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Li-Xin
Zhu, Jing Liu, Ying-Chun Li, Yu-Ying Kong, Yuan Wang, Guang-Di Li, Institute
of Biochemistry and Cell Biology, Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences, Shanghai 200031, China
Caroline Staib, Gerd Sutter, GSF-Institut für Molekulare Virologie,
Trogerstr. 4b, 81675 München, Germany. The first two authors
contributed equally to this paper.
Supported by the National 863 High Technology Foundation of
China, No.863-102-07-02-02, No.2001AA215171 and the project CHN
98/112 (WTZ-Internationales Büro des BMBF).
Correspondence to: Yuan Wang and Guang-Di Li, Institute of
Biochemistry and Cell Biology, Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai
200031, China. wangyuan@server.shcnc.ac.cn
Telephone: +86-21-64374430 Fax: +86-21-64338357
Received 2001-12-05 Accepted 2002-01-23
Abstract
AIM: To study HCV polyprotein processing is important for the
understanding of the natural history of HCV and the design of
vaccines against HCV. The purpose of this study is to investigate
the affection of context sequences on hepatitis C virus (HCV) E2
processing.
METHODS: HCV genes of different lengths were expressed and
compared in vaccinia virus/T7 system with homologous patient serum
S94 and mouse anti-serum ME2116 raised against E.coli-derived
E2 peptide, respectively.Deglycosylation analysis and GNA (Galanthus
nivalus) lectin binding assay were performed to study the post-translational
processing of the expressed products.
RESULTS: E2 glycoproteins with different molecular weights (～75kDa
and ～60kDa)
were detected using S94 and ME2116, respectively.
Deglycosylation analysis showed that this difference was mainly due
to different glycosylation. Endo H resistance and its failure to
bind to GNA lectin demonstrated that the higher molecular weight
form (75kDa) of E2 was complex-type glycosylated, which was readily
recognized by homologous patient serum S94. Expression of
complex-type glycosylated E2 could not be detected in all of the
core-truncated constructs tested, but readily detected in constructs
encoding full-length core sequences.
CONCLUSION: The upstream conserved full-length core coding
sequence was required for the production of E2 glycoproteins
carrying complex-type N-glycans which reacted strongly with
homologous patient serum and therefore possibly represented more
mature forms of E2. As complex-type N-glycans indicated modification
by Golgi enzymes, the results suggest that the presence of
full-length core might be critical for E1/E2 complex to leave ER.
Our data may contribute to a better understanding of the processing
of HCV structural proteins as well as HCV morphogenesis.
Zhu LX, Liu J, Li YC, Kong YY, Staib C, Sutter G, Wang Y, Li GD.
Full-length core sequence dependent complex-type glycosylation of
hepatitis C virus E2 glycoprotein.World J Gastroenterol
2002;8(3):499-504
INTRODUCTION
Hepatitis C virus (HCV), the major cause of post-transfusion and
community-acquired non-A, non-B hepatitis[1, 2], is a
member of the Flaviviridae family[3]. This virus has a
positive-sense, single stranded RNA genome of about 9.6 kb, which
encodes a polyprotein precursor of about 3000 amino acids. The
polyprotein is further processed into various precursors and mature
viral proteins[4, 5]. The structural proteins are encoded
in the order NH2-core-E1-E2-P7, which are processed into
core (C), E1, E2, and P7 by host membrane- associated signal
peptidase(s)[6-11]. The downstream nonstructural region
is processed by a viral metalloprotease and a viral serine protease
located at the N-terminus of NS3[12-17]. The core protein
is thought to constitute the viral capsid with E1 and E2 being the
virus envelope proteins. Numerous studies have shown that E1 and E2
are heavily glycosylated and associate to form a noncovalent
heterodimeric complex[9, 10, 18, 19]. E1 and E2 are
believed to be type I transmembrane proteins with an N-terminal
glycosylated ectodomain and a C-terminal hydrophobic anchor.
The
lack of an efficient in vitro cell culture system for productive HCV
propagation[20-27] and low levels of HCV particles in the
liver tissues or blood of infected patients[28, 29] have
hampered the study of native viral proteins. Fortunately, a variety
of prokaryotic and eukaryotic expression systems have proved useful
for the production and characterization of HCV encoded proteins[8,
9, 11, 30-32]. However, diverse findings have been reported,
regarding the molecular weights of E2, which most likely is a
reflection of the differences in efficiency of HCV polyprotein
processing and post-translational modification achieved in the
particular systems[7-9, 11, 17-19, 30]. In this study,
the E2 expression of recombinant plasmids carrying various length of
HCV C-E1-E2 coding sequences was analyzed in the vaccinia virus/bacteriophage
T7 RNA polymerase expression system[33]. The results
suggest that the upstream conserved core coding sequence is required
for the production of E2 glycoproteins carrying complex-type N-glycans
which react strongly with homologous patient serum and therefore
possibly represent more mature forms of E2.
MATERIALS AND METHODS
Cells and viruses
Human HeLa (ATCC #CCL-2) and monkey BS-C-1 (ATCC #CCL-26)
cells were maintained in Dulbecco's modified essential medium (DMEM/HG)
supplemented with 5% heat inactivated fetal calf serum (FCS) at 37℃
in a 5% CO2 atmosphere. Recombinant vaccinia virus vTT7
that expresses the bacteriophage T7 RNA polymerase gene under the
control of vaccinia virus early/late promoter P7.5 was generated and
propagated as previously described[34]. PFU (plaque
forming unit) titration was performed on BS-C-1 cell monolayers.
Plasmid constructions
The vaccinia virus/T7 promoter expression vector pTM1 was
kindly provided by Bernard Moss (NIH, Bethesda, USA) and all of the
expression plasmids carrying HCV cDNA encoding structural proteins
described below were derived from pTM1. Figure1 depicts the HCV gene
fragment in the expression plasmids. Plasmids pCEH-2 (1-730) and pEH
containing HCV C, E1 and E2 gene of subtype 1b[35] (GENBANK
accession #D10934) were described previously[34].
Briefly, cDNA sequences encoding HCV polyprotein amino acids 192 to
730 were inserted into pTM1 to obtain pEH. HCV sequences encoding
complete C were fused with the E1/E2 sequences of pEH to result in
plasmid pCEH-2(1-730). The latter plasmid served as basis for PCR
cloning to generate plasmids pCE1(1-341), ptCEH-2(108-730),
ptCEH-2(120-730), ptCEH-2(137-730), ptCEH-2(156-730),
ptCEH-2(167-730), pCEH-2(1-661) and pTM1/EH(192-661) for the
expression of 5' or/and 3' truncated HCV sequences encoding HCV
polyprotein amino acids as indicated by numbers in parenthesis.
Plasmid pC-E2tH(1-195/394-661) was generated by deleting E1 coding
sequences from pCEH-2(1-661) and linking polyprotein amino acids
aa194 and aa394 together. Authenticity of HCV cDNA sequences in all
constructed plasmids was confirmed by automatic sequencing.
Figure 1(PDF)Schematic
maps of HCV coding sequences inserted into the plasmid pTM1 and
expressed under transcriptional control of the bacteriophage T7 pol
promoter. Numbers refer to amino acids of the HCV polyprotein.
Transient expression of recombinant genes using vaccinia
virus/T7 RNA polymerase (VV-T7pol)
HeLa cells grown to 80% confluency were infected with
recombinant vaccinia virus vTT7 at a multiplicity of infection (MOI)
of 10 PFU per cell to allow for production of recombinant T7 RNA
polymerase. At 2h post-infection, the inoculum was removed and DNA
of pTM1 based expression plasmids was transfected using DOTAP
liposomal transfection reagent as described by the manufacturer
(Roche Molecular Biochemicals, Mannheim). After 24 hours of
incubation, infected/transfected cells were washed twice with PBS,
harvested by scraping, pelleted upon brief centrifugation, and
resuspended in a small volume of PBS. Cell lysates were prepared by
adding SDS-PAGE sample loading buffer and stored at -80℃
until further analysis.
Western blot analysis
Cell lysates were separated by reductive SDS-PAGE and then
transferred onto nitrocellulose membranes (Schleicher & Schuell).
Blocking was done using 5% fat-free milk powder. For immunodetection
of HCV proteins, blots were incubated with primary antibodies,
washed, and incubated with 1000 fold diluted HRP-protein A (Sigma).
The membranes were then washed again and reactive proteins were
detected using the ECL system (Amersham Phamacia Biotech) according
to the manufacturers' instructions. The primary antibodies used in
this study include: anti-HCV human serum S94 at a dilution of 1:500
(kindly provided by Wang, Y., Beijing University, China), anti-HCV
human serum S268 at a dilution of 1:500 (kindly provided by Lu Z.,
Shanghai Ruijin Hospital, China), anti-E2 mouse polyclonal antibody
ME2116[36] at a dilution of 1:300 (raised
against E. Coli-derived HCV E2 polypeptide aa450 to 565), and
anti-E1 rabbit polyclonal antibody RE1135-C at a dilution
of 1:250 (raised against an E. coli-derived C-terminally truncated
HCV E1 fragment).
Characterization of N-glycans on expressed E2 glycoproteins
For deglycosylation analysis, cell pellets were directly
lysed in denaturing buffer provided by the manufacturer and digested
with PNGase F (NEB) or Endo H (NEB) for 2 hours at 37℃.
The
type of N-glycans on expressed E2 glycoproteins was also analyzed by
testing its ability to bind to GNA (Galanthus nivalus) lectin. HeLa
cells infected with vTT7 and transfected with pCEH-2(1-730) were
collected by scraping, washed in cold PBS, and then lysed with lysis
buffer (50mM Tris-HCl [pH8.0], 150mM NaCl, 0.5% Nonidet P-40, 1mM
PMSF). After centrifugation at 10000g, the supernatant was allowed
to bind to GNA-agarose (Sigma). The flow-through fraction was
collected and the gel matrices were washed with lysis buffer. Bound
proteins were eluted with 1M α-D-mannopyranoside (Sigma) in
lysis buffer. Samples were then analyzed by Western-blotting.
RESULTS
Detection of E2 glycoproteins of different Molecular Weights
using antibodies of distinct origins
The hybrid vaccinia virus/T7 bacteriophage RNA polymerase
expression system was used to study the expression of the HCV
structural proteins. The transient expression products of plasmids
pCEH-2(1-730) and pCEH-2(1-661), which contain HCV cDNA encoding the
structural region terminating at amino acid 730 and 661 of the
polyprotein respectively, were analyzed by Western blot using
polyclonal mouse serum ME2116 raised against E.coli-derived
E2 protein[36]. E2 products with apparent molecular
weights (MWs) of ～60kDa
and ～50kDa
were detected for pCEH-2(1-730) and pCEH-2(1-661) respectively
(Figure 2, lane 1, 3). The apparent molecular weights were higher
than calculated values, which, along with the heterogeneous
appearance of the detected bands, suggested that these were
glycosylated expression products. The lower molecular weight of the
E2 species obtained from expression of pCEH-2(1-661) in comparison
to pCEH-2(1-730) was consistent with the introduced truncation at
the 3' end of the E2 coding sequences leading to the loss of 70
amino acids in the recombinant polypeptide backbone.
Figure 2 Detection
of E2 glycoprotein species of different molecular masses. Transient
expression products were analyzed by Western blot with antibodies of
distinct origin: mouse polyclonal antibody ME2116 (lane
1, 2, 3); HCV patient serum S94 (lane 4, 5, 6); HCV patient serum
S268 (lane 7, 8); rabbit polyclonal antibody RE1135-C
(lane 9, 10, 11). Empty vector pTM1 was used as the negative
control. HCV-specific protein bands are indicated by arrowheads. The
plasmids used for transfection are indicated at the top of the
lanes.
The
expression products were also analyzed with HCV patient serum S94.
Multiple prominent bands were detected for pCEH-2(1-730) and
pCEH-2(1-661), but not for vector plasmid pTM1, representing
expressed HCV structure proteins and possibly some precursors. The
bands of ～75
kDa and ～66
kDa (Figure 2, lane 4, 6), which again consistent with the different
length of E2 coding sequence in both plasmids, should represented
the E2 proteins, although their MWs were higher than that detected
by ME2116. It is worth noting that the analyzed HCV cDNA
originated from the same patient from whom S94 was collected. The
core antigen and some precursors of expression products could also
be detected by another HCV patient serum S268, but we could not
detect the E2-specific 75kDa band (Figure 2, lane 8), which could be
attributed to the high variability of the E2 glycoprotein[37,
38].
The
antibody dependent detection of different E2 glycoprotein species
was surprising. To rule out that the E2 bands of higher MW is the
uncleaved E1-E2 precursors, the expression products from
pCEH-2(1-730) were analyzed with anti-E1 rabbit sera. A heavy band
of about 30kDa was detected, possibly representing multiple forms of
E1 proteins (Figure 2, lane 10), while no E1-specific band with
higher MW was detected. Another possible explanation could be that
E2 polypeptides of varying sizes were synthesized due to incomplete
or irregular processing of the polyprotein. This hypothesis was
abandoned when we subjected recombinant proteins to deglycosylation
with PNGase F prior to Western blot analysis with mouse polyclonal
antibody ME2116. After PNGase F treatment, only one
E2-specific band with apparent molecular weight of 30 kDa was
detected for pCEH-2(1-661) expression products, while an E2-specific
doublet band of 33/34 kDa was detected for pCEH-2(1-730) expression
products (Figure 3), which is consistent with the MWs of calculated
E2 polypeptide backbones. It suggests that difference of the MWs of
E2 species detected by ME2116 and S94 from the same
transient expression products (pCEH-2(1-730): ～60kDa
and ～75kDa,
pCEH-2(1-661): ～50kDa
and ～66kDa)
is mainly due to different N-glycosylation. The detection of a E2
peptide doublet with pCEH-2(1-730) but not with pCEH-2(1-661) is in
agreement with the fact that E2 contains a PKR-eIF2α
phosphorylation site (PKR: RNA-activated protein kinase) at
aa659-670[39], which is largely deleted in the
pCEH-2(1-661) construct.
Figure 3 Deglycosylation
analysis of the expressed HCV E2 proteins with PNGase F. Transient
expression products were subjected to Western blot analysis with ME2116
as the primary antibody. pTM1 transfected cells were used as
control. The plasmids used for transfection are indicated at the top
of the lanes. Samples incubated with deglycosylation buffer were run
in parallel. +: samples digested with PNGase F, -: samples incubated
with PNGase F reaction buffer.
Altogether,
the above results suggest that the 75kDa and 66kDa bands detected by
S94 are HCV E2 glycoproteins of heavier glycosylation and thus
higher molecular weight.
ME2116- and S94- reactive E2 glycoproteins carried
different types of N-glycans
Since ME2116- and S94-reactive E2 species had
polypeptide backbones of the same size, the difference in apparent
molecular weight and antibody reactivity could only be attributed to
differences in the degree and/or type of glycosylation. The glycan
type on different E2 species expressed from pCEH-2(1-730) was then
analyzed by testing their sensitivity to PNGase F and Endo H. PNGase
F hydrolyzes all types of N-glycan chains from glycopeptides and
glycoproteins unless they carry α-1-3 linked core fucose
residues present in insect and plant glycoproteins[40],
while Endo H cleaves only high mannose structures and hybrid
structures on N-linked oligosaccharides of glycoproteins[41].
Figure 4A shows that the ME2116-reactive E2 species was
sensitive to both PNGase F and Endo H digestion. The S94-reactive E2
species disappeared after PNGase F digestion (Figure 4B). It was
difficult to detect the deglycosylated E2 with S94 after PNGase F
treatment, because there were multiple HCV polyprotein precursor
proteins of about 30000 reacted strongly with S94 and unglycosylated
E2 seemed to react weakly with S94 (unpublished data). However, the
highly glycosylated, S94-reactive E2 band remained after Endo H
digestion (Figure 4B). The glycan type of different E2 species
expressed from pCEH-2(1-730) were also analyzed by testing their
ability to bind to GNA lectin. GNA is specific for the non-reducing
end of α-D-mannosyl residue of glycoconjugate and therefore can
be used to probe the presence of high mannose type or hybrid type
glycans on glycoproteins[42, 43]. The ME2116-reactive
E2 species could quantitatively bind to and be eluted from
GNA-agarose, whereas no obvious binding could be demonstrated for
S94-reactive E2 species (Figure 5).
The
resistance of S94-reactive E2 glycoprotein species to Endo H
digestion together with the fact that it could not bind to GNA
indicates that the S94-reactive E2 protein carries complex-type
glycans.
Figure 4 Different
sensitivities of two glycosylated E2 species to PNGase F and Endo H.
Transient expression products of pCEH-2(1-730) were digested with
PNGase F or Endo H, respectively. The digested samples were analyzed
by Western blot with ME2116 (A) and S94 (B).
Empty vector pTM1 was used as negative control.Samples incubated
with deglycosylation buffer were run in parallel. +: samples
digested with Endoglycosidase, -: samples incubated with
Endoglycosidase reaction buffer.The plasmids used for transfection
are indicated at the top of the lanes. Endoglycosidases used in this
study are indicated at the bottom of the lanes. S94-reactive E2
proteins are indicated by arrowheads.
Figure 5 Different
ability of differently glycosylated E2 speicies to bind to
GNA-agarose. HeLa cells infected with vTT7 and transfected with
pCEH-2(1-730) were collected, washed and lysed with lysis buffer.
The cleared supernatant was then allowed to bind to GNA-agarose. The
gel beads were washed and eluted with 1M α-D-mannopyranoside in
lysis buffer. Pre-binding lysate, flow-through and eluant fractions
were analyzed by Western blot analysis. HeLa cells infected with
vTT7 and transfected with pTM1 were served as negative control. The
sera used as primary antibodies are indicated at the bottom of the
lanes. E2 proteins are indicated by arrowheads.
HCV core sequence dependent formation of complex-glycosylated
E2
We also assessed if co-expression of core or E1 coding sequences had
any effect on the production of the E2 proteins, which was readily
recognized by homologous patient serum S94. A set of expression
plasmids containing HCV cDNAs with various deletions in the core
sequence (ORF starting at aa108, aa120, aa137, aa151, or aa167,
respectively) were constructed. The lysates from vTT7-infected and
plasmid DNA transfected HeLa cells were analyzed by Western blot
with either the mouse anti-serum ME2116 or the patient
serum S94 (Figure 6).
Figure 6 Requirement
of the core sequence for the expression of complex-type glycosylated
E2. Expression products of differently truncated HCV structural
genes were analyzed by Western blot analysis. Blots were probed with
ME2116 (A) or with S94 (B).
Empty vector pTM1 was used as negative control. The plasmids used
for transfection are indicated at the top of the lanes.
When
using mouse antibody ME2116, recombinant E2 of ～60kDa
and ～50kDa
could be detected upon expression of all constructs tested (Figure
6A). Patient serum S94 allowed detection of E2 for pCEH-2(1-730) and
pCEH-2(1-661) with full-length core coding sequences (Figure 6B,
lanes 7, 9), similar to that described in Figure 2. In contrast, no
E2 products could be visualized after expression of constructs
containing no or only partial core sequences (Figure 6B, lanes 1-6).
Interestingly, deletion of E1 coding sequences had no significant
effect on the synthesis of S94 detectable E2 protein (Figure 6B,
lane 10). These results suggests that the presence of complete HCV
core sequence is crucial for the expression and/or post-translational
processing of the complex-type glycosylated form of E2.
DISCUSSION
In this study, various constructs of HCV cDNAs placed under
transcriptional control of the bacteriophage T7 promoter were
transiently expressed using vaccinia virus/T7 system. Upon
characterization of the HCV gene products with different antibodies,
two species of E2 with different MWs were identified in the
expression products of the same plasmid. The high molecular weight
forms of E2 were readily recognized by a patient serum, but
displayed weak reactivity with antibodies raised against E. coli
derived E2. These high molecular weight forms of E2 were not likely
produced from inefficient proteolytical processing at the E1/E2
boundary as these proteins were not stained with E1-specific
antibodies. Efficient processing at E1/E2 was confirmed by
deglycosylation analysis. The difference of the MWs of E2 species
detected by S94 and by ME2116 was therefore mainly due to
different N-glycosylation. The S94-reactive E2 glycoproteins, which
were resistant to Endo H digestion and could not bind to GNA, carry
complex-type glycans.
The
specific recognition of the complex-type glycosylated E2 but not the
high-mannose-type glycosylated E2 by homologous patient serum S94
suggested that the former could be a better representation of native
E2 proteins on HCV virions. Similar results were also reported by
Inudoh et al[44]. By comparing the reactivity of
complex-type glycosylated E2 and the high-mannose-type glycosylated
E2 with different patient sera, they demonstrated that the former is
superior in diagnosing HCV infection. Their results and our results
reported here are in concordance with the finding that E2 protein on
patient derived virions contained complex-type sugars indicating
Golgi-specific modification[45].
Expression
of full-length or C-terminally truncated envelop proteins in
eukaryotic cells has demonstrated that E1 and E2 are retained within
the ER membrane system due to the presence of ER-retention signals
in the C-termini of both envelope proteins[46-50].
However, recent study indicates that HCV E2 proteins could also
present in the Golgi apparatus of the stably transfected cell line
expressing HCV C-E1-E2-NS2 fragment. A possible explanation could be
that Martire et al[51] used an HCV gene fragment
including full-length core sequences in their study while structural
protein sequences without full-length core sequences were used to
study the localization of envelop proteins. The results reported
here demonstrated that the complex-type glycosylated, possibly more
mature form of E2 is only detectable upon co-expression of the
complete HCV core coding sequence. Deletion of the first 107
N-terminal core amino acid residues was obviously sufficient to
abrogate production of complex-glycosylated E2. This result suggest
that the core protein might allow for targeting the envelope
glycoproteins to Golgi-specific modification, which could be a key
step in the morphogenesis of HCV virions. HCV-like particles were
observed when HCV cDNA encoding whole core, E1 and E2 was expressed
in baculovirus-insect expression system[52]. After
binding of core to the E1-E2 complex statically located on the ER
membrane, virus-like particles might be formed and the conformation
of E1-E2 complex changed, which could result in the abrogation of
the ER-retention signal for the E1-E2 complex. Then the virus-like
particles might migrate along the secretion pathway, where E2 (and
E1) proteins undergo more complex glycosylation by the Golgi
enzymes.
In
summary, upon expression of recombinant HCV core, E1, and E2
sequences, the E2 proteins of different glycosylations could be
identified. The complex-glycosylated E2 protein might represent a
more mature form of E2 and its formation required the conserved core
coding sequences. Our data may contribute to a better understanding
of the processing of HCV structural proteins as well as HCV
morphogenesis.
ACKNOWLEDGMENT
We thank Wang, Y. and Lu, Z. for providing the HCV clones and
the HCV specific patient sera.
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