Download VP5 autocleavage is required for efficient infection by in vitro

Journal of General Virology (2015), 96, 1795–1800
Short
Communication
DOI 10.1099/vir.0.000116
VP5 autocleavage is required for efficient infection
by in vitro-recoated aquareovirus particles
Shicui Yan,1,2 Jie Zhang,1 Hong Guo,1 Liming Yan,1,2 Qingxiu Chen,1,2
Fuxian Zhang1 and Qin Fang1
Correspondence
Qin Fang
[email protected]
Received 23 January 2015
Accepted 4 March 2015
1
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences,
Wuhan 430071, PR China
2
University of Chinese Academy of Sciences, Beijing, PR China
Grass carp reovirus (GCRV) is a member of the genus Aquareovirus in the family Reoviridae, and
contains five core proteins (VP1–VP4 and VP6) and two outer-capsid proteins (VP5 and VP7) in
its particle. Previous studies have revealed that the outer-capsid proteins of reovirus are
responsible for initiating infection, but the mechanism is poorly understood. Using baculovirusexpressed VP5 and VP7 to recoat purified cores, in vitro assembly of GCRV was achieved in this
study. Recoated GCRV (R-GCRV) closely resembled native GCRV (N-GCRV) in particle
morphology, protein composition and infectivity. Similar to N-GCRV, the infectivity of R-GCRV
could be inhibited by treating cells with the weak base NH4Cl. In addition, recoated particles
carrying an AsnAAla substitution at residue 42 of VP5 (VP5N42A/VP7 R-GCRV) were no longer
infectious. These results provide strong evidence that autocleavage of VP5 is critical for
aquareovirus to initiate efficient infection.
The genus Aquareovirus is a tentative member of the family
Reoviridae, which currently includes 15 proposed genera
(King et al., 2011). Many aquareoviruses have been isolated
from apparently healthy-looking fish, including the finfish
and shellfish species. However, several members within
the genus are identified as pathogens that are capable of
causing serious disease in aquatic animals (Fang et al.,
1989; Ke et al., 1990; Rangel et al., 1999). Grass carp
reovirus (GCRV) has been recognized as the most
pathogenic among the isolated aquareoviruses (Mohd
Jaafar et al., 2008; Rangel et al., 1999). Hence, GCRV
provides a useful model to understand the infection and
pathogenesis of aquareoviruses in general.
the inner capsid or core of the virion. Previous threedimensional structure studies have revealed that the inner
capsid of GCRV is constructed mainly of two proteins, VP3
and VP6, whilst the other three proteins (VP1, VP2 and
VP4) are RNA polymerase-related complex proteins that
possess the enzymic activities necessary for viral transcription and replication (Cheng et al., 2008; Fang et al., 2005).
Similar to the homologous proteins m1 and s3 of MRV in
their structural organization, VP7 is a protective protein
located on top of the VP5 subunits. In contrast to MRVs,
there is no protein equivalent of s1, which sits on the
fivefold vertices of the virion and functions as the cellattachment protein (Cheng et al., 2008; Fang et al., 2005).
The struture of GCRV is a multilayered icosahedral particle
with a diameter of approximately 75–85 nm enclosing a
segmented dsRNA genome. The 11 genome segments
encode seven structural proteins (VP1–VP7) and five nonstructural proteins. Genome sequence analysis and threedimensional structural reconstructions by cryo-electron
microscopy have indicated that there are great similarities
between aquareoviruses and mammalian reoviruses (MRVs)
in protein structure and particle organization (Cheng et al.,
2008, 2010; Fang et al., 2005). Similar to other reoviruses in
the family Reoviridae, the GCRV particle is organized mainly
into two layers: an outer capsid and an inner capsid. Two
hundred trimeric heterodimers of the VP5 and VP7 proteins
make up the outer capsid of the virus particle, whilst the
other five structural proteins (VP1–VP4 and VP6) constitute
Reoviruses are non-enveloped virions that lack a lipidbilayer membrane. Based on their particle organization,
they utilize an entry pathway different from that of
enveloped viruses. To initiate infection in a target cell,
reoviruses must disrupt the host cell membrane through a
mechanism other than the membrane fusion route seen for
enveloped viruses. Previous studies have revealed that MRVs
need to be activated for infection by undergoing stepwise
disassembly of the outer capsid to generate infectious
subvirion particles (ISVP) (Chandran & Nibert, 1998;
Liemann et al., 2002; Sturzenbecker et al., 1987). When
virions are activated to infect host cells, the outermost
capsid protein s3 of MRV is degraded by endosomal
proteases, leaving m1 exposed on the surface. The m1 protein
is found predominantly in the form of two fragments, m1N
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S. Yan and others
(4 kDa) and m1C (72 kDa), following putative autocleavage
between residues Asn42 and Pro43 near the N terminus of
the intact protein (Nibert et al., 1991, 2005). In addition,
infection with intact virions, but not ISVPs, can be blocked
by treating cells with a weak base, such as NH4Cl, that inhibits
the proteolysis of m1 within cells (Chandran & Nibert, 1998;
Jané-Valbuena et al., 1999). Studies on the characteristics of
infectious GCRV particles have demonstrated that complete
digestion of VP7 and partial cleavage of VP5 can lead to
enhanced infectivity of the virus, indicating that VP7 and
VP5 may play important roles in GCRV entry into host cells
during infection (Fang et al., 2008; Zhang et al., 2010).
Recent three-dimensional structural studies of GCRV ISVP
at 3.3 Å resolution revealed that autocleavage of the VP5
protein, which is myristoylated at the N terminus, occurs
during conversion from the dormant to the primed state at
an early stage of viral infection (Zhang et al., 2010). To gain
insight into the molecular mechanism of virus infection and
particle assembly, in vitro assembly of GCRV-like particles
by recoating baculovirus-expressed VP5 and VP7 onto
purified cores was achieved in this study. In addition, the
infection characteristics of recoated WT (R-GCRV or VP5/
VP7 R-GCRV) and mutant (VP5N42A/VP7 R-GCRV)
particles were investigated.
Previous studies reported that the outer-capsid proteins
VP5 and VP7 are required for cell entry during aquareovirus infection, and that putative autocleavage of outercapsid protein VP5 is likely to be critical for virus entry
into host cells (Fang et al., 2008; Zhang et al., 2010). To
determine whether VP5 and/or VP7 were responsible
for aquareovirus entry, a dual-expressed VP5 and VP7
(vAcGCRV-VP5/VP7) and singly expressed VP5 or VP7
(vAcGCRV-VP5 or vAcGCRV-VP7; data not shown)
recombinant baculoviruses were constructed, respectively.
In addition, to determine whether the N42 residue was
responsible for autocleavage of VP5, a mutated VP5
containing an AsnAAla substitution at residue 42 was also
generated (Fig. 1a) and expressed in Spodoptera frugiperda
(Sf9) cells using a recombinant baculovirus (vAcGCRVVP5N42A/VP7). An immunofluorescence assay was carried
out to confirm the subcellular location of VP5 or VP5N42A
with VP7 during recombinant baculovirus infection. As
shown in Fig. 1(b), compared with vAcGCRV-VP5/VP7, the
co-localization of mutant VP5N42A with VP7 in infected Sf9
cells was not changed. To estimate the levels of protein
expression and VP5N/VP5C cleavage, Sf9 cell lysates
containing VP5 or VP5N42A co-expressed with VP7 were
analysed by Western blotting (WB). The data revealed that
the expression of WT VP5 resulted in two VP5-derived
bands, namely intact VP5 and cleaved C-terminal VP5
(termed VP5C). By contrast, in vAcGCRV-VP5N42A/VP7infected cells, no cleaved VP5C could be detected (Fig. 1c).
Moreover, the interactions between VP5 or VP5N42A and
VP7 were investigated by a co-immunoprecipitation assay.
The results showed that WT VP5 or mutant VP5N42A could
efficiently co-immunoprecipitate with VP7 using anti-VP7
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polyclonal antibodies, and VP7 was efficiently co-immunoprecipitated with VP5 or VP5N42A by anti-VP5 polyclonal
antibodies (Fig. 1d) but not by control antibody. These
results indicated that the vAcGCRV-VP5/VP7 and vAcGCRVVP5N42A/VP7 recombinant baculoviruses could express VP5
or VP5N42A and VP7 at high levels in Sf9 cells. Moreover,
both VP5 and VP5N42A could interact with VP7. These
results suggested that the VP5/VP7 and VP5N42A/VP7
recombinant proteins co-expressed in Sf9 cells were suitable
for recoating GCRV cores in vitro.
To achieve the assembly in vitro, GCRV cores with
protruding turrets were purified by CsCl gradient centrifugation and their structure confirmed using transmission electron microscopy (TEM) (Fig. 2a). Cytoplasmic
extracts were also prepared from Sf9 cells infected with
recombinant baculoviruses co-expressing proteins VP5 and
VP7 or singly expressing VP5 or VP7. These extracts were
incubated with purified GCRV cores at 28 uC for 5 h and
the recoated particles purified by CsCl gradient centrifugation. Comparison by TEM of these recoated particles (RGCRV) with native particles (N-GCRV), also purified by
CsCl-based centrifugation, showed that the two types of
particle had indistinguishable morphologies (Fig. 2b, c).
No recoated particles were obtained when purified cores
were incubated with VP5 or VP7 alone, strongly suggesting
that a complex of VP5 and VP7 is required for binding to
and recoating viral cores. To confirm the protein
composition of R-GCRV particles, samples were analysed
using SDS-PAGE (Fig. 2d). Seven structural protein
components were identified whose molecular masses were
consistent with those seen in similar analysis performed on
N-GCRV particles. In addition, WB analysis confirmed
that VP5 and VP7 had specifically bound to the cores and
remained bound throughout the CsCl centrifugation (Fig.
2e). To evaluate the infectivity of recoated particles, they
were titred on Ctenopharyngodon idellus kidney (CIK) cells.
As shown in Fig. 2(f), in common with N-GCRVs, RGCRVs were 105-fold more infectious than cores on a perparticle basis. In contrast to these results, when VP5 or VP7
cell extracts (infected by vAcGCRV-VP5 or vAcGCRVVP7) were singly incubated with cores (VP5 R-GCRV or
VP7 R-GCRV), no infectivity was detected. These results
indicated that infectious R-GCRV could only be produced
by recoating core particles with both VP5 and VP7
proteins.
Cell entry of MRV or aquareovirus has been reported to be
a multistep process involving proteolytic disassembly of
virions into ISVPs (Chandran et al., 1999; Fang et al.,
2008). This proteolysis of intact virions could be inhibited
by treating cells with weak bases, such as NH4Cl (Chandran
et al., 1999). To prove the biological similarity between RGCRVs and N-GCRVs, cells were infected with or without
20 mM NH4Cl treatment. At 24 h post-infection (p.i.),
obvious viral plaques were observed in the mock-treated
cells but not in NH4Cl-treated cells (Fig. 3a). Cell
supernatants were also harvested and virus titres were
assayed. The results showed that more than a 105-fold
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VP5 autocleavage is critical for aquareovirus infection
(a)
VP5
VP5N
VP5C
Native VP5
N42-P43
Mutated VP5
A42-P43
(b)
VP5
VP7
Hoechst
Merger
VP5N42A
VP7
Hoechst
Merger
(c)
vAcGCRVVP5/VP7
GCRV
Mock
vAcGCRVVP5N42A/VP7
VP5
VP5C
VP7
(d)
vAcGCRV-VP5/VP7infected cells
Lysate
IP
Mock
Lysate
IP: VP7
Mock
infected cells
WB: VP5
VP5
VP5C
WB: VP7
VP7
IP: VP5
IP
vAcGCRV-VP5N42A/VP7-
WB: VP7
VP7
WB: VP5
VP5
VP5C
reduction in virus titre was observed with N-GCRV or RGCRV when the cells were treated with NH4Cl (Fig. 3b).
These results indicated that NH4Cl could inhibit the
infection with both N-GCRV and R-GCRV, confirming the
similarity between them.
The infectivity of mutant VP5N42A/VP7 R-GCRV was
evaluated on CIK cells. At 24 h p.i., a large number of
plaques was observed in VP5/VP7 R-GCRV-infected cells
but not in core- or VP5N42A/VP7 R-GCRV-infected cells
(Fig. 3c). To further confirm that recoated particles with
mutant VP5N42A were defective in infection, the virus titres
were examined using both TCID50 and real-time quantitative PCR (qPCR) assays. As shown in Fig. 3(d), the
infectivity of WT R-GCRV was similar to that of N-GCRV.
http://vir.sgmjournals.org
Fig. 1. Identification of recombinant protein
expression in infected cells. (a) Schematic
representation of the VP5 protein and its
mutation at the autocleavage site. The arrow
indicates the putative autocleavage site. (b)
Immunofluorescence assays of VP5 or VP5N42A
and VP7 in vAcGCRV-VP5/VP7- or vAcGCRVVP5N42A/VP7-infected cells. Hoechst dye was
used to stain nuclei and a merged image is
shown on the right. (c) WB analyses of VP5 or
VP5N42A and VP7 in vAcGCRV-VP5/VP7 or
vAcGCRV-VP5N42A/VP7 infected cells. GCRV
infected CIK cells and uninfected Sf9 cells
served as positive and negative controls
respectively. (d) Co-immunoprecipitation assays
of VP5 or VP5N42A with VP7 in infected cells.
In contrast, little or no infectivity was detected with either
control virus cores or mutant VP5N42A/VP7 R-GCRV
particles. In the qPCR assay, the mRNAs of the GCRV core
proteins VP3 and VP6 were detected to assess their
expression levels. High mRNA levels were detected in
WT R-GCRV-infected cells but not in cells infected with
mutant VP5N42A/VP7 R-GCRV, or with virus cores or
cores mixed with mock Sf9 cell lysates (Fig. 3e). In
addition, WB analysis showed that the expression of VP3
and VP6 could be detected in WT R-GCRV-infected cells
but not in mutant VP5N42A/VP7 R-GCRV-infected cells
(Fig. 3f). These results strongly indicated that the WT RGCRVs have similar infectivity to N-GCRV but that
infectivity is lost when the N42 residue is substituted to
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S. Yan and others
(a)
Core
100 nm
(b)
(c)
R-GCRV
N-GCRV
100 nm
100 nm
(d)
e
or
C
V
CR P7
V
G
c V
CR
vA P5/
G
N-V
V
CR
-G
(f)
R
VP1–VP3
8
VP4
VP5
VP5C
VP7
log10 p.f.u. ml–1
VP6
Core
6
R-GCRV
N-GCRV
4
VP5 R-GCRV
VP7 R-GCRV
2
(e)
VP5
VP5C
0
VP7
Fig. 2. Characteristics and infectivity assays of in vitro-recoated particles. (a) TEM image of purified GCRV cores. The boxed
area in the image is enlarged to show details. The arrow indicates the turret. (b, c) TEM images of purified R-GCRV and
N-GCRV. (d, e) SDS-PAGE analysis of protein components and WB analysis of VP5 and VP7 in purified R-GCRV and
N-GCRV. Purified core and vAcGCRV-VP5/VP7 served as controls. (f) Infectious titre of R-GCRV determined by a p.f.u. assay.
The core, N-GCRV, VP5 R-GCRV and VP7 R-GCRV served as controls. Each bar represents the mean±SD derived from three
independent experiments.
A (VP5N42A), suggesting that the autocleavage of VP5 at
aa 42 is indispensable for GCRV to initiate infection.
The baculovirus expression system was used for the highlevel expression of recombinant VP5 and VP7 proteins, and
in vitro assembly using the expressed proteins to recoat
purified GCRV cores was achieved. Infectious R-GCRV
could only be produced by recoating core particles with
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both VP5 and VP7 proteins, and not with VP5 or VP7
singly, indicating that VP7 is required for particle assembly
and cell entry. These findings not only suggest that a VP5–
VP7 complex can be spontaneously assembled onto
purified core particles in stoichiometric amounts, but also
support a proposed structure-based mechanism whereby
VP7 removal triggers a VP5 conformational change and
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Journal of General Virology 96
VP5 autocleavage is critical for aquareovirus infection
(a)
(c)
–
NH4Cl
+
N-GCRV
VP5/VP7
R-GCRV
Core
VP5N42A/VP7
R-GCRV
R-GCRV
(b)
(d)
N-GCRV
N-GCRV+NH4Cl
R-GCRV
8
R-GCRV+NH4Cl
8
log10 TCID50 ml–1
log10 p.f.u. ml–1
10
6
4
2
0
Core
VP5/VP7 R-GCRV
N-GCRV
VP5N42A/VP7 R-GCRV
6
4
2
0
(e)
(f)
107
Relative expression of
core proteins
106
105
VP3
104
1
2
4
VP3
VP6
103
3
102
VP6
101
100
Actin
10–1
1
2
3
4
Fig. 3. VP5 autocleavage is indispensable for efficient infection of aquareovirus. (a) Plaque assays of R-GCRV and N-GCRV in
the absence (”) or presence (+) of NH4Cl. (b) Infectious titres of supernatants harvested at 24 h p.i. in (a) were determined by
a p.f.u. assay. (c) Plaque assay of VP5N42A/VP7 R-GCRV. Core and VP5/VP7 R-GCRV served as controls. (d) Infectious titre of
VP5N42A/VP7 R-GCRV. Core, VP5/VP7 R-GCRV and N-GCRV served as controls. (e, f) Relative expression of VP3 and VP6
in mRNA (e) or protein (f) level calculated by qPCR or WB assay, respectively. Lanes: 1, core; 2, core mixed with Sf9; 3, VP5/
VP7 R-GCRV; 4, VP5N42A/VP7 R-GCRV. Actin was used as a loading control in (f). Data in (b), (d) and (e) represent the
mean±SD derived from three independent experiments.
autoproteolysis, converting a virion from a dormant state
to a primed ISVP (Zhang et al., 2010).
To enter host cells, a reovirus virion needs to transform
into an ISVP that can penetrate the cell membrane, and
this process is acid dependent (Chandran et al., 1999;
Kothandaraman et al., 1998). In this study, the infectivity of
N-GCRV or R-GCRV could be blocked by the weak base
NH4Cl, suggesting that N-GCRVs and R-GCRVs utilize a
similar route to enter host cells. More importantly, RGCRVs with a VP5N42A mutation were shown to be defective
in infectivity and progeny protein expression in infected
cells, indicating that an N42A substitution almost completely blocked the VP5 autocleavage. This result is consistent
with a previous report in MRV where cores recoated with s3
http://vir.sgmjournals.org
and m1 bearing the N42A mutation were defective for
infectivity (Odegard et al., 2004), suggesting that this amino
acid of m1 plays an important role in the cleavage process.
In summary, an in vitro assembly model of aquareovirus
was generated in this study. The results suggest that RGCRV can serve as a tool for investigating virus entry. Based
on the in vitro assembly model of GCRV, the functions of the
outer-capsid proteins VP5 and VP7 in virus entry could be
fully investigated. To our knowledge, this study has also
provided the first experimental evidence for the indispensability of aquareovirus VP5 autocleavage in cell entry by
using cores recoated with a mutated VP5 protein. These
results lay a foundation for further efforts to reveal the
detailed entry mechanism of aquareoviruses.
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S. Yan and others
Acknowledgements
Ke, L. H., Fang, Q. & Cai, Y. Q. (1990). Characteristics of a novel
We express our sincere gratitude to Professor Peng Gong for editing
our manuscript, Juan Liu, Wen Dai and Lanlan Zhang for their work
in constructing recombinant VP5 and/or VP7 baculoviruses, and Ling
Shao for her work in generating mutant VP5 recombinant and the
related VP5N42A and VP7 baculoviruses. This work was supported in
part by grants from the National Natural Science Foundation of
China (31172434, 31372565 and 31370190).
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(2002). Structure of the reovirus membrane-penetration protein, m1,
in a complex with is protector protein, s3. Cell 108, 283–295.
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