Download Interaction between hepatitis delta virus

Journal
of General Virology (1998), 79, 1115–1119. Printed in Great Britain
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SHORT COMMUNICATION
Interaction between hepatitis delta virus-encoded proteins and
hepatitis B virus envelope protein domains
Christophe Hourioux,1, 2 Camille Sureau,3 Francis Poisson,1 Denys Brand,1 Alain Goudeau1
and Philippe Roingeard1, 2
Laboratoire de Virologie EP CNRS 1171 and Laboratoire de Biologie Cellulaire2, Faculte! de Me! decine, 2 bis Boulevard Tonnelle! ,
F-37032 Tours, France
3
Laboratoire de Virologie URA CNRS 1487, Institut de Biologie, 2 Boulevard Henri IV, F-34060, Montpellier, France
1, 2
Hepatitis delta virus (HDV) packaging requires
prenylation of the HDV large protein (p27), as well
as a direct protein–protein interaction between
HDV proteins and hepatitis B virus (HBV) envelope
protein domains. To investigate this interaction, we
have analysed the binding capacity of baculovirusexpressed delta p24 and p27 proteins to synthetic
peptides specific for the HBV envelope. Although a
higher degree of binding was observed with p27,
both p24 and p27 could bind HBV envelope
peptides. One such peptide corresponded to residues 56–80 located in the cytosolic loop of the
small HBV envelope protein, and another corresponded to 23 carboxy-terminal residues of the preS1 specific to the large HBV envelope protein. This
indicates that in addition to p27, p24 may contribute to packaging of HDV through a protein–
protein interaction with HBV envelope domains,
and that an interaction between the pre-S1 polypeptide and delta proteins may play a role in
infectivity.
Hepatitis delta virus (HDV), a satellite of hepatitis B
virus (HBV), can cause fulminant hepatitis and liver cirrhosis
in chronically infected patients (Rizzetto, 1983). The HDV
virion is coated with an outer envelope composed of host
cell-derived lipids and the envelope proteins of HBV. It
surrounds an inner ribonucleoprotein (RNP) complex comprising a circular RNA genome and two proteins bearing the
hepatitis delta antigen (HD Ag) : the small HD Ag (p24, 195
residues) and the large HD Ag (p27, 214 residues) (Wang et al.,
1986 ; Weiner et al., 1988). They are encoded by the HDV
genome and their sequences are identical except for 19
additional residues at the carboxy terminus of the large protein
Author for correspondence : Philippe Roingeard (address 2).
Fax ­33 2 47 36 60 90. e-mail roingeard!med.univ-tours.fr
(Weiner et al., 1988). The small protein, p24, is required for
HDV RNA replication (Kuo et al., 1989) whereas the large
protein, p27, is necessary for virion assembly (Chang et al.,
1991 ; Ryu et al., 1992) and has an inhibitory effect on genome
replication (Chao et al., 1990 ; Glenn & White, 1991). The 19
carboxy-terminal polypeptide residue of p27 contains a CXXX
motif that directs prenylation of the protein. This modification
to p27 results in its targeting to the host cell membranes
(Glenn et al., 1992), but is not sufficient for virion assembly.
Specific binding of delta proteins to HBV envelope proteins
seems to be necessary for the formation of enveloped HDV
(Lee et al., 1995). In vitro studies done with deletion mutants
have suggested that this protein–protein interaction occurs
through the 19 carboxy-terminal residues of p27 (Hwang &
Lai, 1993 ; Lazinski & Taylor, 1993 ; Lee et al., 1994, 1995).
However, this sequence is poorly conserved among various
HDV isolates and its role in protein–protein interaction during
HDV morphogenesis remains uncertain (Lai, 1995). The
envelope of HDV particles is identical to that of HBV. It is
composed of three HBV-encoded proteins designated large (L),
middle (M) and small (S) (Tiollais et al., 1985). From amino to
carboxy terminus, the L protein contains the pre-S1 (119
residues), pre-S2 (55 residues) and S (226 residues) domains.
The M protein contains both pre-S2 and S domains, whereas
the S protein contains only the S region. The S protein is
unique among viral envelope proteins in its capacity to selfassemble with host-derived lipids and to form empty envelope
particles (Simon et al., 1988). The amino terminus of S contains
a type I transmembrane signal and an internal type II signal
which result in the protein traversing the ER membrane at least
twice to form a cytosolic loop and a lumenal domain (Heerman
& Gerlich, 1991). The L protein has a more complex topology.
The pre-S domain is exposed at either the cytosolic or the
lumenal side of the ER membrane (Bruss et al., 1994 ;
Ostapchuck et al., 1994 ; Prange et al., 1992). Although the
three HBV envelope proteins, S, M and L, are found in the
HDV virion in vivo (Bonino et al., 1986), it is clear that the S
protein is sufficient for virion maturation (Sureau et al., 1993 ;
Wang et al., 1991) and that the L protein is important for HDV
infectivity (Sureau et al., 1992, 1993, 1994).
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C. Hourioux and others
Fig. 1. Amino acid sequence of the HBV
envelope (ayw subtype) and control
peptides. The small HBV surface protein
(S, 226 residues) spans the membrane
at its amino-terminal type I and central
type II signals (open boxes), and
probably twice at its hydrophobic
carboxy-terminal region. S exposes a
loop of 50 residues at the cytosolic side
of ER, covered by peptides S1 and S2.
The middle HBV surface protein (M)
contains the S domain plus the 55
residues of the pre-S2 domain, covered
by peptide L5/M. The large HBV surface
protein (L) contains the S plus pre-S2
domains and the 108 residues of the
pre-S1 domain, covered by peptides L1,
L2, L3 and L4, and two overlapping
peptides, L4a and L4b. The four control
peptides are from HIV GP 120 (HIV-P2
and HIV-P4) and HCV NS4 (HCV-9 AND
HCV 24).
The protein–protein interactions involved in the maturation process of HBV have previously been investigated by
determining the binding capacity of purified HBV core particles
to a panel of synthetic peptides mapping the HBV envelope
proteins (Poisson et al., 1997). The 13 carboxy-terminal
residues of pre-S1 and residues 56–80 in the cytosolic loop of
S were found to bind efficiently to HBV core particles. In the
present study, the binding capacity of the p24 and p27 delta
proteins to the same panel of HBV envelope-specific peptides
was analysed.
The synthesis of the HBV peptides has been described
previously (Poisson et al., 1997). Peptides corresponding to
pre-S1, pre-S2 and the cytosolic loop of S are shown in Fig. 1.
Four control peptides were also included in this study : human
BBBG
immunodeficiency virus (HIV)-P2 and HIV-P4 (residues 242–
264 and 411–436, respectively, of HIV gp120) ; and hepatitis
C virus (HCV)-9 and HCV-24 (residues 1710–1728 and
1691–1708, respectively, of HCV genotype II NS4A).
For construction of the p27 and p24 delta protein vectors,
a DNA sequence encoding p24 was cloned into the BamHI and
XbaI restriction sites of the pVL1393 transfer vector (Invitrogen) after DNA amplification by PCR, using a 5« primer
containing a BamHI restriction site sequence and a 3« primer
containing an XbaI restriction site sequence. The appropriate
sequence was PCR-amplified from plasmid pSVLD3, which
contains three copies of the HDV genome encoding the p24
form of the delta protein (Kuo et al., 1989). For cloning of the
p27 DNA coding sequence, in vitro mutagenesis was performed
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HBV envelope/HDV antigen interaction
Fig. 2. Analysis of p24 and p27 delta proteins produced in a baculovirus
expression system. Left :, silver staining of molecular mass markers (M),
HD24A, HD27A and WT baculovirus-infected cell nucleus lysates. Right,
Western blot analysis of the same print reacting with purified human antidelta IgG antibodies.
by the PCR overlap extension method (Higuchi et al., 1988) to
mutate the p24 TAG stop codon to TGG. A PCR-generated
fragment encoding p27 was cloned into the BamHI and XbaI
restriction sites of pVL1393. Two viruses, designated HD24A
and HD27A, were isolated and characterized for their ability to
express the p24 and p27 delta proteins.
For protein expression, SF9 insect cells were cultured in
Grace’s culture medium (Gibco BRL) supplemented with 10 %
foetal calf bovine serum. Cells (10() were infected with HD24A,
HD27A or wild-type (WT) baculovirus at an m.o.i. of 10.
HD27A-infected cells were harvested 2 days post-infection,
whereas HD24A- and WT-infected cells were harvested 3 days
post-infection. Expression of delta proteins p24 and p27 was
detected in infected cells by immunocytochemistry, as previously described (Pol et al., 1989). The intracellular localization
was predominantly nuclear. For purification, the cells were
resuspended in a hypotonic lysis buffer (1 mM Tris pH 7±5,
0±1 mM MgCl , 0±1 % Triton), incubated at 4 °C for 30 min
#
and then lysed with a Dounce homogenizer. Membranes were
removed by centrifugation at 1000 g for 10 min at 4 °C. Pellets
containing the nuclear fraction were resuspended in 1 ml lysis
buffer and then sonicated at 4 °C to disrupt nuclear membranes.
Each step was monitored by light microscopy after toluidine
blue staining. Proteins in each lysate were analysed by 11±5 %
polyacrylamide–SDS gel electrophoresis (PAGE) followed
either by silver staining or transfer to nitrocellulose for
Western blot analysis, using purified human anti-delta IgG
antibodies and peroxidase-conjugated goat anti-human F(ab«)#
(Fig. 2). Staining of 24 and 27 kDa proteins was observed in
the HD24A and HD27A baculovirus-infected cell lysates,
respectively. An additional band at approximately 20 kDa was
detected in both lysates, probably corresponding to a degradation product (Wang et al., 1992). Delta proteins in each
lysate were quantified with a commercial HD Ag ELISA
(Turnout, Organon Teknica) (Roingeard et al., 1992). HD24A
lysate was found to contain four times more delta proteins than
HD27A lysate, while WT lysate was negative.
For the HD Ag–HBV envelope peptide interaction assay,
all peptides were conjugated to BSA with carbodiimide as
previously described (Poisson et al., 1997). Microtitre plate
wells (Maxisorp, Nunc) were coated at 20 µg per well with
BSA-coupled peptides in PBS for 12 h at 4 °C. The coating
efficiency was verified for the different peptides in preliminary
assays using antibodies against pre-S1, pre-S2 and S epitopes
described by Sureau et al. (1992). After coating, the wells
were washed three times with PBS–0±5 % Tween 20 (PBS-TW)
and blocked by addition of 300 µl 1¬ PBS containing 2 %
newborn calf bovine serum (NBCS) and incubation for 45 min
at 37 °C. After three washes in PBS-TW, HD24A, HD27A and
WT lysates were added at a dilution of 1 : 100, 1 : 25 and 1 : 100,
respectively, in 0±1¬ PBS, and the plates incubated for 3 h at
37 °C. In some experiments, 10 % WT lysate was added to the
HD24A and HD27A lysate dilutions. After three washes in
PBS-TW, delta antigen binding was evaluated with the same
reagents as used in the Western blot described above : 100 µl
of a 1 : 10 000 dilution of human anti-HD purified IgG
antibodies in PBS, 5 % NBCS, 5 % BSA, 0±5 % Tween 20 (PBSNBT) was added and the plates incubated for 30 min at 37 °C.
After three more washes, 100 µl peroxidase-conjugated goat
anti-human F(ab«)# (Biosource International) diluted 1 : 10 000
in PBS-NBT was added, and plates were incubated for 30 min
at 37 °C. Plates were then washed three times in PBS-TW, and
100 µl of a mixture containing hydrogen peroxide–o-phenylenediamine was added to each well and left in the dark for
15 min at room temperature. Colour development was stopped
with 1 M H SO and the absorbance was read at 492 nm. The
# %
binding capacity of each peptide for HD24A, HD27A and WT
lysates was expressed as a multiple of the cut-off value (2±5
times the average value obtained with the four control
peptides). All results are summarized in Table 1. Although we
cannot exclude the possibility that the anti-HD IgG could have
induced a dissociation of the delta antigens from coated
peptides, peptides L1, L2, L3, L5}M and S1 did not bind to
delta proteins in this assay. However, peptide S2 (residues
56–80 in the cytosolic loop of S) and peptide L4 (corresponding
to the 23 carboxy-terminal residues of pre-S1) showed binding
activity to HD Ags. Peptide L4a bound very efficiently to both
HD24A and HD27A lysates, whereas peptide L4b had a lower
binding capacity (Table 1). With the exception of peptide L4b,
all peptides bound preferentially to HD27A lysate. This
interaction was specific since addition of 10 % WT lysate in
HD24A or HD27A lysate dilutions did not decrease the signal
(data not shown).
When binding to HBV envelope proteins of p24 versus p27
was determined the highest degree of binding was observed
for p27, although both p27 and p24 bound to peptides
representing different HBV envelope domains (Table 1). These
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C. Hourioux and others
Table 1. Binding of HBV envelope peptides to HD24A, HD27A and WT baculovirusinfected cell nucleus lysates
Results are given as multiples of the cut-off value (cut-off value ¯ 2±5 times the average value obtained with
the four HIV and HCV control peptides for a given lysate). Values presented represent the mean³standard
deviations of four independent experiments performed with all peptides. –, below cut-off value.
Peptide
Lysate
L1
L2
L3
HD24A
HD27A
WT
–
–
–
–
–
–
–
–
–
L4
L4a
L4b
1±2³0±2 6±7³1±1 5±1³1±6
3±5³0±7 10±6³1±5 4±3³1±5
–
–
–
results further suggest that specific binding of the delta
proteins to HBV envelope proteins may occur in domains
other than the 19 carboxy-terminal residues of p27. Whether
these interactions have a function in morphogenesis or
infectivity of HDV particles remains to be elucidated. Early
studies suggested a possible function of the Pro}Gly-rich
region (residues 146–214) of p27 in a possible interaction with
the HBV envelope proteins (Lazinski & Taylor, 1993), but
subsequent experiments have demonstrated the crucial role of
the carboxy terminus of p27 (Hwang & Lai, 1993 ; Chang et al.,
1994 ; Lee et al., 1994, 1995). It should be noted that the 19
carboxy-terminal residues of p27 are poorly conserved among
the different HDV genotypes, and that their role in the
interaction between HDV RNP and HBV envelope proteins
may lie in their contribution to generating a maturationcompetent conformation of p27, rather than an ability to bind
S directly (Lai, 1995). Nevertheless, the present data show that
both p27 and p24 are able to bind to HBV envelope proteins.
Whether binding of p24 to HBV envelope proteins is of
biological significance for HDV packaging into the HBV
envelope remains to be determined. In vitro binding assays
represent a convenient alternative to in vitro expression of
deletion mutants for exploring a protein–protein interaction
required for HDV maturation. Experiments done in tissue
culture indicate the need for p24 and p27 for efficient HDV
maturation. The p27 protein alone was able to mediate only a
low level of HDV packaging, while the restoration of p24
enhanced HDV packaging 3- to 4-fold (Wang et al., 1994).
The S2 peptide that covers the cytosolic loop of the S env
protein at residues 56–80 can bind to HBV core particles
(Poisson et al., 1997) and to delta proteins, as demonstrated in
this study. In vitro studies in which proteins were expressed in
Huh7 cells have shown that the HDV RNP can be coated with
the S protein alone (Sureau et al., 1993 ; Wang et al., 1991).
However, studies with this system may have limitations,
especially when deletion mutants are examined. Our in vitro
binding assay represents a useful alternative for exploring S
HBV protein domains involved in HDV maturation. The
BBBI
L5/M
S1
S2
–
–
–
–
–
–
5±1³1±6
6±0³0±6
–
results presented here suggest that the protein–protein
interaction required for HDV maturation in addition to p27
isoprenylation may involve p24 and the 56–80 residue domain
in the cytosolic loop of the S HBV envelope protein. On the
other hand, the results indicate that delta proteins may also
bind to the carboxy-terminal region of pre-S1 specific to the L
HBV protein, as for HBV core particles, although HBV core
particles preferentially bind the L4b peptide (Poisson et al.,
1997) while delta proteins preferentially bind the L4a peptide.
The biological significance of this binding to pre-S1 remains to
be determined, since the L protein is dispensable for HDV
assembly (Sureau et al., 1993 ; Wang et al., 1991). The L HBV
envelope protein is, however, required for the HDV particles
to be fully infectious (Sureau et al., 1992, 1993, 1994). This
interaction may thus be helpful to ensure infectivity of HDV
particles at the step of virus internalization after adsorption to
the hepatocyte membrane.
We are indebted to Drs John Taylor, Isabelle Turbica and Sophie Le
Pogam for gifts of the pSVLD3 plasmid, HIV and HCV peptides,
respectively. This work was supported by grants from the Ligue
Nationale Contre le Cancer (Comite! de l’Indre & Loire) and from the
Association pour la Recherche sur le Cancer (ARC). Christophe Hourioux
was supported by a fellowship provided by the Fondation Me! rieux.
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Received 28 November 1997 ; Accepted 22 January 1998
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