Article

Primary Human Hepatocytes Are Susceptible to Infection by Hepatitis Delta Virus Assembled with Envelope Proteins of Woodchuck Hepatitis Virus

Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111-2497, USA.
Journal of Virology (Impact Factor: 4.44). 09/2008; 82(15):7276-83. DOI: 10.1128/JVI.00576-08
Source: PubMed
ABSTRACT
Hepatitis B virus (HBV) and hepatitis delta virus (HDV) share the HBV envelope proteins. When woodchucks chronically infected
with woodchuck hepatitis virus (WHV) are superinfected with HDV, they produce HDV with a WHV envelope, wHDV. Several lines
of evidence are provided that wHDV infects not only cultured primary woodchuck hepatocytes (PWH) but also primary human hepatocytes
(PHH). Surprisingly, HBV-enveloped HDV (hHDV) and wHDV infected PHH with comparable efficiencies; however, hHDV did not infect
PWH. The basis for these host range specificities was investigated using as inhibitors peptides bearing species-specific pre-S
(where S is the small envelope protein) sequences. It was found that pre-S1 contributed to the ability of wHDV to infect both
PHH and PWH. In addition, the inability of hHDV to infect PWH was not overcome using a chimeric form of hHDV containing WHV
S protein, again supporting the essential role of pre-S1 in infection of target cells. One interpretation of these data is
that host range specificity of HDV is determined entirely by pre-S1 and that the WHV and HBV pre-S1 proteins recognize different
receptors on PHH.

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JOURNAL OF VIROLOGY, Aug. 2008, p. 7276–7283 Vol. 82, No. 15
0022-538X/08/$08.000 doi:10.1128/JVI.00576-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Primary Human Hepatocytes Are Susceptible to Infection by Hepatitis
Delta Virus Assembled with Envelope Proteins of
Woodchuck Hepatitis Virus
Severin Gudima,
1
Yiping He,
1
Ning Chai,
1
Volker Bruss,
2
Stephan Urban,
3
William Mason,
1
and John Taylor
1
*
Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, Pennsylvania 19111
1
; Department of Virology, University of Goettingen,
Goettingen 37075, Germany
2
; and Department for Molecular Virology, University of Heidelberg, Heidelberg 69120, Germany
3
Received 14 March 2008/Accepted 12 May 2008
Hepatitis B virus (HBV) and hepatitis delta virus (HDV) share the HBV envelope proteins. When wood-
chucks chronically infected with woodchuck hepatitis virus (WHV) are superinfected with HDV, they produce
HDV with a WHV envelope, wHDV. Several lines of evidence are provided that wHDV infects not only
cultured primary woodchuck hepatocytes (PWH) but also primary human hepatocytes (PHH). Surpris-
ingly, HBV-enveloped HDV (hHDV) and wHDV infected PHH with comparable efficiencies; however,
hHDV did not infect PWH. The basis for these host range specificities was investigated using as inhibitors
peptides bearing species-specific pre-S (where S is the small envelope protein) sequences. It was found
that pre-S1 contributed to the ability of wHDV to infect both PHH and PWH. In addition, the inability of
hHDV to infect PWH was not overcome using a chimeric form of hHDV containing WHV S protein, again
supporting the essential role of pre-S1 in infection of target cells. One interpretation of these data is that
host range specificity of HDV is determined entirely by pre-S1 and that the WHV and HBV pre-S1 proteins
recognize different receptors on PHH.
Hepatitis B virus (HBV) still remains a major health prob-
lem, with about 400 million infected people worldwide and
approximately 1 million deaths a year caused by HBV-associ-
ated liver cancer (35). In natural situations people can be
coinfected with hepatitis delta virus (HDV) and HBV, or HBV
carriers can be superinfected with HDV (40). HDV is a sub-
viral agent that uses the envelope proteins of HBV for virion
formation and infectivity (42). The HDV genome is a negative-
sense single-stranded circular RNA, which is known to fold
into a rod-like structure with 74% self-complementarity. The
genome is replicated via RNA-directed RNA synthesis, appar-
ently catalyzed by host RNA polymerase II (41). Three pro-
cessed HDV RNAs accumulate in infected cells: (i) the ge-
nome, (ii) its exact complement, the antigenome, and (iii) a
5-capped and 3-polyadenylated mRNA for the only viral pro-
tein, the delta antigen (Ag), which is essential for replication.
An RNA-editing event, which occurs on antigenomic RNA,
leads to the production of a longer form of Ag that does not
support RNA replication but is required for HDV assembly
(6, 7).
Hepadnaviruses are considered to be highly species specific
due to recognition of species-specific receptors on hepatocytes,
the target of infection. HDV is presumed to use the same
means for attachment and entry as HBV (36) and, therefore, to
share this species specificity. A number of candidates have
been proposed as the HBV and HDV receptors, but none of
them has been shown to be sufficient or even necessary for
infection (35). In addition, the number of the cell surface
receptors used by HBV, HDV, and other hepadnaviruses is
unknown. Woodchuck hepatitis virus (WHV) is a hepadnavi-
rus with many similarities to HBV. Like HBV, the envelope
proteins of WHV can be used to assemble HDV particles (31),
referred to here as wHDV, to distinguish them from hHDV,
HDV with HBV envelope.
It is known that chimpanzees can be infected with HBV
obtained from patient serum or transfected cells (34) and also
with hHDV collected from infected patients (28–30). Further-
more, hHDV from either an infected chimpanzee or trans-
fected cells is able to infect primary chimpanzee and human
hepatocytes (4, 37, 38). hHDV passaged in chimps can infect
WHV-chronic carrier woodchucks (21, 26), and yet, as consid-
ered further in the Discussion, this probably involves the
spread of wHDV. Serum-derived hHDV cannot infect primary
woodchuck hepatocytes (PWH) (39). Also there are no data
supporting the possibility of productive infection of wood-
chucks with HBV (26).
The HDV from an infected woodchuck, wHDV, is able to
infect PWH regardless of the presence of WHV (39) and is
also able to reinfect chimpanzees (20, 29). In contrast, there is
no evidence that WHV itself can infect humans or other pri-
mates (25, 35, 43). Similarly, primary tupaia hepatocytes which
are susceptible to HBV infection are resistant to WHV (17). It
is also known that woodchucks are susceptible to infection with
either WHV (32) or wHDV that has been assembled in cul-
tured cells (22, 23, 31).
In the present study, we have asked if host range suscepti-
bilities of HBV, WHV, and the homologous forms of HDV are
really at the level of receptor recognition. Our results, albeit
with HDV, are consistent with the following interpretations: (i)
* Corresponding author. Mailing address: Fox Chase Cancer Cen-
ter, 333 Cottman Avenue, Philadelphia, PA 19111-2497. Phone: (215)
728 2436. Fax: (215) 728-3105. E-mail: john.taylor@fccc.edu.
† Present address: USDA, Wyndmoor, PA 19038.
Published ahead of print on 21 May 2008.
7276
Page 1
WHV and HBV and the related HDV recognize different host
receptors, (ii) recognition is via pre-S1 (where S is the small
envelope protein), and (iii) WHV is able to recognize a recep-
tor on human cells but not vice versa (that is, HBV cannot
recognize a receptor on woodchuck cells). The failure of WHV
to infect human hepatocytes occurs, by inference, at a step
after receptor recognition.
MATERIALS AND METHODS
hHDV and wHDV. One source of wHDV was serum from woodchucks chron-
ically infected with WHV and then superinfected with HDV (23). Alternatively
wHDV or hHDV was assembled from Huh7 cells cotransfected with pSVLD3 to
initiate HDV replication, together with plasmids expressing WHV or HBV
envelope proteins, using procedures as previously described (16).
For the experiments shown in Fig. 7, assembly was performed using combi-
nations of HBV and WHV envelope-expressing constructs. HBV S envelope
protein was expressed from pSVBX24H (11). WHV S envelope protein was
expressed from pSV24W (11). The HBV large envelope protein (L) protein was
expressed from a construct, pSVL, on which the initiation codons for pre-S2 and
S were mutated to threonine (10). In order to express WHV L protein, the
relevant sequence from pUC119CMVWHV (33) was transferred to vector (also
named) pSVL (Pharmacia) to create pSG322. This construct was not mutated to
change the initiation codons for pre-S2 and S. However, L was driven from a
strong simian virus 40 late promoter, and consistent with the ineffective expres-
sion of M (middle envelope protein) and S proteins, we observed that this
construct by itself was insufficient to achieve detectable assembly of HDV RNA-
containing particles (see Fig. 7A1 and A2). Presumably the WHV promoter for
the M and S mRNA was much less efficient in the Huh7 cells that the simian virus
40 late promoter driving expression of the L mRNA.
PHH and PWH. Primary human hepatocytes (PHH) plated on rat tail collagen
(type I) in a 48-well format were obtained commercially (CellzDirect, Lonza, or
BD Gentest) and infected the day after arrival. PWH were prepared from the
liver of a WHV-positive woodchuck and cryo-preserved (1). This prior infection
is not a concern since in a previous study we showed that wHDV infection was
equally efficient in hepatocytes taken from either naive or WHV-infected animals
(39). After thawing, PWH were resuspended in prewarmed complete hepatocyte
medium (CellzDirect), seeded on 48-well collagen-coated plates (BD Bio-
Science) at 2 10
5
cells/well, and infected the next day.
Peptide inhibitors of virus infection. The features and preparation of the six
peptide inhibitors tested in the experiment shown in Fig. 6 are summarized in
Table 1. The immunoadhesins (5) bearing either WHV pre-S1 or the whole pre-S
were constructed using the WHV sequence of plasmid pUC119CMVWHV.
Virus infection and assays of inhibition. Primary hepatocyte infections were
performed as described previously for PHH (16). Unless otherwise stated, these
were performed at a multiplicity of infection (MOI) of about 50 HDV genome
equivalents (GE) per cell in the presence of 5% polyethylene glycol (PEG). In
order to examine the ability of peptides and immunoadhesins to block infection,
these potential inhibitors were present along with the virus throughout the
incubation with hepatocytes, that is, for 6 to 16 h.
Determination of virus titers and virus infection parameters. HDV RNA
titers were determined by quantitative real-time PCR (qPCR), as previously
described (16). For hepatocyte infections, total RNA was extracted at 6 days
after infection and assayed by qPCR to determine the number of HDV RNA GE
per average cell (5, 16).
Immunostaining. As an alternative assay of hepatocyte infections, at day 6
cells were fixed, permeabilized, and immunostained for Ag, as previously de-
scribed (16). In addition, PHH were counterstained with anti-albumin antibody
(16), and PWH were counterstained with anti--tubulin (a gift from Elena
Pugacheva).
RESULTS
Predicted topology of WHV L protein in relation to that of
HBV. It is known that HDV can be passaged in woodchucks
infected with WHV to produce wHDV, that is, HDV with a
WHV envelope (26, 27). Both WHV and HBV encode three
co-C-terminal envelope proteins (35). Figure 1A shows the
sequence alignment of the L proteins of HBV and WHV. The
two sequences share very little similarity in most of the pre-S1
domain. However, beginning at the C terminus of pre-S1, se-
quence homology increases significantly, and clearly the ma-
jority of identical amino acids are located in the S domains.
For both envelope proteins TMHMM2.0 software (19) was
used to predict folding, including transmembrane domains
(TMD) (Fig. 2). It was assumed that 22 2 amino acids
constitute the average size for each TMD (2). Also taken into
consideration was that the high level of sequence conservation
between S domains of HBV and WHV will lead to significant
structural-functional conservations between these two viral en-
velopes. The modeling took account of the fact that TMD III
does not possess clear signals for breaking the transmembrane
helix and that the well-conserved patch of amino acids IWM
(M/I)W(Y/F)W should be exposed in the cytosolic compart-
ment, consistent with its critical role in interacting with cyto-
plasmic HDV RNP (36). In the structures shown here, the
entire pre-S1 domain is exposed on the luminal side of the
endoplasmic reticulum or the outer side of the viral mem-
brane. However, previous studies of the HBV envelope have
shown that, for a fraction of the envelope proteins, an alter-
native conformation can exist with this region located inside
the virion (36).
In summary, WHV and HBV L proteins are considered to
share the following characteristic elements: (i) four TMD, I to
IV; (ii) two cytosolic loops, a large one located between TMD
I and II and a small one between TMD III and TMD IV; (iii)
an N-terminal pre-S region on the outer side; and (iv) an
external large loop between TMD II and TMD III. The struc-
tures presented in Fig. 2 are in a good correlation with exper-
imental data placing a receptor-interacting site(s) for HBV
near the N terminus of pre-S1 (3, 14) and an HDV RNP-
binding region in the small cytosolic loop (18).
wHDV infects both PHH and PWH. Next, we compared the
ability of wHDV obtained from the serum of an infected wood-
chuck to infect PHH and PWH. Infections were performed in
the presence of 5% PEG, a strategy known to enhance HBV
and HDV infections of PHH (3, 15, 16). At 6 days after infec-
TABLE 1. Soluble peptides containing pre-S sequences of HBV
and WHV L proteins
a
Source of pre-S
sequence and
peptide no.
Extent of pre-S
sequence
b
Type of peptide
preparation
c
Reference
HBV L
1 2–48 Synthetic 14
2 1–119 (pre-S1) Immunoadhesin 5
3 1–174 (pre-S1
pre-S2)
Immunoadhesin 5
WHV L
4 2–87 Synthetic Unpublished
5 1–149 (pre-S1) Immunoadhesin Unpublished
6 1–209 (pre-S1
pre-S2)
Immunoadhesin Unpublished
a
These peptides were tested for their ability to interfere with wHDV and
hHDV infections of PHH and PWH (results are summarized in Fig. 6).
b
The extent of each pre-S sequence corresponds to the positions of the amino
acid (aa) residues relative to the N terminus.
c
The peptides were prepared either by chemical synthesis followed by myris
-
toylation or by expression as immunoadhesins from plasmids transfected into
293T cells (5).
VOL. 82, 2008 HDV INFECTION OF HUMAN HEPATOCYTES 7277
Page 2
tion, HDV replication was assayed by immunostaining to de-
tect hepatocytes positive for Ag. As shown in Fig. 3A, both
PWH and PHH were infected with wHDV. hHDV infected
PHH but not PWH, which is consistent with a previous report
(39). In the three situations where infection was achieved, the
subcellular distributions of Ag were similar, typically with
nucleoplasmic localization and sometimes with a distribution
throughout the cell.
Quantitation of such infections performed at the same MOIs
showed that wHDV infected 1.5 and 2.4% of PHH and PWH,
respectively; that is, the infection levels were comparable. In
contrast, hHDV infected 5.3% of PHH but 0.0003% of
PWH. The fraction of PHH infected with wHDV increased
with the MOI and reached 14% when 5,000 GE of wHDV per
hepatocyte was used. This is comparable to a report that HBV
infection of primary tupaia hepatocytes at an MOI of 10,000
resulted in infection in 25% of cells (12).
As an independent assay of infection, we used real-time
qPCR, as summarized in Fig. 3B. These data confirmed the
immunostaining results. Furthermore, given the increased sen-
sitivity of the qPCR, we also quantitated infections carried out
in the absence of 5% PEG. Again, wHDV was able to infect
both PHH and PWH, while hHDV infected PHH but not
PWH. (Similarly to HBV and hHDV, the infectivity of wHDV
was enhanced in the presence of 5% of PEG.)
Several other isolates of wHDV obtained from different
woodchucks were tested and found to be infectious for both
PWH and PHH. Also, HDV RNA genome replication was
confirmed in both kinds of wHDV-infected hepatocytes by
Northern analysis (data not shown).
Next, we examined the effect of different MOIs using qPCR,
and the results are summarized in Fig. 4. In a range of MOIs
up to 1,000 GE/cell, wHDV readily infected PWH, while the
same cells were resistant to hHDV. However, for PHH we
observed comparable efficiencies of infection with both viruses.
As another parameter of infection, we performed a time
course analysis, again using qPCR, and the results are shown
in Fig. 5. PWH or PHH were infected with hHDV or wHDV
at an MOI of 500. For PHH infections both viruses revealed
time-dependent accumulation of HDV RNA GE/cell in
FIG. 1. Sequence alignment of WHV and HBV L proteins (wL and hL, respectively). The NCBI database accession numbers for WHV and
HBV are AAA46770.1 and AAK58874.1, respectively. Alignment was via the T-Coffee program (24). Arrows are used to indicate the junctions
of pre-S1, pre-S2, and S domains and also the boundaries of the major antigenic loop. Each protein is considered to have four TMD, as indicated
by underlining. Further details are provided in the text and in Fig. 2. Identical (
*
), conserved (:), and semiconserved (.) amino acids are indicated.
FIG. 2. Predicted topologies for WHV and HBV L proteins (wL
and hL, respectively). Protein folding of the sequences presented in
Fig. 1, including prediction of the TMD, was performed using
TMHMM2.0 software (19). Further details are provided in the text.
The four dark cylinders, indicated as I to IV, represent the TMD
inserted in the lipid membrane, indicated by the light shading. For the
extracellular virus the membrane has inside-outside surfaces, while
during intracellular assembly at an endoplasmic reticulum-Golgi mem-
brane these are cytosol-lumen surfaces, respectively. The amino and
carboxy termini are indicated by N and C, respectively. On the outside,
or luminal side, of the membrane are pre-S1, pre-S2, and the major
antigenic loop. On the inside, or cytosolic side, loops of 51 and 8 amino
acids are indicated. The boundaries of the above-mentioned structural
elements are indicated by position numbers of the corresponding
amino acid residues. aa, amino acids.
7278 GUDIMA ET AL. J. VIROL.
Page 3
amounts, at late times, greatly exceeding input MOI, clearly
demonstrating efficient HDV RNA replication (Fig. 5A and
B). wHDV productively infected PWH (Fig. 5C). With
hHDV there was signal detectable on PWH cultures at the
earliest times, but it decreased progressively (Fig. 5D), con-
sistent with the interpretation that no significant replication
took place.
The above studies using different assay procedures demon-
strated that wHDV can infect both PWH and PHH while
hHDV infects PHH but not PWH. While it might be agreed
that these findings are consistent with the idea that the infec-
tion is largely controlled by the presentation of envelope pro-
teins to receptor(s) at the cell surface, the results do not ad-
dress how this presentation can differ and whether or not a
different host receptor(s) is involved.
Inhibition of wHDV and hHDV infections by pre-S peptides.
As an approach to understanding wHDV attachment and entry
into primary hepatocytes, we made use of a panel of six pep-
tides bearing pre-S sequences, with three derived from HBV L
and three from WHV L, as summarized in Table 1. The ratio-
nale was that these peptides could potentially compete with the
virus for binding of the receptor(s) on the hepatocyte surfaces
and thus block the infection. Note that two of the peptides
were chemically synthesized and then myristoylated. The other
four were created as immunoadhesins (5). These potential
inhibitors were tested at a concentration of 50 nM present
during the time cells were exposed to virus, and the results are
summarized in Fig. 6. Previous studies have shown that at 50
nM, the three HBV peptides inhibit infection of PHH by HBV
and hHDV (5, 9, 14). Consistent with this, peptides 1 to 3
inhibited hHDV infection of PHH (Fig. 6B, lanes 1 to 3).
However, under the same conditions these peptides had little
effect on wHDV infections of PHH (Fig. 6A) or PWH (Fig.
6C). Next, we tested WHV peptides 4 to 6. Of these, only the
synthetic peptide (Fig. 6, lanes 4), inhibited infection by
wHDV of PHH (panel A) and PWH (panel C) but had no
FIG. 3. Ability of wHDV and hHDV to infect PHH and PWH. Both types of hepatocytes were infected at an MOI of 300 HDV GE/cell.
(A) Infections were performed in the presence of 5% PEG, and at 6 days, cells with HDV replication were identified by immunostaining to detect
newly synthesized Ag (red). Counterstaining was with DAPI (blue) and for either albumin in PHH or -tubulin in PWH (green). (B) HDV
infections were carried out either without () or with () 5% PEG. Cells were harvested at day 6 postinfection. Total RNA was assayed for HDV
RNA by qPCR as previously described (16), and the infectivity results are expressed as HDV RNA GE/average cell. Graphs represent the average
results of duplicate infections with wHDV (open bars) and hHDV (shaded bars). The results were confirmed in two separate experiments.
FIG. 4. Effect of wHDV and hHDV MOIs on infections of PHH
and PWH. (A) PHH were infected with different MOIs of wHDV
(open circles) or hHDV (shaded circles) in the presence of 5% PEG.
(B) An experiment similar to that in panel A but using PWH. As
described in the legend of in Fig. 3, total RNA was extracted at day 6
and assayed by qPCR, and the infectivity results were expressed as
HDV RNA GE/average cell. The data are the average of duplicate
infections, and the results were confirmed in two separate experiments.
VOL. 82, 2008 HDV INFECTION OF HUMAN HEPATOCYTES 7279
Page 4
effect on infection by hHDV of PHH (panel B). The other two
WHV peptides (lanes 5 and 6) that were presented as immu-
noadhesins failed to inhibit any of the infections. One possible
reason for this is that the sequences added to the N terminus
could no longer interact with the receptor because of intramo-
lecular folding. Such a phenomenon has been reported for
synthetic HBV peptides that tend to loose their inhibitory
potential after exceeding a certain length (14).
Overall, these studies support the interpretation that
wHDV, like hHDV, needs pre-S1 sequences to achieve infec-
tion. More importantly, and consistent with the fact that the
WHV and HBV pre-S1 regions share very little sequence ho-
mology (Fig. 1), we interpret these inhibition studies as evi-
dence that wHDV and hHDV interact with PHH via different
receptors. Furthermore, the receptor used by wHDV on PHH
might be closely related to that used on PWH.
Infection specificity of hepatitis delta virions assembled us-
ing combinations of HBV and WHV envelope proteins. In all of
the above studies the sources of wHDV and hHDV differed
not only in terms of the envelope proteins used but also in the
way the particles were assembled. wHDV was obtained from
infected woodchucks, and hHDV was assembled, as previously
described, using cells transfected to express HBV L, M, and S
envelope proteins and also replicating HDV RNA (16). There-
fore, it was important to prepare wHDV by a transfection
FIG. 5. Time course of wHDV and hHDV infections of PHH and
PWH. Cultures of PHH (A and B) or PWH (C and D) were infected
either with wHDV (open circles) or hHDV (shaded circles) at an MOI
of 500 in the presence of 5% PEG. At 1, 3, 5, and 7 days after infection,
total RNA was extracted and assayed by qPCR, as described in the
legend of Fig. 3. The infectivity results are expressed as HDV RNA
GE/average cell. The data are the average of duplicate infections.
FIG. 6. Ability of soluble peptides to interfere with infections of
PHH and PWH by wHDV and hHDV. Cultures of PHH (A and B) or
PWH (C) were infected with either wHDV (open bars) or hHDV
(shaded bars) at an MOI of 50 in the presence of 5% PEG. The six
soluble peptides explained in Table 1 were tested for their ability at a
concentration of 50 nM to inhibit the infections. As described in the
legend of Fig. 3, total RNA was extracted at day 6 and assayed by
qPCR. Here, the infectivity results are expressed as a percentage of the
infectivity observed in the absence of peptide. The infections were
performed in duplicate, and the results shown are the average of two
separate experiments. We consider a reduction of infectivity to less
than 25% of control values significant.
7280 GUDIMA ET AL. J. V
IROL.
Page 5
strategy similar to that used for the hHDV. To do this, we
expressed in Huh7 cells the WHV L and S envelope proteins in
various combinations, along with a plasmid to initiate HDV
genome replication. As summarized in Fig. 7A1, HDV RNA-
containing particles were released with WHV S in the absence
of WHV L, and as the percentage of WHV L plasmid trans-
fected increased to 100%, the amount of released particles
dropped to undetectable levels. In this respect these results
were similar to studies of assembly using HBV L and S (16).
Next, we tested aliquots of medium collected from the trans-
fected cells for the ability to infect PHH and PWH. After 6
days, the total cell RNAs were extracted, and HDV replication
was quantitated by qPCR. The number of GE produced per
average cell was normalized relative to the input MOI, in
GE/per average cell, to determine what we refer to as the
specific infectivity of the virus on the susceptible cells. The
specific infectivities of the assembled wHDV on PHH and
PWH are shown in Fig. 7B1 and C1, respectively. Note that the
particles assembled with WHV S alone were not infectious,
consistent with the interpretation that pre-S regions are
needed for infectivity. Also, with both cell types the specific
infectivities demonstrated a peak at the same percentage of
WHV L, and these peak values were not significantly different.
These results not only demonstrate that infectious wHDV
can be assembled in transfected cells but also confirm that such
virus can infect both PHH and PWH. That is, the results
obtained with wHDV assembled in animals were extended to
wHDV assembled from transfected cells.
We next extended the study to look for the assembly of
particles with intermolecular combinations of WHV and HBV
envelope proteins. The aim was to determine whether such
particles could be assembled and, if so, whether they would be
infectious on PHH and/or PWH.
First, we considered combinations of WHV L with HBV S
FIG. 7. Assembly and infectivity of the hepatitis delta virions coated with different intermolecular combinations of WHV and HBV envelope
proteins. Pairwise combinations of plasmids expressing the L and S envelope proteins of either WHV (wL and wS) or HBV (hL and hS) were used
in different transfections of Huh7 cells in order to achieve the assembly of hepatitis delta virions with different envelopes. The three sets of assembly
experiments performed consisted of wL plus wS, wL plus hS, and hL plus wS. We used one plasmid to express L and another to express S protein;
the mass amounts of each plasmid were varied, while the total mass was kept constant. The mass ratios are indicated at the bottom of the figure
as the percentage of L-expressing construct, that is, 100 L/(LS). For each transfection the virion-containing medium was harvested for days
7 to 10 and assayed for HDV RNA by qPCR, with results as shown in panels A1 to A3. Aliquots of these media were also used for infection of
PHH (B) and PWH (C). As described in the legend of Fig. 3, total RNA was extracted at day 6 and assayed by qPCR. However, here we divided
these infectivity values (in HDV RNA GE/average cell) by the input MOI (also expressed as HDV GE/average cell). This ratio, which we have
previously referred to as the specific infectivity (16), is a measure of the ability of the virus particles to infect susceptible cells, either PHH (B1 to
B3) or PWH (C1 to C3). Rather than use duplicates of each assembly, we chose to consider additional combinations of L and S. Thus, in panel
A1, the relatively low particle yield at 60% of L content is considered an experimental variation. The critical results in panels A1 to A3 and B1
to B3 were confirmed in a separate, less extensive repeat experiment.
V
OL. 82, 2008 HDV INFECTION OF HUMAN HEPATOCYTES 7281
Page 6
and of HBV L with WHV S and tested for the assembly of
HDV RNA-containing particles. As shown in Fig. 7A2 and A3,
respectively, assembly took place, and as before (Fig. 7A1),
when the proportion of the L protein was increased to 100%,
the assembly dropped to undetectable levels.
Next, we determined the specific infectivities for these par-
ticles on PHH and PWH. Virus assembled with WHV L plus
HBV S infected both cell types, and the peak of specific infec-
tivity was at about the same percentage of WHV L (Fig. 7B2
and C2). Clearly, the presence of HBV S in the particles did
not interfere with the ability to infect PWH (Fig. 7C2), and, if
anything, it enhanced the ability to infect PHH (Fig. 7B2).
Virus with combinations of HBV L with WHV S infected PHH
(Fig. 7B3) but gave no detectable infectivity on PWH (Fig.
7C3). The presence of WHV S in the particles was not suffi-
cient for infection of PWH.
In summary, the use of combinations of WHV and HBV
envelope proteins did not interfere with the assembly of HDV
RNA-containing particles. Further, these particles could be
infectious on primary hepatocytes. And in all cases it was the
origin of the L protein, whether WHV or HBV L, that deter-
mined the specificity of infection on PWH and PHH. Further-
more, we can deduce that it was the origin of the pre-S se-
quences and not that of the S protein that controlled the
infectivity and the species specificity. Incidentally, it could be
noted that in most cases increasing the relative amount of L in
an HDV particle initially achieved greater infectivity but then
led to suppression (Fig. 7B1, B3, C1, and C2).
DISCUSSION
A prior study reported that wHDV could infect PHH (4).
We have confirmed and extended this result, using immuno-
staining (Fig. 3A) and RNA analyses by Northern blotting and
real-time qPCR (Fig. 3 to 7). We used wHDV isolates from
infected animals and also wHDV assembled in vitro from
transfected cultured cells. Different sources of wHDV did have
different titers and probably had different ratios of virions to
subviral particles and also different relative amounts of the
L/M/S envelope proteins. Except for the study shown in Fig. 7,
we did not attempt to control or determine the relative
amounts of L/M/S in the infectious particles. However (again,
except for Fig. 7), the sources of wHDV and hHDV that we
used were able to infect PWH and PHH, respectively, with
comparable efficiencies.
Independent of potential variations between the sources of
virus, we observed that for a given source of wHDV, the in-
fections of PHH and PWH were of comparable extent. In
contrast, the sources of hHDV infected PHH but gave no
detectable infection of PWH.
There is no reported evidence for the productive infection of
PHH by WHV, and this might be considered to be due to a
block at attachment and entry. However, the results presented
here for infection by wHDV favor the possibility that WHV
can enter PHH but is blocked at some postentry step. For
example, WHV enhancers and promoters may not function
correctly in PHH (8).
The virtual inability of hHDV to infect PWH might seem to
be in contradiction to the in vivo observation that hHDV can
be transmitted to a woodchuck in the presence of WHV (26).
However, in our studies we are detecting only a primary infec-
tion without subsequent spread. In contrast, for the in vivo
studies, a rare infection event into a hepatocyte already in-
fected with WHV can lead to the assembly and release of new
HDV. This will be wHDV rather than hHDV, which will be
able to amplify and spread throughout the susceptible hepato-
cytes of the woodchuck liver.
We have demonstrated that for wHDV, as for hHDV (13,
35), the ability to infect a susceptible cell depended upon
sequences within the pre-S1 domain. Evidence for this was
obtained using peptides related to the pre-S1 region of the
envelope proteins of both WHV and HBV (Fig. 6). However,
a comparison of the pre-S1 sequences of WHV and HBV
showed little sequence conservation (Fig. 1). And while se-
quences from the pre-S1 of HBV could block infection of PHH
by hHDV, they did not block the infection by wHDV.
Furthermore, we have exploited the simpler assembly re-
quirements of HDV relative to hepadnaviruses (16, 36) to
achieve the assembly of infectious HDV RNA-containing par-
ticles containing known combinations of WHV and HBV en-
velopes and thus demonstrated that the ability to infect PHH
is provided by HBV or WHV L but not by HBV or WHV S
(Fig. 7).
These findings lead us to suggest that hHDV and wHDV
might use pre-S1 domains to recognize different receptors on
the surface of PHH to achieve infection. Such an interpreta-
tion may be presumptive in that we still do not know the
identity of the receptor(s) used by HBV and HDV for infection
of PHH (13). Nevertheless, we trust that our studies will ulti-
mately contribute to a more complete picture of how hHDV
and wHDV, as well as HBV and WHV, attach to and enter
susceptible hepatocytes.
ACKNOWLEDGMENTS
J.M.T. was supported by grants AI-058269 and CA-06927 from the
NIH and by an appropriation from the Commonwealth of Pennsylva-
nia. N.C. was supported in part by the Elizabeth Knight Patterson
Fellowship.
Constructive comments on the manuscript were given by Glenn Rall
and Richard Katz. We acknowledge assistance from Emmanuelle
Nicolas and the Biochemistry and Biotechnology Facility, Roland
Dunbrack and the Molecular Modeling Facility, Sandra Jablonsky and
the Cell Imaging Facility, and Carol Aldrich.
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