Journal of General Virology (2014), 95, 2820–2830
DOI 10.1099/vir.0.068122-0
Identification and functional analysis of inter-subunit
disulfide bonds of the F protein of Helicoverpa
armigera nucleopolyhedrovirus
Feifei Yin,1,2 Manli Wang,1 Ying Tan,1 Fei Deng,1 Just M. Vlak,3
Zhihong Hu1 and Hualin Wang1
Correspondence
Hualin Wang
[email protected]
1
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences
(CAS), Wuhan 430071, PR China
2
School of Tropical and Laboratory Medicine, Hainan Medical University, Haikou 571101, PR China
3
Laboratory of Virology, Wageningen University, 6708 PB Wageningen, The Netherlands
Received 18 May 2014
Accepted 10 August 2014
The major envelope fusion protein F of the budded virus of baculoviruses consists of two
disulfide-linked subunits: an N-terminal F2 subunit and a C-terminal, membrane-anchored F1
subunit. There is one cysteine in F2 and there are 15 cysteines in F1, but their role in disulfide
linking is largely unknown. In this study, the inter- and intra-subunit disulfide bonds of the
Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) F protein were
analysed by site-directed mutagenesis. Results indicated that in a functional F protein, an intersubunit disulfide bond exists between amino acids C108 (F2) and C241 (F1). When C241 was
mutated, an alternative disulfide bond was formed between C108 and C232, rendering F nonfunctional. No inter-subunit bridge was observed in a double C232/C241 mutant of F1. C403
was not involved in the formation of inter-subunit disulfide bonding, but mutation of this amino acid
decreased viral infectivity significantly, suggesting that it might be involved in intra-subunit
disulfide bonds. The influence of reductant [tris(2-carboxyethyl) phosphine (TCEP)] and free-thiol
inhibitors [4-acetamido-49-maleimidylstilbene 2,29-disulfonic acid (AMS) and 5,59-dithiobis(2nitrobenzoic acid) (DTNB)] on the infectivity of HearNPV was tested. The results indicated that
TCEP greatly decreased the infection of HzAm1 cells by HearNPV. In contrast, AMS and DTNB
had no inhibitory effect on viral infectivity. The data suggested that free thiol/disulfide isomerization
was not likely to play a role in viral entry and infectivity.
INTRODUCTION
The Baculoviridae is a family of large, enveloped, DNA
viruses that are pathogenic to arthropods (Federici, 1997).
The family is divided into four genera based on molecular
phylogenetic analysis: Alphabaculovirus, Betabaculovirus,
Gammabaculovirus and Deltabaculovirus (Jehle et al., 2006).
The genus Alphabaculovirus is further subdivided into two
groups (I and II) based on their host and major envelope
fusion protein (EFP) (Herniou et al., 2001; Pearson et al.,
2000). The major EFPs, GP64 and F protein, are significant
for the infectivity of baculoviruses. Group I alphabaculoviruses utilize GP64 as their EFP, whereas group II
alphabaculoviruses, and beta- and deltabaculoviruses, exploit
the F protein as their EFP (Blissard & Wenz, 1992; IJkel et al.,
2000; Long et al., 2006; Pearson et al., 2000; Yin et al., 2008).
A functional degenerated F homologue (F-like protein) was
One supplementary table and one supplementary figure are available
with the online version of this paper.
2820
identified in group I alphabaculoviruses (Lung et al., 2003;
Wang et al., 2008a).
The baculovirus F proteins possess common features of
class I viral fusion proteins. During the maturation of
virions, the F proteins are cleaved post-translationally at
their furin cleavage site, and the individual N-terminal
F2 subunit and C-terminal subunit F1 are connected by a
disulfide bond (IJkel et al., 2000; Long et al., 2006; Lung
et al., 2002; Yin et al., 2008). It is generally believed that
disulfide bonds and their dynamic rearrangement are
critical for the accurate folding and transportation of
proteins during synthesis and maturation (Day et al., 2006;
Segal et al., 1992). In mature proteins, disulfide bonds play
essential roles in stabilizing the structure required for their
biological activity (Maar et al., 2012; Markovic et al., 1998;
McCaffrey et al., 2012). Mutation of certain cysteine residues
in the extracellular domain of the F protein of human
respiratory syncytial virus (HRSV) abolished or dramatically
reduced cell surface expression and fusion activity of
the protein (Day et al., 2006). Cysteine residues in the
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Identification of inter-subunit disulfide bonds of HaF
ectodomain of influenza virus haemagglutinin (HA) are
critical for efficient folding of the protein during synthesis
and stabilize the structure of the matured protein (Segal
et al., 1992). For baculovirus GP64 protein, the monomers
are linked by a single intermolecular disulfide bond to form
a trimer. Disruption of the intermolecular bridge resulted in
inefficient transport of GP64 to the cell surface and in the
loss of the ability to rescue a gp64-null Autographa californica
multicapsid nucleopolyhedrovirus (AcMNPV). In addition,
the six conserved intramolecular disulfide bonds are all
indispensable for GP64-mediated membrane fusion (Li &
Blissard, 2010).
Membrane fusion mediated by baculovirus F proteins is
triggered by acidification of the late endosome (IJkel et al.,
2000; Pearson et al., 2000). Whether other factors play a
role in the activation and conformational change during
fusion of baculovirus F protein has not been explored. One
potential mechanism to facilitate the conformational change
required for membrane fusion is disulfide bond isomerization as suggested by studies of various virus membrane
fusion proteins (Abou-Jaoudé & Sureau, 2007; Fenouillet
et al., 2007; Jain et al., 2007, 2009). One or multiple disulfide
bonds in the human immunodeficiency virus (HIV) envelope protein are cleaved by cell-surface-associated protein
disulfide isomerase (PDI) after binding to the CD4 receptor
– a step which is a prerequisite for the subsequent virus–
cell fusion event. Inhibition of the activity of PDI using
membrane-impermeable inhibitors, such as free-thiol reagent
5,59-dithiobis(2-nitrobenzoic acid) (DTNB), can inhibit
membrane fusion and viral infection (Barbouche et al.,
2003; Fenouillet et al., 2007; Ryser et al., 1994). For Sindbis
virus, where intact disulfide bonds of the envelope proteins
are critical for the stability and function of the viral
envelope, treatment with reducing reagents induces virusmediated fusion and addition of DTNB during infection
inhibits virus entry (Abell & Brown, 1993; Anthony et al.,
1992). A further case of disulfide reduction during membrane fusion mediated by a viral envelope protein was
observed in the case of Newcastle disease virus (NDV). Free
thiols are produced in the NDV F protein during fusion and
are critical for efficient virus entry into cells. Inhibitors of
cell surface-associated thiol/disulfide isomerase, including
DTNB, bacitracin and anti-PDI antibody, block F-proteinmediated cell–cell fusion and virus entry (Jain et al., 2007,
2009). The envelope glycoproteins of the pestivirus bovine
viral diarrhea virus also undergo disulfide reduction, which
destabilizes the viral envelope during endocytosis to become
fusogenic at endosomal acidic pH by an unknown
mechanism (Krey et al., 2005).
Baculovirus F proteins have a low overall similarity (20–
40 % amino acid identity) (Wang et al., 2010). However,
alignment of F proteins from group II alphabaculovirus
(Ha133, Ld130 and Se8), betabaculovirus (Agse25, Px26
and Cp31) as well as F-like proteins from group I alphabaculovirus (Ac23 and Op21) revealed that the positions of
11 cysteine residues were highly conserved and the position
of one cysteine residue was relatively conserved in the
http://vir.sgmjournals.org
protein (Fig. 1a) (Rohrmann & Karplus, 2001). An enlarged
schematic diagram of the F protein of Helicoverpa armigera
nucleopolyhedrovirus (HearNPV) (HaF), which is a group
II alphabaculovirus, is shown in parallel in Fig. 1(a) to
indicate the cysteine residues selected for mutagenesis in this
study. Most of these cysteine residues are located in the
membrane-anchored F1 subunit and only one of these is
located in the F2 subunit (HaF C108). In F1, except for one
highly conserved cysteine residue located at the N terminus
(HaF C241) and another located in the cytoplasmic tail
domain (CTD) (HaF C623), all the other eight cysteines
cluster upstream of the transmembrane domain (TMD)
(HaF C358, 365, 393, 403, 418, 436, 471 and 495) and are
separated from the N-terminal conserved cysteine residue by
an intervening polypeptide. A relatively conserved cysteine
residue (HaF C232) can also be found in F1 located either
upstream or downstream of the N-terminally conserved
C241 (Fig. 1a).
To identify which cysteines form the inter-subunit disulfide
bond of HaF, representative cysteine residues including
C108, C232, C241 and one of the cysteines (C403) from the
cysteine cluster upstream of the TMD were selected for sitedirected mutagenesis, and the functions of mutated HaF
proteins were investigated (Fig. 1a). Cysteines in the CTD
were not selected because in general in F proteins they are
fatty acid acylated and not involved in disulfide formation
(Schultz et al., 1988). Furthermore, we investigated whether
or not thiol/disulfide isomerization of HaF was important
for viral entry by using membrane-impermeable thiol/
disulfide exchange inhibitors. The results showed that an
inter-subunit disulfide bond of HaF is formed between C108
in the F2 subunit and C241. When C241 is mutated, C232
then forms an inter-subunit disulfide bond with C108, but
the infectivity is lost. Free thiol/disulfide isomerization of
HaF is not likely to a play a role in viral entry and infectivity.
However, the surface-exposed disulfide bonds of HaF are
important for the infectivity of HearNPV to HzAM1 cells.
RESULTS
Functional analysis of cysteine residues of HaF
The cleavage of baculovirus F proteins by furin results in
two disulfide-linked subunits (Long et al., 2006; Pearson
et al., 2000; Westenberg et al., 2002; Yin et al., 2008). To
identify the cysteine residues that participate in the formation of inter-subunit disulfide bonding in HaF, cysteine
residues at positions 108, 232, 241 and 403 were mutated to
a non-bulky glycine residue individually, and a double
mutation at both C232 and C241 was also constructed (Fig.
1a). Mutated HaF genes as well as the WT HaF gene were
transposed into the HaBacDF bacmid, which resulted
in recombinant bacmids HaBacDF-HaFC108G, HaBacDFHaFC232G, HaBacDF-HaFC241G, HaBacDF-HaFC232/241G,
HaBacDF-HaFC403G and HaBacDF-HaF (Fig. 1b, left).
These mutants also expressed EGFP as a visible marker
to follow infection.
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F. Yin and others
(a)
SP
F2
F1
FP
TMD
Ha133
Group II
alphabaculovirus
Ld130
Se8
Agse25
Betabaculovirus
Px26
Cp31
Ac23
Op21
FP HR1 HR3
HR2 TMD CTD
F1
677
F2
531
568
607
629
1
33
SP
174
197
199
225
246
259
Group I
alphabaculovirus
HaF (Ha133)
(b)
GmR
Tn7R
TetR egfp
HaF locus
GmR
SV40 Tn7L
poly A
Mini-attTn7
Polyhedrin locus
bHa HaF
C626
C628
C632
C633
C637
G
WT/MT HaF
pOp166
C471
C495
GG
C393
C403
C418
C436
C232
C241
G
C358
C365
C108
C19
Cleavage
Tn7R
WT/MT HaF
pOp166
SV40 Tn7L
poly A
Mini-attTn7
Polyhedrin locus
bAcMNPV
Fig. 1. (a) Schematic representation of baculovirus F proteins (adapted from Rohrmann & Karplus, 2001) and enlarged
schematic diagram of HaF. The relative positions of the predicted signal peptide (SP), fusion peptide (FP), heptad repeats
(HR1, HR2 and HR3), TMD, CTD and proteolytic cleavage site (arrow) are indicated. The positions of highly conserved and
relatively conserved cysteine residues are indicated by bold dashed lines; non-conserved cysteine residues are indicated by
normal dashed lines. Mutated cysteine residues are underlined. Ha133, Ld130, Se8, Agse25, Px26, Cp31, Ac23 and Op21
represent F proteins of Helicoverpa armigera nucleopolyhedrovirus (HearNPV; GenBank accession number AF271059),
Lymantria dispar multiple nucleopolyhedrovirus (AF081810), Spodoptera exigua multiple nucleopolyhedrovirus (AF169823),
Agrotis segetum nucleopolyhedrovirus (AY522332), Plutella xylostella granulovirus (AF270937), Cydia pomonella granulovirus
(U53466), Autographa californica nucleopolyhedrovirus (L22858) and Orgyia pseudotsugata multiple nucleopolyhedrovirus
(U75930), respectively. (b) The strategy to generate the recombinant f-null HearNPV bacmid (left) and the gp64-null AcMNPV
bacmid (right). The WT HaF gene and mutated (MT) HaF genes listed were inserted into the polyhedrin locus by Tn7-mediated
transposition. All the genes were under the control of the Op166 promoter (pOp166). Mutations in HaF included HaFC108G,
HaFC232G, HaFC241G, HaFC403G and HaFC232/241G.
To analyse the function of cysteine residues, recombinant
bacmids were transfected into HzAM1 cells and supernatants were used to infect another batch of cells. Green
fluorescence was detected to indicate the success of
transfection and the spread of infectious budding virus
(BV). Fluorescence was detected in all of the transfected
cells (Fig. 2a–g), indicating successful transfer of recombinant bacmids. At 5 days post-infection (p.i.), infectious
BVs were observed in cells infected with the supernatants
of cells transfected with HaBacDF-HaFC232G, HaBacDFHaFC403G and HaBacDF-HaF (Fig. 2j, m, n). No secondary
2822
infection was detected in cells exposed to supernatants
that were collected from cells that were transfected with
HaBacDF-HaFC108G or HaBacDF-HaFC241G (Fig. 2i, k). For
vHaBacDF-HaFC232/241G, although infection was observed,
the spread of fluorescence was restricted to a limited
number of adjacent cells (Fig. 2l). During the amplification
process, BVs of vHaBacDF-HaFC232G, vHaBacDF-HaFC403G
and vHaBacDF-HaF could be propagated easily in HzAM1
cells; however, the amplification of vHaBacDF-HaFC232/241G
failed in all the five attempts. The results demonstrated that
C108 and C241 were crucial for the function of the HaF gene
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Journal of General Virology 95
Identification of inter-subunit disulfide bonds of HaF
Infection
Transfection
Transfection
(a)
HaFC108G
(b)
(c)
Infection
f-null
HaFC232G
(h)
(i)
(j)
HaFC241G
(d)
HaFC232/241G
(e)
HaFC403G
(f)
(g)
(k)
(l)
(m)
(n)
HaF
Fig. 2. Transfection and infection assays of the recombinant f-null
HearNPV bacmids for viral propagation. (a–g) The f-null HearNPV
bacmid and recombinant bacmids with mutant HaF (as indicated)
were transfected into HzAm1 cells. (h–n) At 5 days posttransfection, supernatants were used to infect HzAM1 cells after
clarification. Transfected and infected cells were observed under a
fluorescence microscope.
in rescuing the infectivity of the f-null HaBacmid. As C108
is the only cysteine residue in the F2 subunit of the mature
form, it is reasonable to speculate that C108 participates
in the inter-subunit disulfide linkage between HaF1 and
HaF2.
Inter-subunit disulfide bond formation in HaF
mutants
As some recombinant HearNPVs with cysteine-mutated
HaF failed to produce infectious BVs for further analysis,
all of the mutated HaF genes as well as the WT HaF gene
were transposed into an AcBacmid carrying a GP64 gene to
investigate inter-subunit disulfide bonds in mutated HaF
proteins. The resultant recombinant bacmids were designated AcBac-HaFC108G, AcBac-HaFC232G, AcBac-HaFC241G,
AcBac-HaFC232/241G, AcBac-HaFC403G and AcBac-HaF
(Fig. 1b, right). Recombinant bacmid DNAs were transfected into Sf9 cells, and BVs carrying both AcGP64 and
HaF were purified from the supernatant of infected cells
and subjected to Western blot analysis (Fig. 3). Under
reducing conditions with antibodies specific for the membrane-anchored subunit HaF1, only a band of ~59 kDa was
detected for all BVs (Fig. 3a, lanes 1–6). The molecular mass
of the bands was consistent with the size of HaF1, indicating
proper expression and cleavage of all HaF mutants (Long
et al., 2006). Under non-reducing conditions, in which disulfide bonds were not disrupted, a band of 80 kDa was observed
http://vir.sgmjournals.org
in vAcBac-HaFC232G (Fig. 3b, lane 2), vAcBac-HaFC241G
(Fig. 3b, lane 3), vAcBac-HaFC403G (Fig. 3b, lane 5) and
vAcBac-HaF (Fig. 3b, lane 6), which corresponded to the
size of F2 subunit linked with the F1 subunit (designated
F1+2) (Long et al., 2006). For both vAcBac-HaFC108G (Fig.
3b, lane 1) and vAcBac-HaFC232/241G (Fig. 3b, lane 4), bands
corresponding to only the size of F1 were observed,
indicating the absence of any disulfide linkage between F1
and F2, but also incorporation into BVs as such. The result
for vAcBac-HaFC108G was consistent with the supposition
that C108 is the residue used in the linking the F2 subunit to
the F1 subunit. Although the C241 residue is essential for
HaF to rescue the infectivity of vHaBacDF (Fig. 2d, k), an
inter-subunit disulfide linkage was detected in the HaFC241G
protein (Fig. 3b, lane 3). The presence of the AcMNPV EFP
GP64 and the major capsid protein VP39 in BVs of the
recombinants was confirmed by SDS-PAGE under reducing
conditions followed by Western blot analysis using antibodies against AcMNPV GP64 and VP39, respectively (Fig.
3c, d). It seemed that the expression of HaF or mutated HaF
proteins disturbed the expression or insertion of GP64.
When equal amounts of infectious BVs were analysed based
on the capsid protein (Fig. 3d), higher levels of HaF or
mutated HaF proteins correlated with a lower GP64 quantity
in the same virus (Fig. 3a–c).
BVs of vHaBacDF-HaFC232G, vHaBacDF-HaFC403G and
vHaBacDF-HaF harvested from the supernatants of
infected HzAM1 cells were also analysed in parallel (Fig.
3e, f, lanes 7–9), and the circumstances of the major intersubunit disulfide bond formation were consistent with that
of HaF expressed in WT AcMNPV BVs (Fig. 3b, lanes 2, 5
and 6). For vAcBac-HaFC108G under non-reducing conditions, besides the monomer of the F1 subunit, bands of
~120 and 180 kDa were observed, the apparent size of
which was consistent with a possible dimer and trimer,
although this would need to be confirmed (Fig. 3b, lane 1),
suggesting intermolecular disulfide linkages of F1 subunits.
It is possible that in the absence of C108 in the F2 subunit,
most C241 residues form intermolecular disulfide linkages
with cysteine residues of another F1 instead of forming
intramolecular disulfide bonds with other cysteine residues
within F1. Bands with higher molecular mass ~160 kDa,
which might be a dimer of F1+2, were also observed in
HaFC241G and HaF; for HaFC232G such a band was at
150 kDa (Fig. 3b, f). The original blots are shown in Fig. S1
(available in the online Supplementary Material).
Production of infectious BVs and genomic DNA
To evaluate the role of cysteine residues in HaF in BV
production in more detail, one-step growth curve analysis
was performed on vHaBacDF-HaFC232G, vHaBacDFHaFC403G and vHaBacDF-HaF (control). HzAM1 cells
were infected with the respective BVs at m.o.i. 10 and
supernatants were collected at the indicated time points.
Titres of the progeny virus were determined by end-point
dilution assays (EPDAs). Statistical analysis showed that at
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2823
vA
c
Ba
vA c-H
cB
a
ac F C1
08
vA
H
cB aF C G
a
23
2G
vA c-H
aF
cB
C
ac
24
1G
-H
vA
a
FC
cB
23
ac
2/
-H
24
vA
1G
a
FC
cB
4
03
ac
G
-H
aF
vH
aB
ac
vH
F
aB -Ha
FC
ac
23
vH
F2G
H
aB
ac aF C
40
F3G
H
aF
F. Yin and others
(a) kDa
(e)
170
130
95
72
Anti-HaF1
reducing gel
F1
55
(b)
(f)
170
130
Anti-HaF1
non-reducing gel
95
F1+2
72
F1
55
(c)
1
2
3
4
5
6
Anti-GP64
VP39
Anti-VP39
7
8
9
48, 72 and 96 h p.i., infectious BV production of the three
recombinant viruses was significantly different (P,0.05)
(Fig. 4). Initially, at 24 h p.i., the BV production of
vHaBacDF-HaFC232G was significantly higher than the
control virus vHaBacDF-HaF (P50.046), whilst no significant difference was found between vHaBacDF-HaFC403G
and the control virus (P50.96) (Fig. 4). These results
indicated that the mutation at C232 increased the ability of
HaF to rescue the infectivity of an f-null HaBacmid. In
contrast, mutation at C403 impaired infectious viral
production.
To further investigate whether the differences in viral titre
were due to variation in genomic DNA production or in
viral infectivity, quantitative real-time (qRT)-PCR analysis
was performed. Primer pair Halef8-F/Halef8-R was
designed to amplify a 64 bp fragment within the conserved
LEF-8 gene of HearNPV. Samples collected at 72 h p.i. in
the one-step growth curve assay were evaluated. The results
showed that genomic DNA production of vHaBacDFHaFC232G and vHaBacDF-HaFC403G was approximately
equivalent, and ~1 log unit higher than that of the control
vHaBacDF-HaF (Table 1). For vHaBacDF-HaFC403G,
1 TCID50 unit was equivalent to 1.86104 copies of
viral genomic DNA, and for vHaBacDF-HaFC232G and
vHaBacDF-HaF 1 TCID50 unit was equivalent to ~103
copies of viral genomic DNA (Table 1). When corrected for
the presence of viral genomes, there was no significant
difference in productivity between vHaBacDF-HaFC232G
2824
and vHaBacDF-HaF (P50.057), but the productivity of
vHaBacDF-HaFC403G was 10-fold lower than vHaBacDFHaFC232G and vHaBacDF-HaF (P,0.001).
109
108
Titre (TCID50 units ml–1)
(d)
GP64
Fig. 3. Western blot analysis of protein
expression in recombinant virions collected
from infected cells. (a, b) The expression and
post-translational cleavage of HaF mutants
HaFC108G (lane 1), HaFC232G (lane 2),
HaFC241G (lane 3), HaFC232/241G (lane 4),
HaFC403G (lane 5) and HaF (lane 6) in
recombinant AcMNPV BVs were detected
under reducing conditions (a) and non-reducing conditions (b) with anti-HaF1 antiserum.
(c, d) The expression levels of GP64 and
nucleocapsid protein VP39 (internal control) in
recombinant AcMNPV BVs were examined
with anti-GP64 and anti-VP39 antiserum,
respectively. (e, f) The vHaBacDF-HaFC232G,
vHaBacDF-HaFC403G and vHaBacDF-HaF
BVs were analysed in parallel. The positions
of F1 and F1+2 are indicated.
107
106
105
vHaBacΔF-HaFC232G
104
vHaBacΔF-HaFC403G
103
vHaBacΔF-HaF
102
0
24
48
72
96
Time (h p.i.)
Fig. 4. One-step growth curve analysis of infectious BV
production. HzAM1 cells were infected at m.o.i. 10 with either
vHaDF-HaFC232G, vHaDF-HaFC403G or vHaDF-HaF BVs.
Supernatants were harvested at the indicated time points postinfection and infectious BV yield was determined by EPDAs in
HzAM1 cells. Data represent the mean±SD titres from triplicate
infections.
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Journal of General Virology 95
Identification of inter-subunit disulfide bonds of HaF
Table 1. qRT-PCR analysis of BV genomic DNA production and comparison of viral infectivity
BVs of vHaDF-HaFC232G, vHaDF-HaFC403G and vHaDF-HaF sampled in one-step growth curve infections at 72 h p.i. were collected and genomic
DNA was extracted. Copies of genomic DNA were determined by qRT-PCR based on a standard curve. The infectivity of each sample was
calculated independently by comparing the copies of genomic DNA with the viral titre and the values represent the mean±SD infectivity of the
three samples.
Recombinant virus
Genomic DNA (copies ml”1)
Titre of recombinant virus
(TCID50 units ml”1)
Viral infectivity (DNA copies
per TCID50 unit)
vHaBacDF-HaFC232G
vHaBacDF-HaFC403G
vHaBacDF-HaF
6.3±0.9961011
2.0±0.4861011
2.3±0.4761010
3.8±0.536108
1.2±0.336107
7.3±1.566107
1650±43.2
18 133±3751.8
763±291.5
Inhibitory effect of thiol/disulfide exchange
inhibitors on the infectivity of HearNPV to HzAm1
cells
To investigate the role of cysteines and thiol/disulfide
exchange during viral entry, infection assays were conducted in the presence of inhibitors of thiol/disulfide
isomerization. Three membrane-impermeable inhibitors
were chosen in our study: tris(2-carboxyethyl) phosphine
(TCEP) is a reducer, and both 4-acetamido-49-maleimidylstilbene 2,29-disulfonic acid (AMS) and DTNB are freethiol reaction reagents. All three drugs were used at 1 and
2.5 mM, i.e. concentrations that were reported not to cause
cytotoxicity (Abou-Jaoudé & Sureau, 2007). To check the
effect of selected drugs on cell metabolism and viral
replication, control experiments were conducted in which
the drugs were added to the cell supernatant after 2 h of
virus exposure to the cells. Cells were then incubated with
the drugs for 2 h, after which the medium was replaced
with Grace’s medium containing 10 % FBS. The proportion of infected cells was quantified 24 h p.i. using flow
cytometry. The infection rates of groups did not show any
dramatic difference compared with the group without any
inhibitors (~95 % for TCEP, 112 % for DTNB and 115 %
for AMS), indicating that none of the drugs interfered with
cell metabolism or HearNPV replication. To investigate the
role of thiol/disulfide exchange at the stage of viral entry,
HzAM1 cells were incubated with vHaBacDF-HaF in the
presence of TCEP, DTNB or AMS and left for a 2 h virus–
cell interaction period. The inocula were then removed and
fresh Grace’s medium with 10 % FBS was added. The
infection rate of the control, which was not treated with
any inhibitor, was set as 100 %. As shown in Fig. 5(a),
TCEP dramatically reduced viral entry in a dose-dependent
manner, indicating a significant inhibitory effect, in
contrast to the situation when free-thiol reaction reagents
(DTNB and AMS) were used, where no obvious inhibitory
effect was observed. These results indicated that disulfide
bonds, but not free thiol groups, play critical roles in the
entry of HearNPV into HzAM1 cells.
The roles of surface-exposed disulfide bonds and free-thiol
groups on virion particles were investigated by treating
virions with individual inhibitors for 2 h at room temperature. After treatments, viruses were diluted 100-fold in
http://vir.sgmjournals.org
culture medium and inoculated to HzAm1 cells. At 2 h p.i.,
the supernatants were removed and replaced with fresh
medium. The inhibition relationships were very similar to
the above assays in that TCEP showed a significant inhibitory effect on viral infectivity, whilst neither DTNB nor
AMS reduced viral infectivity compared with the control
(Fig. 5b). These results implied that it was not the free thiol
groups on the surface of vHaBacDF-HaF BVs, but the
disulfide bonds within HaF that were important for viral
infectivity. To investigate whether cell surface-associated
PDI is involved in viral entry, HzAM1 cells were treated
with individual drugs for 2 h at 27 uC. Inhibitors were then
removed, and the cells were inoculated with vHaBacDFHaF and left for a 2 h to allow cell–virus interaction. As
shown in Fig. 5(c), no significant difference was observed
between drug- and mock-treated groups, indicating that no
disulfide bonds or free-thiol groups on the surface of host
cells were necessary for viral entry.
DISCUSSION
As a member of the class I EFPs, the F protein of baculovirus is cleaved post-translationally into an N-terminal F2
subunit and a C-terminal, membrane-anchored F1 subunit
by the cellular protease furin, and the two F subunits
are linked by a disulfide bond (Westenberg et al., 2002).
Cysteine residues are highly conserved among baculovirus
F proteins, suggesting that they are important to the
structure and function of the proteins. In this study, the
identity and importance of disulfide bonds of HaF were
investigated by mutagenesis of cysteine codons to glycine
codons and the functions of the altered F proteins were
analysed via rescue of the infectivity of an f-null HaBacmid.
The role of thiol/disulfide exchange in viral entry and
infectivity was also explored using membrane-impermeable
thiol/disulfide exchange inhibitors.
The results demonstrated that a functional disulfide bond
was formed between the F1 and F2 subunits of HaF between
the only cysteine residue, C108, in the F2 subunit and
C241, the most N-terminally conserved cysteine residue in
the F1 subunit (Fig. 6a, solid line). Mutation of C108 in F1
completely abolished disulfide bond formation and the
generation of infectious BVs. When C241 was mutated,
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2825
F. Yin and others
Co-inoculation of virions
with inhibitors
(a)
120
TCEP
DTNB
Infected cells (%)
100
AMS
80
60
40
20
0
Control
1 mM
2.5 mM
Concentration of inhibitor
120
120
TCEP
AMS
80
60
40
TCEP
DTNB
100
DTNB
Infected cells (%)
Infected cells (%)
100
AMS
80
60
40
20
20
0
Pre-treatment of cells
with inhibitors
(c)
Pre-treatment of virions
with inhibitors
(b)
Control
1 mM
2.5 mM
Concentration of inhibitor
0
Control
1 mM
2.5 mM
Concentration of inhibitor
Fig. 5. Inhibition assay of HearNPV with thiol/disulfide exchange inhibitors. (a) Inhibitory effect of inhibitors on viral entry. HzAm1
cells were incubated with vHaBacDF-HaF at m.o.i. 1 in Grace’s medium in the presence of TCEP, DTNB or AMS at
concentrations of 1 or 2.5 mM for 2 h. The inocula were then removed and fresh Grace’s medium with 10 % FBS was added.
(b) Infection assay with vHaBacDF-HaF virions pre-treated with inhibitors prior to inoculation. vHaBacDF-HaF particles were
treated with TCEP, DTNB or AMS at concentrations of 1 or 2.5 mM for 2 h at room temperature, and then 1 : 100 diluted in
culture medium and inoculated to HzAm1 cells at m.o.i. 1. At 2 h p.i., the supernatants were removed and replaced with fresh
medium. (c) Infection assay with HzAM1 cells pre-treated with inhibitors. HzAM1 cells were incubated with each inhibitor at
concentrations of 1 or 2.5 mM for 2 h at 27 6C. Supernatants with inhibitors were removed before inoculation with vHaBacDFHaF. After a 2 h cell–virus exposure period, the inocula were replaced with normal Grace’s culture medium. For all these assays,
the infection rates were quantified at 24 h p.i. by flow cytometry. Each infection was performed in triplicate and the infection rate
without inhibitors was set as 100 %.
however, the inter-subunit disulfide bond between F1 and
F2 was not destroyed, but the resulting protein failed to
rescue the infectivity of the f-null HaBacmid. Inter-subunit
disulfide bonds were no longer detected with the double
mutation at both C241 and C232 (Fig. 6e). These results
indicate a partial rescue effect of C241 by C232 in the
formation of the inter-subunit disulfide bond.
Interestingly, a mutation at C232 increased infectious BV
production by more than five times (Fig. 4, Table 1).
Whether C232 plays a role in the synthesis of HaF and
whether the linkage between C108 and C232 exists in
the WT virus are currently unknown. Considering the
existence of the residue in similar positions in almost all
baculovirus F proteins, even in the F-like protein of group I
2826
nucleopolyhedroviruses (Fig. 1a), we propose that C232
plays an important role in F protein synthesis and/or
function. Its potential importance needs to be addressed
further.
The organization of cysteine residues and functional motifs
of the baculovirus F protein shares large similarities with
paramyxovirus F proteins when compared with other class
I viral EFPs, such as influenza virus HA and HIV-1 envelope proteins (Fig. 6a, g) (Baker et al., 1999; Iwata et al.,
1994; Lamb & Jardetzky, 2007). Assignment of disulfide
bridges in the F protein of Sendai virus, a member of the
family Paramyxoviridae, demonstrated that cysteine residues clustered upstream of the TMD form intra-subunit
disulfide bonds and were supposed to contribute to the
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Journal of General Virology 95
Identification of inter-subunit disulfide bonds of HaF
Baculovirus F protein (HaF) and mutants
BVs
S S
S S
F2
HaF
C
CCCCCCCC
Functional
(a)
SP
FP HR1 RH3
F2
HaFC108G
C
CC
HR2 TM CTD
F1
CCC CCCCC
Deficient
(b)
FP HR1 RH3
SP
HaFC232G
S
F2
S
C
(c)
HR2 TM CTD
F1
GC
CCCCCCCC
Functional
FP HR1 RH3
SP
HaFC241G
(d)
S
F2
S
C
Deficient
F1
CCCCCCCC
FP HR1 RH3
HR2 TM CTD
F1
F2
C
(e)
HR2 TM CTD
CG
SP
HaFC232/241G
GG
CCCCCCCC
Deficient
SP
FP HR1 RH3
S
S
HaFC403G
F2
(f)
F1
CC
S
S
C
Functional
HR2 TM CTD
F1
CC
SP
CCCCCCCC
FP HR1 RH3
HR2 TM CTD
Paramyxovirus F protein (Sendai virus F)
F2
S
S
F1
(g)
SP
FP HR1
HR2 TM CTD
Fig. 6. Schematic diagram of inter-subunit disulfide bonds of (a) HaF and (b–f) mutated HaF on BVs, and (g) comparison with
paramyxovirus F protein. The black circles on the BVs indicate functional HaF; white circles indicate deficient HaF, but with
inter-subunit disulfide bonds; triangles indicate deficient HaF without any inter-subunit disulfide bonds. The relative positions of
the signal peptide (SP), fusion peptide (FP), heptad repeats (HR1, HR2 and HR3), TMD (TM), CTD are indicated. The
confirmed disulfide bonds are shown by solid lines and the proposed disulfide bonds are shown by dashed lines. The mutations
of cysteine to glycine are indicated by stars in mutated HaFs.
formation of a bunched structure (Iwata et al., 1994).
Investigation of cysteine residues of the F protein of HRSV,
a member of the subfamily Pneumovirinae within the
family Paramyxoviridae, revealed that most of the cysteine
residues in the cysteine cluster are critical for folding,
transport and fusion activity of the protein (Day et al.,
2006). For HaF, the mutation at C403, which is one of the
cysteine residues in the cysteine cluster, decreased viral
infectivity .10 times as compared with WT HaF (Figs 4
and 6f). It is reasonable to speculate that cysteine residues
clustered upstream of the TMD of HaF1 form intra-subunit
disulfide bonds, and play important roles in the synthesis,
processing and functional structural stability of HaF.
For several viruses, such as NDV, which enter cells by
fusion of the viral membrane to the plasma membrane, the
activation of the fusogenicity depends on the precise thiol/
http://vir.sgmjournals.org
disulfide rearrangements (Abell & Brown, 1993; Jain et al.,
2007, 2009). For other viruses, such as influenza virus,
although the conformational changes needed to activate
fusogenicity are triggered mainly by the acidic milieu, the
exchange of thiol/disulfide groups still plays certain roles
in the process of membrane fusion (Carr et al., 1997;
Markosyan et al., 2001). No disulfide linkage was detected
in the oligomerization of the baculovirus F protein and the
protein was detected by chemical cross-linking to form
homotrimers in which the monomers are associated by
non-covalent interactions (Long et al., 2006).
In the current study, free-thiol groups in F proteins were
not thought to play important roles in viral entry and
infectivity, as evidenced by inhibition assays with free-thiol
reaction reagents (Fig. 5a, b). These results reduce the
likelihood that thiol/disulfide exchange plays a critical role
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2827
F. Yin and others
in the conformational change of F proteins. The observation
that inhibition of the activity of cell surface-associated PDI
did not inhibit viral entry provides further support for this
hypothesis (Fig. 5c). However, there is a possibility that
functional free-thiol groups or disulfide bonds of PDI on
the host cell plasma membrane are not accessible to the
impermeable inhibitors. The observation that the reductant
TCEP dramatically reduces the infectivity of HearNPV is
possibly due to the breakage of disulfide bonds that are
essential for the structural stability of HaF. Further detailed
studies are needed to address whether thiol/disulfide
exchange plays a role in baculoviral entry.
primer pair used to generate the C-terminal domain was HaFC108GCF/HaF-R (Table S1). The other HaF gene mutants, HaFC232G,
HaFC241G and HaFC403G, were amplified similarly with the respective
primers listed in Table S1. The two PCR products were then annealed
together and primer pair HaF-F/HaF-R was used to generate
complete mutated HaF genes. Mutated HaF gene fragments were
then cloned into pGEM-T Easy vector, and designated pT-HaFC108G,
pT-HaFC232G, pT-HaFC241G and pT-HaFC403G. The double-cysteine
mutated HaF gene HaFC232/241G was constructed according to the
method described for single-residue mutation, but using pTHaFC232G as template, and the primers were the same as used for
generating the HaFC241G gene mutant. All of the HaF gene mutants
were verified by sequencing and then inserted into pFastBac1-Op166
to generate donor plasmids.
The high conservation of cysteine residues is a striking
feature of baculovirus F proteins. In this study, we
demonstrated that an inter-subunit disulfide bond of the
F protein subunits of HearNPV is formed by C108 and
C241. C232, the location of which is not highly conserved
amongst baculovirus F proteins, appears to form a disulfide
bond with C108 in the absence of C241, but this does not
compensate functionally for the natural C108–C241
disulfide bond. We also detected a cysteine residue, C403,
which may have the potential to form intra-subunit disulfide bonds in the F1 subunit on the basis of the structural
similarity with paramyxovirus F proteins (Dutch et al.,
2000). The observation that free-thiol groups are not
involved in the F-protein-mediated entry of baculovirus in
insect cells indicates that successful entry of BV is entirely
dependent on the inter- and intra-subunit sulfur bridging.
These results may also contribute to a further understanding of the structure–function relationship of the baculovirus F protein. Crystallization of a baculovirus F protein is
required to substantiate the structure–function claims as
discussed in this paper.
Mutated HaF genes and the WT HaF gene under the control of the
Orgyia pseudotsugata multiple nucleopolyhedrovirus GP64 gene
promoter (Op166) were transposed into both HaBacDF (Fig. 1b)
and AcBacmid (Fig. 1c) by Tn7-mediated transposition according to
the Bac-to-Bac Baculovirus Expression System manual (Gibco-BRL).
Colonies resistant to both tetracycline and kanamycin were further
confirmed by PCR with M13/pUC-F and M13/pUC-R primers.
METHODS
Insect cells, virus and reagents. The HzAM1 and Sf9 cell lines
were cultured at 27 uC in Grace’s medium (Gibco-BRL) supplemented with 10 % FBS, pH 6.0. The HearNPV bacmid (HaBacHZ8) and
f-null bacmid HaBacDF used in the experiment were constructed
previously in our laboratories (Wang et al., 2003; Wang et al., 2008b).
TCEP and DTNB were purchased from Sigma. AMS was purchased
from Invitrogen Molecular Probes.
Transfection and infection assay. Samples of 3 mg of each
recombinant HearNPV bacmid DNA were transfected into 56105
HzAM1 cells using 12 ml Lipofectin reagent (Invitrogen). Supernatants
were harvested at 5 days post-transfection. After centrifugation at
1000 g for 5 min, 0.5 ml supernatant was used to infect another batch
of HzAM1 cells. The percentage of transfected and infected cells was
monitored using fluorescence microscopy. For BV amplification,
5.06106 HzAM1 cells were infected at m.o.i. 0.1 TCID50 units per
cell and the supernatant was collected at 5 days p.i. Each of the
transfections was performed in triplicate. The transfection and
infection assays of recombinant AcBacmids were performed on Sf9
cells using a similar procedure.
Western blot analysis. BVs used for Western blot analysis were
collected from infected cell culture supernatants and purified by
ultracentrifugation through a 25 % sucrose cushion. Samples were
disrupted in Laemmli buffer for electrophoresis under reducing
conditions or in buffer containing 10 mM Tris/HCl (pH 6.8), 0.5 %
SDS, 10 % glycerol, 50 mM iodoacetamide and 0.001 % bromophenol
blue for electrophoresis under non-reducing conditions. Proteins were
separated by 10 % SDS-PAGE and then electroblotted onto ImmobilonP membranes (Millipore). Western blot analysis was performed with
various polyclonal antibodies: anti-HaF1 (diluted 1 : 500; Wang et al.,
2008b), anti-GP64 (diluted 1 : 2000; Wang et al., 2008a) and anti-VP39
(diluted 1 : 1000; Wang et al., 2008a). Detection of antibody binding was
visualized by alkaline phosphatase-conjugated goat anti-rabbit secondary antibodies (NovoGene Biosciences). The final signals were detected
with nitro-blue tetrazolium and BCIP.
Construction of recombinant bacmids. For the construction of
donor plasmids, the HaF gene was PCR amplified from the
HaBacHZ8 template with primers HaF-F and HaF-R using Pyrobest
DNA polymerase (Takara). The PCR product was cloned into pGEMT Easy vector (Promega) to generate pT-HaF. Following sequence
confirmation, the HaF gene fragment was digested away from pT-HaF
and then inserted into pFastBac1-Op166 (Wang et al., 2008b) to
generate donor plasmid pFastBac1-Op166-HaF. Mutated HaF genes
were constructed by overlapping extension PCR as reported by Ho
et al. (1989). Briefly, mutated HaF genes were first PCR amplified as
two fragments with pT-HaF as template. Each of the N-terminal
domains was amplified with primer HaF-F and the N-terminal reverse
mutagenic primer. For the C-terminal domain, PCR was performed
with C-terminal forward mutagenic primer and HaF-R. For example,
the primer pair used to generate the N-terminal domain of the
mutated HaFC108G gene was HaF-F/HaFC108G-NR (Table S1) and the
2828
One-step growth curve analysis. To perform one-step growth
curve analysis, 56105 HzAM1 cells were incubated with vHaDFHaFC232G, vHaDF-HaFC403G or vHaDF-HaF at m.o.i. 10 TCID50 units
per cell for 1.5 h at 28 uC. Cells were then washed for three times with
Grace’s medium before addition of 2 ml fresh Grace’s medium
(supplemented with 10 % FBS). Supernatants were harvested at 0, 12,
24, 48 and 72 h p.i., and, after clarification by centrifugation,
infectious BV yields were determined by EPDAs using HzAM1 cells.
The infection experiments were performed in triplicate, and the mean
BV titres were analysed using one-way ANOVA (SPSS) with virus
type and time as factors.
qRT-PCR. To determine the differences of genomic DNA production
among recombinant viruses by qRT-PCR, primers Halef8-F and
Halef8 were designed to amplify a 64 bp fragment within the LEF-8
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Journal of General Virology 95
Identification of inter-subunit disulfide bonds of HaF
gene. Aliquots of 100 ml supernatant were collected in each sample of
the one-step growth curve infections at 72 h p.i. and genomic DNA
was extracted as described by Wang et al. (2010). qRT-PCR was
carried out with EvaGreen fluorescent dye (Biotium) using 40 cycles
of 95 uC for denaturation and 53 uC and 72 uC for annealing/
extension.
Infection of HzAm1 cells with thiol/disulfide exchange inhibitors. To test the effect of selected drugs on cell metabolism and viral
replication, cells were inoculated with vHaBacDF-HaF at m.o.i.
1 TCID50 unit per cell for 2 h, and drugs were then added to the
supernatant and incubated for another 2 h. The supernatant was then
replaced with fresh Grace’s medium and left for 24 h before
quantification by flow cytometry. The inhibitory effects of TCEP,
DTNB and AMS on viral infectivity of vHaBacDF-HaF were tested
during different stages of infection. (i) Inhibitory effect of inhibitors
on viral entry. HzAm1 cells were incubated with vHaBacDF-HaF at
m.o.i. 1 TCID50 unit per cell in the presence of each inhibitor at a
final concentration of 1 or 2.5 mM and left for 2 h. (ii) Pre-treatment
of virions with inhibitors. vHaBacDF-HaF BVs were pre-treated with
each inhibitor for 2 h at room temperature, and then diluted 100-fold
in culture medium and inoculated to HzAm1 cells at m.o.i. 1 TCID50
unit per cell. (iii) Pre-treatment of cells with inhibitors. HzAm1 cells
were pre-incubated with each inhibitor for 2 h at 27 uC; supernatants
with inhibitors were then removed and the cells were inoculated with
vHaBacDF-HaF at m.o.i. 1 TCID50 unit per cell. Infections without
any inhibitor were set up as control. For all the infections, after a 2 h
cell–virus exposure period at 27 uC, the inocula were removed and
normal Grace’s medium with 10 % FBS was added. The infection
rates were quantified at 24 h p.i. by flow cytometry.
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ACKNOWLEDGEMENTS
Jain, S., McGinnes, L. W. & Morrison, T. G. (2007). Thiol/disulfide
This work was supported by grants from the National Natural Science
Foundation of China (31125003, 31100120, 31370191, 31130058 and
31200124), National Basic Research Program of China (2010CB530100)
and Programme Strategic Scientific Alliances between China and The
Netherlands (2008DFB30220). The authors would like to thank Dr Basil
M. Arif for the scientific editing of the manuscript. We thank Professor
Kai Yang for providing anti-VP39 antiserum, Dr Xiulian Sun for
statistical analysis and Ms Yanfang Zhang for cell culture.
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