoforms (Supplementary Figure S1), the expression of viral RNA within the cell was
19
apparently delayed and not appeared until 12 hpi in cells with knockdown of Hsc70
20
isoform D (Fig. 9). Since the efficiency of JEV binding to C6/36 cells did not show
21
significant difference (Fig. 10a), there seems to be different effects among the Hsc70
22
isoforms in C6/36 cells in response to JEV infection. In contrast, virus penetration
23
efficiency was significantly reduced to
Journal of General Virology Heat shock cognate protein 70 isoform D is required for clathrin-dependent endocytosis of Japanese encephalitis virus in C6/36cells --Manuscript Draft-Manuscript Number:
JGV-D-14-00014R1
Full Title:
Heat shock cognate protein 70 isoform D is required for clathrin-dependent endocytosis of Japanese encephalitis virus in C6/36cells
Short Title:
Hsc70D in clathrin-dependent endocytosis of JEV
Article Type:
Standard
Section/Category:
Animal - Positive-strand RNA Viruses
Corresponding Author:
Wei-June Chen, Ph.D. Chang Gung University Kwei-San, Tao-Yuan, TAIWAN
First Author:
Ching-Kai Chuang, Ph.D.
Order of Authors:
Ching-Kai Chuang, Ph.D. Tsong-Han Yang, MS Tien-Huang Chen, Ph.D. Chao-Fu Yang, MS Wei-June Chen, Ph.D.
Abstract:
Japanese encephalitis virus (JEV), one of encephalitic flaviviruses, is naturally transmitted by mosquitoes. During the infection, JEV generally enters host cells via receptor-mediated clathrin-dependent endocytosis that requires the involvement of the 70 kDa heat shock protein (Hsp70). Heat shock cognate protein 70 (Hsc70) is one member of the Hsp70 family and is mainly constitutive; thus, it may be expressed at physiological condition. In C6/36 cells, Hsc70 is up-regulated in response to JEV infection. Since Hsc70 shows no relationship with viruses attaching to the cell surface, it probably does not serve as the receptor according to our results in the present study. In contrast, Hsc70 is evidently associated with virus penetration into the cell and resultant acidification of intracellular vesicles. It suggests that Hsc70 is highly involved in clathrin-mediated endocytosis, particularly at a late stage of viral entry into host cells. Furthermore, we found that Hsc70 is composed of at least three isoforms, including B, C, and D. Of them, the isoform D is the one that helps JEV to penetrate C6/36 cells via clathrin-mediated endocytosis. This study provides relevant evidence that sheds light on the regulatory mechanisms of JEV infection in host cells, especially on the process of clathrin-mediated endocytosis.
Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation
Manuscript Including References (Word document) Click here to download Manuscript Including References (Word document): JGV 14 MS.doc
1 2
Short title: Hsc70D in clathrin-dependent endocytosis of JEV
3
Heat shock cognate protein 70 isoform D is required for clathrin-dependent endocytosis of Japanese encephalitis virus in C6/36cells
4 5 6 7 8 9 10 11 12 13 14 15 16
Ching-Kai Chuang1,#, ¶, Tsong-Han Yang1,#, Tien-Huang Chen1,§, Chao-Fu Yang1, and Wei-June Chen1,2,* 1
Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan 33332, Taiwan
2
Department of Public Health and Parasitology, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan 33332, Taiwan
17 18
# These authors contribute equally to this work.
19
¶ Current address: Department of Plant Pathology, University of Kentucky,
20 21 22 23 24 25 26 27 28 29 30 31 32 33
Lexington, KY 40546-0312, USA. § Current address: Research and Diagnostic Center, Centers for Disease Control, Ministry of Health and Welfare, Taipei 10050, Taiwan. * To whom correspondence should be addressed: [email protected].
Based on the sequences of Hsc70 reported from Aedes albopictus (C6/36 cells), three isoforms (B, C, and D) were identified in this study. Their nucleotide sequences has been deposited to the database in NCBI with accession numbers KJ701753, KJ701754, and KJ701755 for isoforms B, C, and D, respectively.
1
1
Abstract
2
Japanese encephalitis virus (JEV), one of encephalitic flaviviruses, is naturally
3
transmitted by mosquitoes. During the infection, JEV generally enters host cells via
4
receptor-mediated clathrin-dependent endocytosis that requires the involvement of the
5
70 kDa heat shock protein (Hsp70). Heat shock cognate protein 70 (Hsc70) is one
6
member of the Hsp70 family and is mainly constitutive; thus, it may be expressed at
7
physiological condition. In C6/36 cells, Hsc70 is up-regulated in response to JEV
8
infection. Since Hsc70 shows no relationship with viruses attaching to the cell surface,
9
it probably does not serve as the receptor according to our results in the present study.
10
In contrast, Hsc70 is evidently associated with virus penetration into the cell and
11
resultant acidification of intracellular vesicles. It suggests that Hsc70 is highly
12
involved in clathrin-mediated endocytosis, particularly at a late stage of viral entry
13
into host cells. Furthermore, we found that Hsc70 is composed of at least three
14
isoforms, including B, C, and D. Of them, the isoform D is the one that helps JEV to
15
penetrate C6/36 cells via clathrin-mediated endocytosis. This study provides relevant
16
evidence that sheds light on the regulatory mechanisms of JEV infection in host cells,
17
especially on the process of clathrin-mediated endocytosis.
18 19
2
1
INTRODUCTION
2
Endocytosis is a physiological process by which molecules translocate from outside
3
into the intracellular environment of eukaryotic cells (Conner &Schmid, 2003). In
4
most cases, the formation of clathrin-coated vesicles (CCVs) is necessary for
5
endocytosis, specifically clathrin-mediated endocytosis, serving as the endocytic
6
portal into cells (McMahon & Boucrot, 2011). Generally, molecules are packaged into
7
vesicles with the aid of a clathrin coat, followed by a sequence of ligand attachment,
8
invagination, bud formation, and fission (Ungewickell & Hinrichsen, 2007). The
9
efficiency of this process is increased when receptors for extracellular molecules to be
10
internalized are located at the clathrin-coated pit (CCP), which has been implicated as
11
the major site of internalization for receptor-bound ligands (Anderson, 1989) from
12
which free CCVs are released into the cytosol (Rappoport et al., 2004). In turn, the
13
receptor-mediated endocytosis of extracellular molecules requires the involvement of
14
clathrin that is highly dynamic and can be recycled after each round of endocytosis
15
(McMahon & Boucrot, 2011; Merrifield et al., 2005).
16
The infection of host cells via clathrin-mediated endocytosis has been extensively
17
utilized by a variety of viruses including vesicular stomatitis virus (VSV) (Sun et al.,
18
2005), hepatitis C virus (HCV) (Blanchare et al., 2006), hepatitis B virus (HBV)
19
(Cooper & Shaul, 2006), respiratory syncytial virus (RSV) (Gutiérrez-Ortega et al.,
20
2008), human immunodeficiency virus (HIV) (Daecke et al., 2005), and Ebola virus
21
(Bhattacharyya et al., 2010). Orthobunyavirus, one of the encephalitic arboviruses, is
22
also reported to infect mammalian host cells via clathrin-mediated endocytosis as its
23
primary mechanism (Hollidge et al., 2012). This mechanism for infecting host cells is
24
also implemented by various mosquito-borne flaviviruses (Acosta et al., 2008; Chu &
25
Ng, 2004). Among them, Japanese encephalitis virus (JEV) which infects host (either
26
mammalian or mosquito) cells via receptor-mediated endocytosis (Andoh et al., 1998; 3
1
Nawa, 1998) has also been confirmed to be clathrin-dependent (Chai et al., 2013;
2
Yang et al., 2013), except for certain unusual cases (Kalia et al., 2013).
3
Although the mechanism is sophisticated, clathrin-mediated endocytosis requires
4
the involvement of the 70 kDa heat shock protein (Hsp70) (Chang et al., 2002). The
5
Hsp70 family includes Hsp70 and Hsc70, which are molecular chaperones with
6
critical roles in protein folding and trafficking in all eukaryotic cells (Takayama et al.,
7
1999). They may also help to protect cells from a wide array of stresses like elevated
8
temperature and infection (Morano, 2007). Within the Hsp70 family, Hsp70 genes are
9
usually inducible whereas Hsc70 genes are mainly constitutive and usually expressed
10
in the absence of stress (Yamashida et al., 2004). Hsc70s are highly conserved in
11
structure from prokaryotes to eukaryotes (Boorstein et al., 1994). In general, Hsc70
12
can be involved in folding/unfolding and assembly/disassembly of macromolecular
13
structures (Elefant and Palter, 1999; Jiang et al., 2005) as well as anti-apoptotic
14
activity (Powers et al., 2008). Thus, Hsc70 expression is usually beneficial for cells
15
by resisting injury under conditions of minimal stress (Sens et al., 1997).
16
Among viruses using clathrin-dependent endocytosis as the entry mechanism for
17
infecting host cells, the influenza virus M1 protein is known to bind Hsc70 and is
18
required for the efficient production of the virus in infected cells (Watanabe et al.,
19
2006). Hsc70 also plays a role in outer capsid disassembly during cell entry by
20
reoviruses (Ivanovic et al., 2007), further indicating that HSc70 helps a virus infect
21
host cells and successfully replicate via interaction(s) with viral protein(s). In
22
rotaviruses, the Hsc70 protein interacts with the viral VP5 at a post-attachment step
23
during viral entry (Zárate et al., 2003). Even in mosquito cells, a 74 kDa protein likely
24
to be Hsc70 that binds JEV was identified from uninfected C6/36 cells based on an
25
analysis with a co-immunoprecipitation (Co-IP) assay and mass spectrometry (Re et
26
al., 2007). Meanwhile, Hsc70 is also one of the proteins interacting with DENV-2 in 4
1
the membrane fraction, suggesting that it may function to concentrate viral particles
2
and allow efficient interactions between primary receptors and the virus on the cell
3
surface (Paingankar et al., 2010). Accordingly, it was proposed that Hsc70 may play a
4
role as a penetration receptor that mediates JEV entry into C6/36 cells (Re et al.,
5
2007). Nonetheless, the function of Hsc70 is still ambiguous and remains to be
6
clarified for the detail.
7
Based on an EST library previously established in our lab (Chen et al., 2011), a
8
significantly higher number of Hsc70 gene copies were found in DENV-2-infected
9
C6/36 cells. In addition, Hsc70 was further confirmed to be responsive to JEV
10
infection in C6/36 cells according to a follow-up preliminary study. Therefore, the
11
purpose of this study was to investigate in detail how significant this protein is in JEV
12
infection of C6/36 cells. More importantly, we further identified specific Hsc70
13
isoforms in this study and elucidated their different roles in the process of JEV
14
infection to mosquito cells.
15
5
1
RESULTS
2
Expression of Hsc70 is required for JEV infection of C6/36 cells
3
Using the semi-quantitative RT-PCR Hsc70 was repeatedly shown to be upregulated
4
at 12 hpi at the mRNA level in C6/36 cells infected by JEV (MOI = 5) (Fig. 1a).
5
Meanwhile, quantitative RT-PCR revealed there was an increase of some 1.6 folds
6
(Fig. 1b). According to the result of Western blot, expression of Hsc70 was also
7
slightly increased at protein level (Fig. 1c). Through RNA interference (RNAi)
8
derived from a microRNA-based system, ca. 21% of the Hsc expression was revealed
9
by qRT-PCR compared with that detected from cells without knockdown of HSC70
10
(Fig. 2a). The lower level of the Hsc70 was also shown in Hsc-knockdown C6/36
11
cells according to the result from semi-quantitative RT-PCR (Fig. 2b). In the
12
meantime, decreased expression of the Hsc70 protein can clearly be seen in the result
13
from the Western blot analysis in Hsc70-knockdown cells compared to controls (Fig.
14
2c). Upon gene knockdown in C6/36 cells, intracellular viral RNA assessed by
15
RT-PCR was clearly detected as early as 6 hpi in cells without Hsc70 knockdown but
16
not until 12 hpi in cells with Hsc70 knockdown (Fig. 3a). Likely, the virus entered the
17
cells earlier when Hsc70 was not knocked down, implying this chaperone may be
18
essential for JEV entry into mosquito cells. The profile of viral infection in such cells
19
was further monitored by IFA with antibodies against the viral NS3 protein during the
20
infection, showing a large proportion of cells were eventually infected by JEV at 6 hpi
21
in C6/36 cells without the knockdown of Hsc70 (C6/36 or miN). However, the viral
22
NS3 protein was evidently reduced in Hsc70 knockdown cells (Fig. 3b), indicating the
23
necessity of Hsc70 for virus entry. Collectively, Hsc70 could be a crucial advantage
24
allowing JEV propagation in C6/36 cells.
25 26
Hsc70 affects JEV infection in C6/36 cells at the stage of entry 6
1
In C6/36 cells infected by JEV, virus titers in cells with Hsc70 knockdown were
2
evidently lower than in controls (C6/36 and miN) at 24, 48, and 72 hpi, respectively
3
(Fig. 4a). It revealed that Hsc70 plays a role in determining viral growth in C6/36
4
cells although other factors may also be involved as limited reduction of the virus titer
5
was shown in the present result. To investigate the role of Hsc70 on specific stages
6
during JEV infection, C6/36 cells with (miHsc70) or without (C6/36 or miN)
7
knockdown of Hsc70 were inoculated with the virus at MOI = 50 and incubated for 1
8
h at 4 ℃ or 28 ℃, respectively. Using IFA to detect the viral E protein,
9
approximately equal amounts of the viral antigen were observed to bind on the
10
surfaces of cells either with or without Hsc knockdown at 4 ℃ (Fig. 4b). In contrast,
11
less viral E antigen appeared in cell cytoplasm with Hsc70 knockdown when those
12
cells were incubated at 28 ℃ (Fig. 4b) compared to controls. Measurement by
13
qRT-PCR revealed that the amount of intracellular viral RNA was significantly
14
decreased to a level of ~60% in Hsc70-knockdown cells when incubated at 28 ℃ (p
15
< 0.05, Student’s t-test), however, it did not change evidently at 4 ℃ (Fig. 4c). It
16
suggests that JEV penetration into, but not binding onto, C6/36 cells is highly
17
regulated by Hsc70.
18 19
Hsc70 is involved in the endocytosis of JEV by C6/36 cells
20
Texas red conjugated with avidin was used as an endocytosis tracer to confirm the
21
role of Hsc70 in the endocytosis of C6/36 cells either knockdown of Hsc70 or not
22
when incubated at 28 ℃. Results show that tracer-containing vesicles evidently
23
appeared in the cells of controls (C6/36 and miN groups) after exposure to the tracer
24
for 1 min compared to Hsc70-knockdown cells (Fig. 5a). This suggests that tracer
25
internalization during the entry stage was closely associated with the involvement of
26
Hsc70. In C6/36 cells inoculated with JEV to replace the treatment with the tracer, 7
1
vesicles formed in response to viral inoculation were easily detected by the assay for
2
cellular uptake of the endocytic tracer at the initial step, i.e., 0 min post-inoculation, in
3
all groups. However, vesicles were not shown in control group cells but remained as
4
vesicle-like structures in cells with Hsc70 knockdown at 30 min post-inoculation (Fig.
5
5b). This implies that Hsc70 is important for viral particles up-taken by the cell to
6
form and particularly to escape the virus-containing vesicles or endosomes that are
7
usually formed in response to infection by the virus.
8 9
Formation of acidified endosomes in C6/36 cells is dependent on Hsc70
10
The acidification of virus-containing endosomes is an essential step in virus
11
un-coating for releasing the viral genome into the cytoplasm for replication. This can
12
be demonstrated by staining using acridine orange (AO), a fluorescent dye, that shows
13
orange in color for the newly-formed acidified vesicles or endosomes after
14
endocytosis. The results showed that acidified vesicles were prevalent in control
15
group cells (C6/36 and miN), whereas they were dramatically reduced to a relatively
16
low level in cells with Hsc70 knockdown (Fig. 6), suggesting that efficient
17
endocytosis of JEV in C6/36 cells is highly regulated by Hsc70.
18 19
Hsc70 is associated with clathrin-mediated endocytosis of JEV in C6/36
20
cells
21
Clathrin-coated vesicles (CCVs) are usually formed during the process of
22
clathrin-mediated endocytosis, which actually occurs in the JEV infection of C6/36
23
cells. To further prove the role of Hsc70 in this phenomenon, the clathrin light chain
24
(a component of the clathrin triskeleton) fused with RFP (CLC-RFP) was
25
overexpressed in C6/36 cells for monitoring the formation and localization of CCVs.
26
When C6/36 cells were inoculated with JEV (MOI = 50) and incubated at 28 ℃ for 8
1
5 and 30 min, respectively. The results show that CCVs tagged with RFP (CCVs-RFP)
2
were evidently decreased in control group cells at 30 min post-infection compared to
3
those cells infected for 5 min whereas the change was insignificant in
4
Hsc70-knockdown cells (Fig. 7). It indicates that clathrin is normally dissociated from
5
CCVs for recycling, likely back to the plasma membrane in C6/36 cells infected by
6
JEV. It creates an advantage for viral RNA to release from the formed CCVs. In
7
contrast, CCVs may be retained and gradually accumulated in the cytoplasm,
8
blocking the formation of more endosomes containing ingested viral particles due to
9
the knockdown of Hsc70 in C6/36 cells.
10 11
JEV infection to C6/36 cells is decided by the specific Hsc70 isoform
12
Based on the sequences of Hsc70 from Aedes albopictus (C6/36 cells), three isoforms
13
(B, C, and D) were identified in this study. Their nucleotide sequences were retrieved
14
from the database in NCBI by accession numbers KJ701753, KJ701754, and
15
KJ701755 for isoforms B, C, and D, respectively. As shown in the results of RT-PCR,
16
all Hsc70 isoforms, particularly D, were elevated in C6/36 cells infected by JEV
17
infection at MOI = 5 for 12 h (Fig. 8). Using dsRNA to knock down individual Hsc70
18
isoforms (Supplementary Figure S1), the expression of viral RNA within the cell was
19
apparently delayed and not appeared until 12 hpi in cells with knockdown of Hsc70
20
isoform D (Fig. 9). Since the efficiency of JEV binding to C6/36 cells did not show
21
significant difference (Fig. 10a), there seems to be different effects among the Hsc70
22
isoforms in C6/36 cells in response to JEV infection. In contrast, virus penetration
23
efficiency was significantly reduced to
