JVI Accepts, published online ahead of print on 12 March 2014 J. Virol. doi:10.1128/JVI.00430-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Envelope proteins derived from naturally integrated hepatitis B virus DNA support assembly and
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release of infectious hepatitis delta virus particles.
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Natalia Freitasa, Celso Cunhab, Stephan Mennec and Severin O. Gudimaa#
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Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical
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Center, Kansas City, Kansas, USAa; Medical Microbiology Unit, Center for Malaria and Tropical
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Diseases, Institute of Hygiene and Tropical Medicine, New University of Lisbon, Lisbon, Portugalb;
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Department of Microbiology and Immunology, Georgetown University Medical Center,
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Washington, DC, USAc.
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Running title: HBV integrant-derived envelope proteins
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#Address correspondence to Severin O. Gudima,
[email protected].
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Word counts: Abstract (250 words); text (6766 words)
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ABSTRACT
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A natural subviral agent of human hepatitis B virus (HBV), hepatitis delta virus (HDV) requires
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only the envelope proteins from HBV in order to maintain persistent infection. HBV surface
27
antigens (HBsAgs) can be produced either by HBV replication, or from integrated HBV DNA
28
regardless of the replication. The functional properties of the integrant-generated HBsAgs were
29
examined using two human HCC-derived cell lines Hep3B and PLC/PRF/5 that contain HBV
30
integrants, but do not produce HBV virions and have no signs of HBV replication. Both cell lines
31
were able to support HDV replication, and assembly/egress of HDV virions. Neither of the cell lines
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was able to produce substantial amounts of the PreS1-containing HDV particles. HDV virions
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assembled in PLC/PRF/5 cells were able to infect primary human hepatocytes, while Hep3B-derived
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HDV appeared non-infectious. These results correlate with the findings that the entire open reading
35
frame (ORF) for the large (L) envelope protein that is essential for infectivity was present on HBV
36
RNAs from PLC/PRF/5 cells, while the L ORF that was truncated and fused to inverted pre-core
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sequences was found using RNAs from Hep3B cells. This study demonstrated for the first time that
38
at least some of HBV DNA sequences naturally integrated during infection can produce functional
39
small and large envelope proteins capable of the formation of infectious HDV virions. Our data
40
indicated that in vivo chronic HDV infection can persist in the absence of HBV replication (or when
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HBV replication is profoundly suppressed) if functional envelope proteins are supplied from HBV
42
integrants.
43 44
IMPORTANCE. The study addresses the unique mechanism of HDV persistence in the absence of
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ongoing HBV replication; advances our understanding of HDV-HBV interactions; and supports the
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implementation of treatments directly targeting HDV for HDV/HBV-infected individuals.
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47
INTRODUCTION
48
Hepatitis delta virus (HDV) is a significant human pathogen with approximately 20 million chronic
49
carriers worldwide. HDV is a natural subviral agent of human hepatitis B virus (HBV) that requires
50
from its helper hepadnavirus only the envelope proteins in order to form the virions and infect
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hepatocytes via HBV receptor. In infected livers, HDV co-exists with HBV. Chronic HBV infection
52
remains a main risk factor of hepatocellular carcinoma (HCC) and is associated with more than half
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of all HCC cases (1-4). Concomitant HDV infection is able to inflict additional liver damage
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associated with accelerated liver disease, cirrhosis, liver failure and HCC (5-11). Treatment with
55
alpha-interferon is beneficial only for a subset of HDV carriers. There are no treatments in clinical
56
practice that directly target HDV, and practically all anti-HBV drugs do not block HDV infection (5,
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12-13). In livers chronically infected with HBV, as many as 90% of hepatocytes may appear free of
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HBV replication markers. HBV-infected individuals can support HDV infection regardless of the
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presence of HBV replication markers. Often HDV/HBV carriers exhibit relatively high HDV levels,
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while HBV levels remain low or undetectable. In HDV/HBV-infected human liver, a subpopulation
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of hepatocytes was positive only for HBV, a second subpopulation was positive only for HDV, and
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another subpopulation was positive for both viruses. Thus, a number of HDV-infected hepatocytes
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apparently may not display markers of HBV replication. In addition, HDV is able to suppress HBV
64
replication (2, 14-24). The above observations indicate that HDV and HBV, while being present in
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the same liver, may not necessarily be present in the same cell. Furthermore, the data indicate that (i)
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HDV may not depend on the ongoing HBV replication and (ii) chronic HDV infection may actually
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persist in absence of HBV replication, if another alternative source of HBV envelope proteins is
68
available.
69
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The envelope proteins can be produced from integrated HBV DNA independently of hepadnavirus
71
replication. During chronic HBV infection, a significant number of hepatocytes contains HBV
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DNA integrants. Normal hepatocytes that are apparently free of HBV replication markers, but still
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express HBV envelope proteins can appear as the result of resolved HBV infection or via immune-
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mediated selection. In addition, HCC cells may apparently no longer support HBV replication, but
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still could support the production of envelope proteins from HBV integrants (3, 14-16, 25-27). In
76
this study, we examined the hypothesis that HDV can persist in the absence of HBV replication (or
77
when HBV replication is profoundly suppressed) if functional envelope proteins are supplied from
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naturally integrated HBV DNA. Such mechanism of HDV persistence was not explored previously.
79
A potential obstacle here is that deletions, insertions and rearrangements were often observed in
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integrated HBV DNA (28-33). In addition, the functional properties of HBV integrant-derived
81
surface antigens (HBsAgs) remain to be fully evaluated (16, 29-31, 34-35).
82 83
We examined two human cell lines derived from HBV-induced HCCs, Hep3B and PLC/PRF/5
84
(Alexander cells) that bear HBV integrated DNA, but do not produce HBV virions and display no
85
markers of HBV replication (29-31, 36). Both cell lines were able to support HDV replication,
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assembly and secretion of HDV virions. HDV virions produced by PLC/PRF/5 cells (HDV-PLC)
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were able to infect primary human hepatocytes (PHH). However, Hep3B-derived HDV (HDV-
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Hep3B) appeared non-infectious. Our results support the hypothesis that at least some of natural
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HBV integrants produce functional L (large) and S (small) envelope proteins (the presence of which
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represents the minimal requirements for the formation of infectious virus particle) and thus can
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facilitate HDV persistence regardless of ongoing HBV replication. Our findings also suggest that
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HDV is likely more independent and more significant pathogen than it is currently assumed, and that
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HDV/HBV carriers will likely benefit from treatments directly targeting HDV.
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MATERIALS AND METHODS
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Plasmids, cell lines and transfection. The LMS vectors, pNF-HBV-LMS-B4, pNF-HBV-LMS-C5,
97
pNF-HBV-LMS-D1, pNF-HBV-LMS-D3 and pNF-HBV-LMS-F1, were constructed to express the
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L, middle (M) and S envelope proteins that belong to natural variants of HBV genotypes B, C, D
99
and F (accession numbers: D00330, AF411411 (M-minus variant), AB205127, V01460 and
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X69798, respectively). For each LMS vector, the number next to HBV genotype name refers to the
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number of a particular sequence among the variants of a given HBV genotype in the assembled
102
collection of HBV sequences of genotypes A-I. The HBV variants D1, B4 and C5 were acquired
103
during the chronic stage of HBV infection. For the variants D3 and F1, the information about their
104
origin was not available ((37-40) and Abe K, personal communication). The HBV sequences were
105
inserted into XhoI/XbaI sites of the pSVL vector (Pharmacia). Each HBV insert begins at the natural
106
start codon of the L, contains the entire L coding sequence along with 3'-untranslated region that
107
bears the post-transcriptional regulatory element (PRE) and ends at about one hundred nucleotides
108
downstream of the PRE. For reference, in a typical HBV genotype A sequence, the PRE occupies
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the positions 1151-1684 (41-43). Hep3B (Hep3B2.1-7) and PLC/PRF/5 cells (29-31) were
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purchased from ATCC. To initiate HDV replication and assembly, Hep3B and Alexander cells were
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transfected with the plasmid pSVLD3 (44). Each type of the reference HDV virions was produced
112
by co-transfection of the Huh7 cells with a 1:1 mixture of pSVLD3 and one of the above LMS
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vectors, using the Fugene HD reagent (Roche) according to the instructions of the manufacturer.
114
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Immunoprecipitation (IP). The immunoprecipitaion experiments were conducted using the method
116
that was described previously and employs Pansorbin (Calbiochem) (45). The rabbit polyclonal
117
"anti-matrix" antibodies were generated by GenScript. They were raised against the synthetic
118
peptide that spans the amino acid residues 91-
119
IPPPASTNRQSGRQPTPISPPLRDSHPQAMQWNSTAFH-128 of the PreS region of the L
120
envelope protein (genotype A of HBV) and contains the conserved matrix domain (underlined) (45-
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46). According to the software predicting the antigenic sites in proteins (bips.u-strasbg.fr/EMBOSS/
122
and mobyle.pasteur.fr/cgi-bin/portal.py?#forms::antigenic), the above peptide harbors a single
123
antigenic determinant TPISPPLRDS, which is located within the PreS1 domain. Thus, it was
124
anticipated that the IP with the "anti-matrix" antibodies would target specifically only the PreS1-
125
containing HDV particles. The virions that contain the PreS1 domain of the L envelope protein
126
(which interacts with HBV receptor (45)) on the outer surfaces are considered as potentially
127
infectious. In a separate study, we examined the specificity of the "anti-matrix" antibodies. Two
128
types of HDV virions were examined. First type of the virions (HDV-D1 LMS) was assembled
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using the vector expressing the L, M and S proteins of the HBV variant D1 (i.e., pNF-HBV-LMS-
130
D1). The second type (HDV-D1 L-minus) was prepared using the L-minus version of the above
131
mentioned vector, which was modified to express the M and S, but not the L envelope protein. The
132
"anti-matrix" antibodies were compared to the S26 mouse monoclonal antibodies that recognizes
133
QDPR sequence in the PreS2 domain (47), and therefore was able to bind the L and M proteins.
134
Using HDV-D1 L-minus virions, it was found that the "anti-matrix" antibodies (as compared to the
135
S26 antibody) were unable to immunoprecipitate about 78% of the L-M+S+ virions. This result
136
suggested that the "anti-matrix" antibodies recognized very poorly the M protein, which was
137
exposed on the outer surfaces of the particles. Using HDV-D1 LMS virions, it was deduced that the
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fraction of the L-containing particles in the virions that were pulled down during the IP procedure by
139
the "anti-matrix" antibodies was at least 83%, while the rest of precipitated virions (about 17% or
140
less) may consist of the particles that bore the PreS2 sequences in the context of the M protein,
141
while the L was absent (i.e., L-M+S+ virions) (data not shown). Because the PreS1-HDVs
142
represented the predominant majority of the virions that were immunoprecipitated by the "anti-
143
matrix" antibodies, the contribution of the virions that contain the M, but not the L protein can be
144
considered as not significant. The virions that were immunoprecipitated by the "anti-matrix"
145
antibodies were designated as the PreS1*-HDVs, because these antibodies demonstrated significant
146
preferential, but not exclusive specificity for the PreS1-bearing virus particles. The use of the
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asterisk indicates that the virions, which were immunoprecipitated by the "anti-matrix" antibodies,
148
were predominantly the PreS1-HDVs with an addition of not significant amounts of the particles
149
that bear the M, but not the L protein. The PreS1*-HDVs served, therefore as acceptable
150
approximation for the fraction of virions that are potentially infectious. The ratio of 3 µl of "anti-
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matrix" antibodies per 10 µl of the concentrated HDV stock (prepared using polyethylene glycol
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(48)) was found to be optimal in order to achieve the best performance of the IP procedure. In
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addition, the optimized IP procedure has been tested using different HDV stocks that were prepared
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employing the envelope proteins of seventeen different HBV variants. Regardless of the presence of
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the variant-specific amino acid residues that were not identical to those in the region of the above
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peptide sequence (pos. ## 91-128 in HBV genotype A), the IP procedure was able to pull down the
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PreS1*-HDVs with the efficiency that was very similar for different HDV stocks tested. It appears,
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that approximately 90% of all the PreS1*-HDVs (and in some cases >90%) were precipitated. In
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addition, no correlation was observed between the amounts of the immunoprecipitated PreS1*-
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HDVs and the presence of particular non-identical residues (or their combinations) in the region
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spanned by the above peptide (not shown). Therefore, it has been concluded that "anti-matrix"
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antibodies recognize the envelope proteins of different HBV variants with comparable efficiency.
163 164
RNA isolation and real-time PCR assay (qPCR). Total RNA from cells or virions (including
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Pansorbin-bound particles) was isolated with TRI reagent (Molecular Research Center) and then
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treated with DNase (Life Technologies) prior to further analysis. The genomic (G) HDV RNA was
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measured using qPCR as previously described (48). The copy numbers were quantified using a 10-
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fold dilution series of in vitro transcribed and gel-purified unit-length G HDV RNA standard in the
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range of 20-2x106 genome equivalents (GE) of HDV, and considering that one million of HDV
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RNA molecules equals to one picogram of the RNA standard.
171 172
Infection of primary human hepatocytes (PHH) in vitro. The PHH were purchased in the format
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of 48-well plates from Life Technologies. HDV virions were concentrated approximately 100-fold
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before the infection, using polyethylene glycol (PEG). The infections of PHH were performed in
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presence of 5% PEG 8000 (Sigma) as described previously (48). Infected PHH were assayed at nine
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days post-infection.
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Immunofluorescence (IF). The procedure is described in detail elsewhere (48-49). PHH were
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fixed at room temperature with 4% paraformaldehyde for 30 min, washed two times with phosphate
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buffer saline (PBS) and then permeabilized using 0.2% Triton X-100 solution in PBS. Mouse
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monoclonal antibody against human alpha-tubulin (Sigma) and polyclonal rabbit antibodies against
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the delta antigens were used to stain PHH. Nuclear DNA was stained using 1 μg/ml DAPI solution
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(Sigma). An inverted microscope Nikon TE2000-U with 20x objective was used for analysis of
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stained PHH.
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Analysis of HBV integrant-derived RNAs using PCR, cloning and sequencing. To examine the
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presence of specific HBV sequences on the RNAs harvested from not transfected Hep3B or
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Alexander cells, the primer A, 470-CGGGCAACATACCTTGATAATCCAGA-445; primer B,
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2813-ATTTTGTGGGTCACCATATTCTTGGGAACAA-2843; primer C, 3186-
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TCTAAGAGACAGTCATCCTCAGGC-3209; primer D, 138-GGGACCCTGCACCGAACATG-
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157; primer E1 (E1 was used only for the material from Alexander cells), 2878-
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CCTCTCGAGTGGGGGAAGAAT-2898; primer E2 (E2 was used only for the material from
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Hep3B cells), 2878-GCAAAGGCATGGGGACGAAT-2898; and primer F, 1886-
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CAAGGCACAGCTTGGAGGCTT-1866 were used. Six PCR assays were conducted using the
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pairs of primers, BA, CA, DA, E1A, E2A and FA. The primer R, 752-
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ACCACATCATCCATACTGAAAGCC-726 was used for reverse transcription (RT). The primers
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B and A were also used for sequence analysis of the region coding for 211 N-terminal amino acids
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of the L protein. The primers, G (801-ATTGTAACAGCGGTATAAAGGGA-740) and H (413-
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CTGCTGCTATGCCTCATCTTCTT-435) were utilized to analyze the sequences coding for the
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amino acids 95-209 in the S domain. For the latter assay, primer K (873-
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GTTTAGGGAATAACCCCATCTCTTCGT-847) was used for cDNA synthesis. The nucleotide
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numbering is according to HBV genotype A (41-43). The PCR products BA and GH were cloned
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into the Topo-vector II (Life Technologies) and the resulting clones were sequenced using M13
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universal primers.
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To address further the functionality of the integrant-derived envelope proteins, total RNA from
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Alexander cells was used and the entire open reading frame for the L envelope protein was
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amplified by RT-PCR using the following primers: (i) for cDNA synthesis, primer M (1190-
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TCAGCAAACACTTGGCACAG-1171); (ii) for the outer (first) PCR, primers B and N (1120-
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GAAAGGCCTTATAAGTTGGCGAGAA-1096); (iii) for the inner (second) PCR, primers P (starts
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with an adaptor sequence containing SalI site (underlined) and continues with HBV-specific
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sequence: TCTAGTCGAC-2854-ATGGGGCTTTCTTGG-2868) and Q (starts with an adaptor
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sequence containing NheI site (underlined) and continues with HBV-specific sequence:
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GTAGCTAGC-1084-GCTTAGCTTGTATACATGCATATA-1061). The resulting product of the
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nested PCR spanning the entire open reading frame (ORF) of the L protein was digested with SalI
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and NheI restriction enzymes and cloned into pSVL vector (Pharmacia) using XhoI/XbaI sites.
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Next, to ensure the export of the mRNA into cytoplasm and its proper translation, the fragment of
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the HBV genotype G genome (because the initial sequencing of the cloned ORFs for the L protein
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revealed that they belong to the genotype G of HBV) that starts immediately downstream of the L
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ORF, includes the post-transcriptional regulatory element (PRE) and finishes about 100 nucleotides
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downstream of the PRE (42-43), was introduced immediately next to the 3′-end of the ORF of the L
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protein that was cloned into pSVL using the material from Alexander cells. To amplify the above
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fragment, the plasmid pNF-HBV-LMS-G1 that is a vector that expresses the L, M and S envelope
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proteins of the HBV genotype G variant 1 (accession number AF405706) was used. The primers for
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this amplification were S, 1032-CCAATGTGGTTACCCTGCCT-1047, and T (starts with an
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adaptor sequence containing BlpI site (underlined) and continues with HBV-specific sequence:
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GCTTAGC-1788-GCCTACAGCCTCCTA-1773). Prior to the cloning into the construct bearing the
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sequences coding for the entire ORF of the L (from PLC/PRF/5 cells), the PCR product ST was
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digested with Eco91I. After completion of the cloning and sequencing, two sequences initially
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derived from RNA harvested from Alexander cells were selected and designated as ORF52 and
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ORF62. Based on these sequences, two LMS vectors expressing ORF52 or ORF62 were designated
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as pNF-LMS-ORF52 and pNF-LMS-ORF62. These two LMS vectors were subsequently used to
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assemble two virus types, HDV-PLCORF52 and HDV-PLCORF62 as described above using a co-
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transfection strategy of Huh7 cells.
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RESULTS
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HDV assembly in Hep3B and Alexander cells. Hep3B cells apparently contain at least two HBV
238
integrants, while PLC/PRF/5 cells bear at least seven HBV inserts. Neither of the cell lines produces
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HBV nor exhibits markers of HBV replication (29-31). Since a main substrate of integration is a
240
double-stranded linear (DSL) HBV genome, the production of the pre-genomic and pre-core RNAs
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from such integrant is not anticipated, while mRNAs encoding the L, M and S envelope proteins can
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be transcribed as HBV-host hybrids harboring host sequences at 3’-end immediately prior to the
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poly(A) tail. The functional properties of the polypeptides translated from these integrant-derived
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mRNAs are yet to be fully evaluated (16, 29-31, 34-35). Both cell lines, Hep3B and Alexander cells,
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were transfected with the plasmid pSVLD3 (44) to initiate HDV replication. As a control, Huh7
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cells were transfected simultaneously with pSVLD3 and vector pNF-HBV-LMS-D3 that expresses
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functional L, M and S of HBV genotype D variant.
248 249
The qPCR analysis of intracellular accumulation of HDV G RNA between days 3 and 17 post-
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transfection revealed that both Hep3B and Alexander cells supported HDV replication levels
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comparable to that of Huh7 cells (Fig.1A). All three cell lines facilitated assembly and secretion of
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HDV (Fig. 1B). At three days post-transfection, HDV secretion levels were comparable in all cell
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lines. Between day 5 and 11 post-transfection, HDV virions were produced most efficiently by Huh7
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cells. During this time period, Hep3B cells were producing about 2-12 times less of HDV virions
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(HDV-Hep3B). Starting day 13, Hep3B cells displayed levels of HDV secretion approximately 2-5
256
times higher than Huh7 cells. Between days 5-17, as compared to Huh7 cells, Alexander cells
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secreted about 4-121 times less of HDV (HDV-PLC), which were the lowest HDV yields observed
258
among the three cell lines.
259 260
Assembled HDV-Hep3B and HDV-PLC virions were then compared to the reference HDV types,
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each of which was produced in Huh7 cells by co-transfection with pSVLD3 and LMS vector
262
expressing the envelope proteins of a particular natural HBV variant. Those were HDVs coated
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with the envelope proteins of HBV genotype: (i) B (HDV-B4); (ii) C (HDV-C5); (iii) D (HDV-D1)
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and (iv) F (HDV-F1). Thus, all of HDV types, including HDV-Hep3B and HDV-PLC, contained the
265
same HDV ribonucleoprotein inside the virions and differed only by the origin of HBV envelope
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proteins employed for assembly. Prior to further analysis, all the above HDV stocks were
267
concentrated about 100-fold using polyethylene glycol (48). The results of comparison of the
268
concentrated virus stocks are summarized in Table 1. The HDV-F1 displayed the highest total
269
concentration of HDV virions. The total concentration of HDV-D1 virions was approximately five-
270
fold lower. For the HDV-Hep3B and HDV-C5, the total HDV levels were 11.7% and 5.7%,
271
respectively, as compared to that of HDV-F1. The concentrations of HDV-B4 and HDV-PLC were
272
1.1% and 2.1%, respectively. Therefore, the total HDV yields facilitated by HBsAgs expressed in
273
Hep3B and Alexander cells were within the range observed for the envelope proteins of natural
274
HBV variants. The fraction of the PreS1-containing and therefore potentially infectious virions
13
275
(because the N-terminus of the PreS1 domain harbors the receptor-binding region (50)) was
276
analyzed using the immunoprecipitation (IP) with the "anti-matrix" antibodies against the peptide
277
harboring conserved HBV matrix domain (3, 46, 50). As described in Materials and Methods, the
278
"anti-matrix" antibodies displayed strong preferential, but not exclusive specificity to the PreS1. In a
279
separate study, it was determined that the virions immunoprecipitated using the "anti-matrix"
280
antibodies were predominantly (greater than 80%) the PreS1-containing HDVs along with not a
281
significant fraction (less than 20%) of the L-M+S+ virions (not shown). The HDV virions pulled
282
down with the "anti-matrix" antibodies were designated as the PreS1*-HDVs, indicating that
283
predominant majority of the precipitated virions were the PreS1-HDVs. The PreS1*-HDVs
284
quantification therefore served as acceptable approximation for the content of the potentially
285
infectious HDV virions. The results of the IP-based quantification of PreS1*-HDVs are presented in
286
Table 1. The highest fractions of the PreS1*-HDVs were found in HDV-D1 and HDV-F1, 32.1%
287
and 27.3%, respectively. For HDV-B4 and HDV-C5, the percentages of PreS1*-HDVs were 16.1%
288
and 12.4%, respectively. The lowest yields of PreS1*-HDVs were measured in HDV-Hep3B (0.5%)
289
and HDV-PLC (1.0%). The percentages of the PreS1*-HDVs measured for HBV-Hep3B and HDV-
290
PLC were not significantly higher than the cut-off value of the sensitivity of the IP procedure that
291
was determined using HDV coated with the S envelope protein only (i.e., 0.2%).
292 293
Infectivity of HDV virions coated with the envelope proteins produced from integrated HBV
294
DNA. Infectivity of HDV-Hep3B and HDV-PLC was compared to that of HDV-B4, HDV-C5,
295
HDV-D1 and HDV-F1 using in vitro infection of primary human hepatocytes (PHH). Four
296
independent infections were conducted using four different PHH lots. For the experiments detecting
297
HDV-positive hepatocytes using the immunofluorescence (IF), the multiplicity of infection (MOI)
14
298
was in the range of 25-100 total HDV virions per hepatocyte. HDV-infected cells were detected
299
using rabbit polyclonal antibodies against recombinant small delta antigen (48). The representative
300
IF images are shown in Figure 2. No HDV-positive cells were observed during all infections among
301
PHH inoculated with HDV-Hep3B virions (Fig. 2A). Therefore, HDV-Hep3B was considered non-
302
infectious. Unlike HDV-Hep3B, HDV-PLC (Fig. 2B) was infectious on all occasions. The HDV-
303
PLC was able to infect between 0.43% and 1.96 % of PHH (MOI 25-50). Similarly, HDV-positive
304
PHH were observed during all infections for HDV-B4, HDV-C5, HDV-F1 and HDV-D1 (Figs. 2C,
305
2D, 2E and 2F, respectively). For HDV-D1, the percentage of infected PHH was in the range of
306
16.60% to 28.25% (MOI 100). For HDV-F1, the fraction of infected hepatocytes was 3.71 - 23.41%
307
(MOI 100). For HDV-B4 (MOI 30) and HDV-C5 (MOI 50-100), the observed fractions of infected
308
cells were 0.52 - 6.18% and 1.94 - 18.99%, respectively. In agreement with previous observations
309
(48), two typical patterns of the delta antigen (δAg) subcellular localization in infected PHH were
310
observed (Fig. 2). The major pattern was nucleoplasmic staining, and the minor one included an
311
additional staining in cytoplasm. Next, the extent of HDV replication in infected PHH was measured
312
by qPCR that specifically assayed a number of HDV genomic RNA copies in isolated total cellular
313
RNA (48). Based on qPCR data, the specific infectivity (SI) values were calculated. The SI reflects
314
the number of HDV genomes produced as a result of infection per average hepatocyte, which is
315
normalized by the PreS1*-MOI (a number of the PreS1*-HDVs per hepatocyte used in inoculum).
316
The average SI determined for HDV-D1 was the highest and was used as 100% value. All other SI
317
numbers were expressed relatively to the SI of HDV-D1 (Fig. 3). The HDV-F1 and HDV-C5 had
318
intermediate SI levels of 57.3% and 33.5% respectively, while HDV-B4 displayed a relatively low
319
number of 6.4%. For HDV-PLC, the average SI value observed was 8.8%. As expected, HDV-
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320
Hep3B had the lowest SI number of 1.4%. Overall, the generated data demonstrated that HDV-PLC
321
was infectious, while Hep3B appeared to be non-infectious.
322 323
To examine further the infectivity of HDV-PLC, the entire open reading frame (ORF) for the L
324
envelope protein was PCR-amplified using total RNA from not transfected Alexander cells and
325
cloned into the vector that enables the expression of the L, M and S proteins after the transfection.
326
Two different ORF sequences were used to produce HDV-PLCORF52 and HDV-PLCORF62 virus
327
stocks using the above described co-transfection strategy of Huh7 cells. The total amounts of HDV
328
virions/µl in the concentrated stocks of HDV-PLCORF52 and HDV-PLCORF62 were 3.51x105 and
329
7.97x105, respectively. These values correspond to 4.0% and 9.2%, respectively, as compared to that
330
of HDV-F1 (see Table 1 for comparison), and are similar to the total yield of HDV as facilitated by
331
a natural variant of HBV genotype G (accession number AF405706), which was 7.53x105 GE/µl (in
332
a concentrated stock) (not shown). The measured percentages of the PreS1*-HDVs were 9.3% for
333
HDV-PLCORF52 and 33.0% for HDV-PLCORF62. The increased (as compared to HDV-PLC
334
(Table 1)) fractions of PreS1*-HDVs are likely a result of efficient L expression from a strong SV40
335
late promoter located upstream of the cloned ORFs in the expression vectors. Given that the
336
expression of the S in both vectors occurred from authentic HBV promoter, the above results
337
suggest that in Alexander cells, the S proteins per se are likely not deficient in formation and egress
338
of the L-S complexes, and low percentage of the PreS1-HDVs in HDV-PLC at least in part could be
339
explained by low rates of the L production/accumulation. At MOI of 100, HDV-PLCORF52 and
340
HDV-PLCORF62 infected 4.17% and 10.42% of PHH, respectively, which is consistent with the
341
higher percentage of the PreS1-HDVs in inoculum. The SI values for HDV-PLCORF52 and HDV-
342
PLCORF62 were 24.84% and 10.42%, respectively, as compared to that of HDV-D1 (see Fig. 3 for
16
343
comparison). These results suggest that the L proteins encoded by the ORF52 and ORF62 likely
344
have no obvious defects in supporting HDV infectivity. Since these SI values were similar or less
345
than three-fold higher than that of HDV-PLC (Fig. 3), it is possible that HDV-PLC consist of
346
virions, individual infectivities of which do not differ significantly.
347 348
Analysis of RNA sequences transcribed from integrated HBV DNAs. Next, we conducted
349
further examination of HBV RNAs transcribed from the integrated DNAs using RT-PCR, followed
350
by cloning and sequencing in order to better understand the results of the infectivity assays. Several
351
different PCR assays were developed to examine the presence of specific HBV sequences on
352
mRNAs accumulated in untransfected Hep3B and Alexander cells (Fig. 4). The primers A, B and R
353
spanned the sequences that are conserved among different HBV genotypes, A-I. The design of the
354
other primers, C, D, F, E1 and E2, incorporated the information about HBV integrants found in
355
Alexander cells and Hep3B cells (30, 34-35, 51) and our data acquired by partial cloning/sequencing
356
of RNA sequences accumulated in the above cell lines (not shown). The PCR products BA, E1A,
357
CA and DA were observed for RNAs harvested from Alexander cells, which was consistent with the
358
presence of the 5′-UTR and sequences coding for the entire PreS1and PreS2 regions and for a
359
considerable N-terminal portion of the S domain (Figs. 4A and 4B). In contrast, using total RNA
360
from Hep3B cells, the absence of the BA PCR product and the presence of the E2A, CA, DA and
361
FA products was observed (Figs. 4C and 4D), suggesting that the 5′-UTR and sequences coding for
362
the N-terminus of the PreS1 are not expressed in Hep3B cells. The results presented in Fig. 4 and
363
subsequent examination of the sequences, which were amplified by the FA PCR assay using RNA
364
harvested from Hep3B cells (Fig. 5), were consistent with the description of an integrant in Hep3B
365
cells that has (i) a large deletion that includes the PreS1 promoter, the 5′-UTR and the N-terminus of
17
366
the PreS1, and (ii) a rearrangement, which resulted in the joining of the remainder of truncated
367
PreS1 domain to the inverted pre-core/core sequences (Fig. 4D and (30)). Interestingly, the junction
368
site mapped on RNAs accumulated in Hep3B cells, the pre-core 1819(1818)-PreS1 2877(2878) (the
369
positions in parentheses are shown, because the second T of the TT dinucleotide at the junction site
370
could belong to either pre-core or to the PreS1) was not the exact match for the junction that was
371
determined previously on the DNA integrant, which was the pre-core 1815-PreS1 2875 (Fig. 5 and
372
(30)). All these data (Figs. 4 and 5) argue against expression of the L protein in Hep3B cells and are
373
consistent with HDV-Hep3B being non-infectious. The existence of the PCR product FA for RNA
374
from Hep3B cells (Fig. 4C) suggests that transcription of the RNA bearing the inverted pre-
375
core/core sequences joined to the truncated PreS1 domain was driven by unknown cellular promoter.
376 377
We further analyzed (i) the sequences of the N-terminal part of the L envelope protein that contains
378
HBV receptor-binding site, and also (ii) the sequences of the S domain, at and around the HDV-
379
binding site (HDV-BS) that is essential for HDV assembly (45, 52). As described above, the intact
380
sequences coding for the PreS1were found only on RNAs from Alexander cells, and not on RNAs
381
from Hep3B cells (Fig 4). Fig. 6A shows the N-terminal region of the L (amino acids 1-211)
382
determined using HBV RNAs from Alexander cells. No deletions or other rearrangements were
383
observed. The closest match to the most abundant sequence was the variant of the genotype G
384
(accession number BAG50284.1, 88% sequence identity). The most abundant sequence found in
385
Alexander cells (7/14 sequences) was identical to the PLCORF52, and one of the least abundant
386
sequences was the match for the PLCORF62 (1/14 sequences), which means that PLCORF52 and
387
PLCORF62 together represent the majority (8/14) of RNA sequences recovered from Alexander
388
cells. Therefore, our data regarding the infectivity of HDV-PLCORF52 and HDV-PLCORF62 are
18
389
likely representative for the majority of HDV-PLC virions. In addition, taking into consideration (i)
390
the above mentioned total yields of HDV virons as facilitated by the PLCORF52 and PLCORF62,
391
and (ii) the fact that PLCORF52 and PLCORF62 correspond to the majority of sequences recovered
392
from Alexander cells, it became apparent that majority of the L mRNAs in Alexander cells encode
393
the envelope proteins that are likely not defective in supporting HDV assembly. Within the N-
394
terminus of the PreS1 (up to the position 86 for HBV genotypes bearing the PreS1 domain of 119
395
amino acids (45)) that is critical for the infectivity of HBV and HDV, all the recovered sequences
396
are identical, which suggests that if the variants of the L envelope protein presented in Fig. 6A,
397
would have facilitated different levels of HDV infectivity, this should have been mediated by the
398
residues outside of the N-terminus of the PreS1. Curiously, PLCORF52 and PLCORF62 within the
399
entire PreS1 differ by a single amino acid at position 101 (the position that is dispensable for HDV
400
infectivity (45, 53)), in which the former has K, and the latter - G. Given the variety of recovered
401
RNA sequences, it is conceivable that in Alexander cells, more than one HBV integrant facilitates
402
production of the L envelope protein.
403 404
The alignment presented in Fig. 6A allows us to conduct an additional analysis of the sequences that
405
were found Alexander cells in terms of their ability to react with the "anti-matrix" antibodies. As
406
compared to the sequence of the peptide that was originally used to produce the "anti-matrix"
407
antibodies, there are a few residues present in the alignment that are not identical to the above
408
mentioned peptide sequence. Clearly V(90) and T(100) (the numbering is for a typical genotype G;
409
see Fig. 6A) do not influence the interactions with the "anti-matrix" antibodies. They both are
410
present on the PLCORF62 sequence, for which we already determined a relatively high (i.e., 33.0%)
411
percentage of the PreS1*-HDVs in the HDV-PLCORF62 virus stock. Only 3/14 sequences bear the
19
412
unique L(120) residue, while a very abundant non-identical residue K(101) is present on 10/14
413
sequences shown in the alignment. As stated above, this is the only position, in which the non-
414
identical residues were found in the sequences PLCORF52 (contains K(101)) and PLCORF62
415
(bears G(101)) within the entire PreS1. Therefore, the change G(101)K has been introduced into the
416
PLCORF62 sequence to create the PLCORF62G(101)K mutant. When the two newly made virus
417
stocks, HDV-PLCORF62 and HDV-PLCORF62G(101)K were assayed by IP with the "anti-matrix"
418
antibodies, practically no differences were observed. The percentage of the PreS1*-HDVs in HDV-
419
PLCORF62 in the new experiment was measured as 37.5%, while for HDV-PLCORF62G(101)K,
420
the number was 34.8%. The result suggest that there was no defect in the recognition by the "anti-
421
matrix" antibodies of the PreS1*-HDVs in the stock HDV-PLCORF62G(101)K. It also became
422
apparent that the observed differences in the percentages of the PreS1*-HDVs between HDV-
423
PLCORF52 and HDV-PLCORF62 are not related to the above mentioned K residue at pos 101. In
424
addition, using the data presented in the Fig. 5, the sequences of the C-terminus of the PreS1 were
425
deduced from the corresponding nucleotide sequences found in Hep3B cells. Within the region that
426
is recognized by the "anti-matrix" antibodies, no residues were found that could potentially alter the
427
interactions with the "anti-matrix" antibodies (not shown). Therefore, it was interpreted that if the L
428
protein would have been expressed in Hep3B cells, it would be easily identified by the IP using the
429
"anti-matrix" antibodies. Overall, considering these new findings and the abovementioned data
430
regarding the analysis of different HBV variants that was conducted in a separate study, it was
431
concluded that in Hep3B and Alexander cells there were no considerable number of sequences
432
found that would not be efficiently recognized by the "anti-matrix" antibodies.
433
20
434
Next, we examined the sequences coding for amino acids 95-209 of the S domain spanning the
435
important for infectivity region of major antigenic loop (MAL) and HDV-BS (45, 52). In Alexander
436
cells (Fig. 6B), twelve sequences were 99% identical to another HBV sequence (FJ692561.1, HBV
437
genotype A, subtype A1). All the sequences had a single substitution A(166)G in MAL. One
438
sequence had a substitution Q(101)R in the C-terminus of the second transmembrane domain, one –
439
a change W(191)R in the C-terminus of a third transmembrane domain, and another one – a
440
substitution Y(200)C in HDV-BS (the numbering was considering the N-terminal position of the S
441
protein (S domain) as the number one). The functional consequences of these changes are yet to be
442
determined. Another sequence from Alexander cells was identical to the above 12/16 sequences in
443
the region of positions 95-209, but it had a single nucleotide deletion immediately downstream of
444
the residue 209, which would result in the appearance of non-HBV amino acid sequence
445
downstream of this position (Fig. 6B). In Hep3B cells (Fig. 6C), eleven sequences were identical to
446
previously reported HBV sequence (GQ477489.1, HBV genotype A, subtype A2), and displayed no
447
mutations in the MAL and HDV-BS. The twelfth sequence had an insertion T(113)TT and a unique
448
deletion, which may lead to translation of the non-HBV sequences starting the position 161 and a
449
possible production of a hybrid protein. The above deletion also results in four premature stop
450
codons downstream the position 160, which could explain low abundance of this sequence. In
451
addition, the latter sequence may indicate mRNA transcription from a different HBV integrant.
452
Overall, the majority of sequences recovered from both cell lines had unchanged HDV-BS,
453
including three tryptophans that are critical for HDV assembly (1, 52). These findings are in
454
agreement with the observation that both cell lines support assembly and secretion of HDV. The
455
data also suggest that low efficiency of HDV secretion by Alexander cells (Fig. 1 and Table 1) is not
456
a result of compromised interaction of the HDV-BS with HDV ribonucleoprotein. It also appears
21
457
that the differences in infectivity between HDV-Hep3B and HDV-PLC were not mediated by the
458
sequences of the MAL, which has the ability to influence the overall infectivity of the virions (52),
459
and likely reflect the absence of the L protein in HDV-Hep3B virions (Figs. 4 and 5, and (30, 54)).
460 461
DISCUSSION
462
HDV persistence depends on the availability of functional L and S envelope proteins of HBV in
463
order to maintain assembly and infectivity of the virions (50, 52). Since HDV requires only the
464
envelope proteins from HBV, the ongoing HBV replication may not be a prerequisite of
465
maintenance of persistent HDV infection. The following observations support this notion: HDV-
466
infected individuals may have high levels of HDV replication and significantly decreased or
467
undetectable levels of HBV replication; most of anti-HBV drugs that successfully suppress HBV
468
replication do not block HDV infection; HDV can suppress HBV replication; and HDV-infected
469
hepatocytes may appear free of the signs of HBV replication (2, 5, 17, 19-23). A production of
470
HBsAgs from integrated HBV DNA can be an alternative to replication source of the envelope
471
proteins. HBV DNA integration occurs throughout the course of infection. Since DSL HBV genome
472
is a main substrate for integration, the pre-core and pre-genomic HBV RNAs are not expected to be
473
transcribed from the integrants, while synthesis of mRNAs for the envelope proteins can occur
474
regardless of HBV replication (3, 16). Therefore, hepatocytes in livers, where HBV infection is
475
resolved or HBV replication is significantly suppressed (by a drug treatment, for example) still may
476
be able to support persistent HDV infection by providing HBV envelope proteins from naturally
477
integrated DNA. However, the functional properties of HBV integrant-derived envelope proteins
478
were not fully evaluated previously, and no data exist regarding their ability to support the
479
production of infectious virions (16, 29-31, 34-35). In the current study, using two HCC-derived cell
22
480
lines that bear HBV integrated DNA and have no signs of HBV replication (29-31), we for the first
481
time demonstrated that at least some of HBV DNA integrant(s) express functional envelope proteins
482
that facilitate assembly of infectious HDV.
483 484
As expected, after transfection with HDV-expressing construct, Hep3B and Alexander cells
485
supported replication, assembly and release of HDV (Fig. 1 and Table 1). The immunoprecipitation
486
results suggested very low yield of the PreS1-containing HDV virions for both cell lines (Table 1).
487
Regardless of the considerable levels of HDV replication, Alexander cells facilitated lowest yield of
488
HDV virions as compared to transfected Huh7 and Hep3B cells (Fig. 1). The sequencing data
489
revealed that the majority of the integrant-derived HBV mRNAs in both cell lines code for the
490
functional HDV-binding sequence (Fig. 6). Alexander cells likely have a problem with efficiency of
491
the L production, and not with the egress of the S-L complexes, since when we expressed the
492
sequences of the ORF52 and ORF62 from the plasmids with a strong promoter for the L mRNA, a
493
9.3 times higher and 33 times higher yield of the PreS1*-HDVs, respectively, as compared to HDV-
494
PLC was achieved. This issue, however, is outside the scope of the current study.
495 496
Virions secreted from Alexander cells were able to infect PHH, while Hep3B-derived HDV
497
appeared non-infectious (Figs. 2 and 3). Our analysis of HBV RNAs expressed in Hep3B and
498
PLC/PRF/5 cells suggests the likely reasons for that. Using RT-PCR, we found on RNAs
499
accumulated in Alexander cells, the presence of the sequences coding for the entire PreS1 and PreS2
500
regions along with a considerable N-terminal part of the S domain. Taken together with the fact that
501
Alexander cells support assembly and egress of HDV virions and considering the results of the
502
infectivity assays, we conclude that Alexander cells express at least functional L and S proteins in
23
503
quantities that are sufficient to facilitate the production of infectious HDV. Unlike for Alexander
504
cells, our results suggest that the L protein is likely not produced in Hep3B cells, since our assay did
505
not find full-size intact sequences coding for the PreS1 on the accumulated HBV RNAs, which was
506
consistent with HDV-Hep3B being non-infectious. Our data are consistent with previous reports
507
describing that (i) Alexander cells contain at least one integrant bearing the entire ORF for the L
508
protein, which could facilitate the production of the L; (ii) while in Hep3B cells, five different HBV-
509
specific transcripts were detected, of which three (one predominant and two minor) were likely
510
initiated at the PreS2/S promoter and the other two minor transcripts might have cover most of the
511
PreS region (while no evidence was obtained that the latter two RNAs might bear the intact and
512
complete PreS1 domain). In addition, in the integrant that was earlier characterized in Hep3B cells,
513
the sequences coding for the N-terminus of the PreS1 were deleted and the reminder of the PreS1
514
domain was fused to the inverted pre-core/core sequences (30, 34-35, 51, 55).
515 516
The variants D1, B4 and C5 used in this study were collected from chronic carriers of HBV, and the
517
envelope proteins of at least two of them, D1 and C5 supported considerable levels of HDV
518
infectivity (Fig. 3). This observation may indicate that if there could be a tendency of diminishing of
519
overall infectivity of HDV particles over the course of HDV infection, such possible decrease of
520
HDV infectivity may not be mediated by functional deficiency of available HBV envelope proteins.
521
Further studies are needed to examine, whether during chronic stage of HDV infection, HBV
522
envelope proteins are able to support considerable levels of HDV infectivity.
523 524
Overall, our study for the first time demonstrated that in cells of HBV-induced HCC at least some
525
HBV integrant(s), which were naturally incorporated into the host DNA during the infection,
24
526
express functional L and S envelope proteins competent for facilitating assembly of infectious HDV
527
virions in the absence of ongoing HBV replication. The obtained results provide therefore a novel
528
information regarding the functionality of the integrant-derived HBV envelope proteins and the
529
mechanisms of HBV-HDV interactions. As expected and in agreement with the previous reports
530
demonstrating the alterations in integrated HBV DNAs (29-30, 35, 51), not all integrants were able
531
to support the production of functional HBV envelope proteins. Thus, none of the integrants in
532
Hep3B cells was able to support the production of infectious HDV. In Alexander cells, only one out
533
of seven so far described HBV integrants (51) apparently may facilitate the production of the
534
functional L protein that is essential for the virions' infectivity. The generated data further support
535
our hypothesis that HDV persistent infection in vivo may not require ongoing HBV replication, as
536
long as functional HBsAgs are supplied from the integrated HBV DNA. Furthermore, our results (i)
537
suggest that HDV is likely more independent and more significant pathogen than it is currently
538
appreciated; and (ii) support the implementation of medical interventions that directly target HDV to
539
be used along with anti-HBV drugs for HDV/HBV carriers.
540 541
In addition, taken together our recently published data that HDV in vivo infects the cells of
542
hepadnavirus-induced HCCs (56) and the results of the current study, we conclude that that HBV-
543
induced HCCs are likely able to support the entire HDV life cycle, including, attachment, entry,
544
trafficking to the replication sites and assembly and egress of new infectious HDV virions.
545
Therefore, our study further advanced our understanding of the mechanism of the relationship
546
between HDV infection and HCC.
547 548
ACKNOWLEDGEMENTS
25
549
SG and SM were supported by NIH grant NCI R01CA166213. SG was also supported by NIH
550
grants NIAID R21AI099696, NIAID R21AI097647, NCRR P20RR016443, by the University of
551
Kansas Research Institute Bridging Grant and by the University of Kansas Endowment Association.
552
We thank Stefan Wieland, Frank Chisari, Kenji Abe, Yu-mei Wen and Stephan Schaefer, for
553
providing us with constructs harboring sequences of different HBV variants. We acknowledge help
554
of Megan Dudek.
555 556
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line (PLC/PRF/5). Dev. Biol. Stand. 54:81-92.
654
37. Galibert F, Mandart E, Fitoussi F, Tiollais P, Charnay P. 1979. Nucleotide sequence of
655
hepatitis B virus genome (subtype ayw) cloned in E. coli. Nature. 281:646-650.
656
38. Okamoto H, Tsuda F, Sakugawa H, Sastrosoewignjo RI, Imai M, Miyakawa Y,
657
Mayumi M. 1988. Typing hepatitis B virus by homology in nucleotide sequence:
658
comparison of surface antigen subtypes. J. Gen. Virol. 69: 2575-2583.
30
659
39. Lin X, Yuan ZH, Wu L, Ding JP, Wen YM. 2001. A single amino acid in the reverse
660
transcriptase domain of hepatitis B virus affects virus replication efficiency. J. Virol. 75:
661
11827-11833.
662
40. Takashi K, Brotman B, Usuda S, Mishiro S, Prince AM. 2000. Full-genome sequence
663
analyses of hepatitis B virus (HBV) strains recovered from chimpanzees infected in the wild;
664
implications for an origin of HBV. Virology. 267:58-64.
665
41. Kramvis A, Kew M, Francois G. 2005. Hepatitis B genotypes. Vaccine. 23:2409-2423.
666
42. Panjaworayan N, Roessner SK, Firth AE, Brown CM. 2007. HBVRegDB: Annotation,
667
comparison, detection and visualization of regulatory elements in hepatitis B virus
668
sequences. Virol. J. 4:136.
669
43. Panjaworayan N, Payungporn S, Poovorawan Y, Brown CM. 2010. Identification of an
670
effective siRNA target site and functional regulatory elements, within the hepatitis B virus
671
posttranscriptional regulatory element. Virol. J. 7:216.
672 673 674 675 676 677 678
44. Kuo M, Chao M, Taylor J. 1989. Initiation of replication of the human hepatitis delta virus
genome from cloned DNA: role of delta antigen. J. Virol. 63:1945-1950. 45. Blanchet M, Sureau C. 2007. Infectivity determinants of the hepatitis B virus pre-S domain
are confined to the N-terminal 75 amino acid residues. J. Virol. 81:5841-5849. 46. Bruss V. 1997. A short linear sequence in the pre-S domain of the large hepatitis B virus
envelope protein required for virion formation. J. Virol. 71:9350-9357. 47. Sominskaya I, Bichko V, Pushko P, Dreimane A, Snikere D, Pumpens P. 1992.
679
Tetrapeptide QDPR is a minimal immunodominant epitope within the preS2 domain of
680
hepatitis B virus. Immunol. Lett. 33:169-172.
31
681
48. Gudima SO, He Y, Meier A, Chang J, Chen R, Jarnik M, Nicolas E, Bruss V, Taylor J.
682
2007. Assembly of hepatitis delta virus: particle characterization including the ability to
683
infect primary human hepatocytes. J. Virol. 81:3608-3617.
684
49. Gudima SO, He Y, Chai N, Bruss V, Urban S, Mason W, Taylor J. 2008. Primary human
685
hepatocytes are susceptible to infection by hepatitis delta virus assembled with envelope
686
proteins of woodchuck hepatitis virus. J. Virol. 82:7276-7283.
687 688 689 690
50. Glebe D, Urban S. 2007. Viral and cellular determinants involved in hepadnaviral entry.
World J. Gastroenterol. 13:22-38. 51. Shamay M, Agami R, Shaul Y. 2001. HBV integrants of hepatocellular carcinoma cell
lines contain an active enhancer. Oncogene. 20:6811-6819.
691
52. Sureau C. 2006. The role of the HBV envelope proteins in the HDV replication cycle. p.
692
113-131. In: Casey JL (ed). Hepatitis delta virus. Current Topics in Microbiology and
693
Immunology. 1st ed. vol 307. Springer-Verlag, Berlin, Heidelberg, Germany.
694
53. Gudima SO, Meyer A, Dunbrack R, Taylor J, Bruss V. 2007. Two potentially important
695
elements of the hepatitis B virus large envelope protein are dispensable for infectivity of
696
hepatitis delta virus. J. Virol. 81:4343-4347.
697
54. Chou CK, Su TS, Chang C, Hu CP, Huang MY, Suen CS, Chou NW, Ting LP. 1989.
698
Insulin suppresses hepatitis B surface antigen expression in human hepatoma cells. J. Biol.
699
Chem. 264:15304-15308.
700
55. Su TS, Lin LH, Chou CK, Chang C, Ting LP, Hu C, Han SH. 1986. Hepatitis B virus
701
transcripts in a human hepatoma cell line, Hep 3B. Biochem. Biophys. Res. Commun.
702
138:131-138.
32
703
56. Freitas N, Salisse J, Cunha C, Toshkov I, Menne S, Gudima SO. 2012. Hepatitis delta
704
virus infects the cells of hepadnavirus-induced hepatocellular carcinoma in woodchucks.
705
Hepatology. 56:76-85.
706
57. Valenzuela P, Quiroga M, Zalvidar J, Gray P, Rutter WJ. 1980. The nucleotide sequence
707
of hepatitis B viral genome and identification of the major viral genes. p. 57-70. In: Fields
708
BN, Jaenisch R, Fox CF (eds). Animal Virus Genetics. Academic Press, New York, NY,
709
USA.
710
58. Blanchet M, Sureau C. 2006. Analysis of the cytosolic domains of the hepatitis B virus
711
envelope proteins for their function in viral particles assembly and infectivity. J. Virol.
712
80:11935-11945.
713 714 715 716 717 718 719 720 721 722 723 724 725
33
726
Table 1. Parameters of the concentrated virus stocks of different HDV types
727
-------------------------------------------------------------------------------------------------------------------------
728
HDV typee
729
Total yielda
Total yield
PreS1*-HDVc
PreS1*-HDV
(GE/μl)
(%b)
(GE/μl)
(%d)
730
-------------------------------------------------------------------------------------------------------------------------
731
HDV-PLC*
(1.80+/-0.41)x105
2.1
(1.72+/-0.43)x103
1.0
732
HDV-Hep3B**
(1.02+/-0.06)x106
11.7
(4.64+/-0.29)x103
0.5
733
HDV-D1***
(1.59+/-0.46)x106
18.3
(5.10+/-1.57)x105
32.1
734
HDV-F1****
(8.71+/-0.85)x106
100.0
(2.38+/-0.32)x106
27.3
735
HDV-B4***
(9.28+/-2.18)x104
1.1
(1.49+/-0.89)x104
16.1
736
HDV-C5***
(4.98+/-1.93)x105
5.7
(6.19+/-2.11)x104
12.4
737
-------------------------------------------------------------------------------------------------------------------------
738
The HDV stocks were concentrated using PEG as described in Materials and Methods. The data in
739
Table 1 reflects the averages obtained by analysis of several independent preparations of the virus
740
stocks (* stands for seven independent preparations,
741 742
***
stands for four independent preparations , a
****
**
stands for three independent preparations,
stands for two independent preparations).
, Total yield values represent the numbers of HDV genome-containing particles (i.e. the
743
numbers of HDV genome equivalents (GE)) per one microliter of the stock for any given type of
744
HDV virions. All virions contained the S protein, while only a fraction of potentially infectious
745
virions contained the molecules of the L protein with the PreS1 domain exposed on the outer
746
surfaces.
747 748
b
, The highest total HDV yield observed for HDV-F1 type was used as 100% value to
compare the efficiency of the virions’ production for the other HDV types.
34
749
c
, The PreS1*-HDV values represent the amounts of the virions that were
750
immunoprecipitated using the "anti-matrix" antibodies (HDV GE/µl of the stock). The anti-matrix
751
antibodies displayed strong preferential specificity in binding to the PreS1 sequences, and the
752
PreS1*-HDV values serve as a good approximation for the quantification of potentially infectious
753
(L-containing) particles. The asterisk indicates that the predominant majority of precipitated
754
particles were the PreS1-HDVs (greater than 80%), and the rest (less than 20%) were apparently L-
755
M+S+ virions (see Materials and Methods).
756 757 758
d
, The percentage of the Pres1*-HDVs shows the fraction of the PreS1*-HDVs relatively to
the total number of secreted virions for any given HDV type. e
, HDV-Hep3B are HDV virions assembled in Hep3B cells. HDV-PLC are HDV virions
759
assembled in PLC/PRF/5 cells. Other HDV virion types were assembled in Huh7 cells using the co-
760
transfection approach described in Materials and Methods. HDV-B4 are virions coated with the
761
envelope proteins of HBV genotype B, variant 4. HDV-C5 are the virions coated with the envelope
762
proteins of HBV genotype C, variant 5. HDV-F1 are the virions assembled with the envelope
763
proteins of HBV genotype F, variant 1. HDV-D1 are the virions assembled with the envelope
764
proteins of HBV genotype D, variant 1.
765
To calculate copy numbers of genomic (G) HDV RNA, the in vitro transcribed gel-
766
purified unit-length G HDV RNA standard was used in the G HDV strand-specific qPCR assay (48).
767
In each qPCR assay, each RNA sample was analyzed in triplicate. The standard errors of the mean
768
for both kinds of measurements, total HDV yield and concentration of the PreS1*-HDVs are
769
indicated in parentheses.
770 771 772
35
773
FIGURE LEGENDS
774
Fig 1. Replication of hepatitis delta virus (HDV) genome and assembly of HDV virions in cells
775
transfected with the plasmid pSVLD3. Hep3B (Hep3B2.1-7) and PLC/PRF/5 (Alexander) cells
776
were transfected with plasmid pSVLD3 to initiate HDV replication (44). As a control, Huh7 cells
777
were transfected simultaneously with equal mass amounts of the vectors pSVLD3 and pNF-LMS-
778
D3 that expresses the L, M and S proteins of HBV (genotype D, variant D3, accession number
779
V01460). During each transfection, each time point has been performed in duplicate. Panel A.
780
Replication of HDV genomes. Total RNA was isolated from transfected cells at days 3, 5, 7, 9, 11,
781
13, 15 and 17 post-transfection and analyzed by HDV-specific qPCR (48). The vertical axis
782
represents the levels of HDV genomic (G) RNA accumulation (Log10(GE of HDV G RNA/μg of
783
total RNA)) in Huh7 cells (open circles), Hep3B cells (black triangles) or in Alexander cells (open
784
rectangles). Panel B. Assembly of HDV virions. Total RNA was isolated from the media of the
785
transfected cells at days 3, 5, 7, 9, 11, 13, 15 and 17 post-transfection and analyzed by qPCR for G
786
RNA of HDV (48). The vertical axis represents the levels of HDV G RNA accumulation in the
787
media (Log10(GE of HDV G RNA/ml of media)) of Huh7 cells (open circles), Hep3B cells (black
788
triangles) or Alexander cells (open rectangles). In case of Hep3B and Alexander cells, each number
789
represents an average value obtained by analysis of two independent transfections. In each qPCR
790
assay, each RNA sample has been analyzed in triplicate. The standard errors of the mean are
791
indicated in both panels.
792 793
Fig 2. Detection of primary human hepatocytes (PHH) infected with different types of HDV
794
virions using immunofluorescence. At day nine post-infection, PHH were fixed, permeabilized
795
and analyzed for HDV-infected cells by immunofluorescence, using rabbit polyclonal antibodies
36
796
raised against recombinant small delta antigen (δAg) (48). Red staining is for the δAg accumulated
797
in infected PHH. For detection of the cellular α-tubulin (green staining), the mouse monoclonal
798
antibodies (Fisher Scientific) were used. Blue staining (DAPI) represents nuclear DNA. The panels
799
A-F represent PHH infected with: A – HDV assembled in Hep3B cells (HDV-Hep3B); B – HDV
800
assembled in PLC/PRF/5 cells (HDV-PLC); C – HDV coated with the envelope proteins of HBV
801
genotype B, variant B4 (HDV-B4); D – HDV coated with the envelope proteins of HBV genotype
802
C, variant C5 (HDV-C5); E – HDV coated with the envelope proteins of HBV genotype F, variant
803
F1 (HDV-F1); and F – HDV coated with the envelope proteins of HBV genotype D, variant D1
804
(HDV-D1).
805 806
Fig 3. Specific infectivity of different HDV types. The specific infectivity (SI) reflects the number
807
of new HDV genomes per hepatocyte produced as a result of infection, which is normalized by the
808
PreS1*-MOI. The PreS1*-MOI is the multiplicity of infection that represents the number of the
809
PreS1*-HDVs per hepatocyte used in inoculum. The numbers of HDV genomes in inoculum and in
810
total RNA harvested from infected primary human hepatocytes were determined using HDV-
811
specific qPCR assay (48). The Figure represents the averaged data of four independent infection
812
experiments (HDV-B4 was assayed in three infections out of four). The SI average number for
813
HDV-D1 was the highest and was used as 100% value. All other SI numbers are presented as the
814
percentage values relatively to the SI of HDV-D1. The SI, % values are shown as horizontal bars.
815
The SI percentage scale is shown at the bottom. The name of HDV type is indicated at the left hand
816
side next to the corresponding SI bar. The SI, % values are also indicated as numbers at the right
817
hand side of each bar. The bar reflecting the SI, % for HDV-PLC is lightly shaded, while the
818
corresponding bar for HDV-Hep3B has a dark shade. The standard errors of the mean are indicated.
37
819 820
Fig 4. Analysis of the presence of specific HBV regions on integrant-derived RNAs
821
accumulated in Hep3B and PLC/PRF/5 cells. Total RNA from not transfected Alexander cells or
822
Hep3B cells was extracted with TRI reagent, reverse transcribed using the primer R (shaded
823
pentagon), and then assayed for the presence of the specific HBV sequences related to the
824
production of the envelope proteins using six different PCR assays, which employed the pairs of
825
primers FA, BA, E1A, E2A, CA and DA. The numbering for the primers' locations and for other
826
indicated HBV-specific boundaries is based on the genomic organization of a typical genotype A of
827
HBV (41). Primers A, B, and R were based on the sequences conserved among different HBV
828
genotypes, A-I. Primers C, D, E1 (E1 was used only for the material from Alexander cells), E2 (E2
829
was used only for the material from Hep3B cells) and F were designed based on information (i) that
830
we acquired by partial sequencing of the RNAs from Hep3B and Alexander cells and (ii) that was
831
previously published regarding HBV integrants found in the above cell lines (30, 34-35, 51). The
832
primer B is placed in the conserved 5′-untranslated region (UTR) of the L mRNA. The occurrence
833
of the PCR product BA should reveal the presence of the PreS1 domain and the most part of the
834
open reading frame for the L protein on RNA regardless of the origin of HBV sequence, because the
835
PCR assay BA is based on the sequences conserved among different HBV genotypes, A-I. Panel A
836
represents the analysis of total RNA harvested from Alexander cells. Panel C represents the PCR
837
analysis of total RNA harvested from Hep3B cells. On Panels A and C, the lanes marked as "M"
838
are double-stranded DNA markers with selected molecular weights (in base pairs) indicated on the
839
left hand side of the gel images. The PCR assays, when cDNA synthesis was performed (i) in the
840
presence of the reverse transcriptase or (ii) in the absence of the reverse transcriptase (negative
841
controls) are indicated at the bottom of the gel images. The types of the PCR assay that were
38
842
conducted are indicated on the top of the images above the corresponding lanes. For example, BA
843
indicates that the PCR using the B and A primers was conducted. Note that on Panel A, one of the
844
PCR assays was E1A (specific for RNAs from Alexander cells), while on Panel C, it was replaced
845
by E2A PCR (specific for RNAs from Hep3B cells). The sizes of the observed PCR products BA,
846
E1A (or E2A), CA, DA and FA are indicated on the right hand side. Note that FA PCR product was
847
not observed on Panel A, as expected (see diagram on Panel B). The results shown on Panel A
848
indicate (i) the presence of the 5′-UTR, sequences coding for the PreS1, PreS2 and for a
849
considerable N-terminal part of the S domain on RNAs accumulated in Alexander cells; (ii) as well
850
as the absence of the rearrangements in the assayed area on the corresponding integrated HBV
851
DNA. The PCR products observed in Panel A are consistent with the diagram presented in Panel B.
852
Panel B represents the locations and orientations of the primers relatively to non-rearranged HBV
853
integrant, which is the likely template for the synthesis of HBV RNAs producing the L, M and S
854
envelope proteins in Alexander cells. The diagram is consistent with the description of one of the
855
integrants found in Alexander cells, which has no alterations or rearrangements in the assayed area,
856
bears the entire L open reading frame (51), and therefore likely facilitates the production of at least
857
functional L and S proteins. The left hand side position of the integrant is shown as approximately
858
at nucleotide 1832, which reflects the fact that the main substrate for integration is double-stranded
859
linear (DSL) HBV DNA genome (16). On Panel C, the BA PCR product was not observed. The
860
results shown on Panel C are consistent with (i) the absence of the 5′-UTR and the sequences coding
861
for the N-terminus of the PreS1 on RNAs expressed in Hep3B cells, and (ii) the
862
deletion/rearrangement in the corresponding integrated DNA that resulted in the loss of (a) the
863
PreS1 promoter, (b) 5′-UTR, (c) N-terminus of the PreS1 and (d) a considerable region upstream of
864
the PreS1 promoter, which led to the fusion of the remainder of the PreS1 to the inverted pre-
39
865
core/core sequences. The occurrence of the FA PCR product strongly suggests that the
866
corresponding RNA sequences were transcribed using unknown cellular (non-HBV) promoter. The
867
PCR products observed in the Panel C are consistent with the diagram presented in Panel D. Panel
868
D represents of the locations and orientations of the primers relatively to integrated HBV DNA,
869
which is the likely template for the synthesis of HBV RNAs encoding the envelope proteins in
870
Hep3B cells. The diagram is consistent with the description of the integrant found in Hep3B cells,
871
which has a deletion/rearrangement that resulted in the loss of the PreS1 promoter, N-terminus of
872
the PreS1, and fusion of the remainder of the PreS1 sequences to the inverted pre-core/core
873
sequences. The positions of the pre-core and PreS1 sequences at the junction site are shown in the
874
integrant as previously described (30).
875 876
Fig 5. Sequences at and around of the inverted pre-core/truncated PreS1 junction site found
877
on RNAs from Hep3B cells. The FA PCR product obtained by RT-PCR procedure (see the legend
878
of Fig. 4) using the total RNA from Hep3B cells was cloned into the Topo-vector II, and the
879
resulting plasmids were sequenced to reveal the sequences at and around of the inverted pre-
880
core/truncated PreS1 junction site. The alignment obtained using the Clustalw2 software
881
(http://www.ebi.ac.uk/Tools/msa/clustalw2/) reflects the comparison of seven different sequences
882
generated by the above cloning to Valenzuela's HBV sequence (accession number X02763, (57)).
883
The reference nucleotide sequence (X02763) is shown on the top. The numbering is as for a typical
884
HBV genotype A genome (41). The nucleotide positions are indicated at the top row. The
885
nucleotides identical to the reference sequence are shown as dashes, non-identical nucleotides are
886
shown as single letters. At the bottom, identical nucleotides are marked as the stars, purine to purine
887
substitutions are marked by dots, and A to T change is indicated by a colon. The number of identical
40
888
sequences obtained by the above cloning is indicated on the left hand side next to each sequence.
889
The obtained sequences demonstrate the deletion and rearrangement, which resulted in the junction
890
of the inverted pre-core/core sequences to the truncated PreS1 region. The Figure demonstrates the
891
fragment of the inverted pre-core nucleotide sequences (positions 1865-1819(1818)) joined to the
892
fragment of the truncated open reading frame for the L envelope protein (shown are the nucleotide
893
positions 2877(2878)-3221/1-208). Note, that because the second T of the TT dinucleotide at the
894
junction site could belong to either pre-core or to the PreS1, the adjoining positions are shown as
895
1819(1818) for the pre-core and 2877(2878) for the PreS1. The junction site that was mapped on the
896
expressed RNA, pre-core 1819(1818)-PreS1 2877(2878) is not the exact match to the junction that
897
was mapped previously on the DNA integrant and was pre-core 1815-PreS1 2875 (30).
898 899
Fig 6. Examination of amino acid sequences encoded by HBV mRNAs transcribed from the
900
integrated viral DNAs. Total RNA was extracted from Hep3B or Alexander cells and analyzed by
901
RT-PCR, followed by cloning and sequencing in order to examine two regions. The first region
902
spans the PreS1, PreS2 and the N-terminus of the S domain. The second region covers areas at and
903
around of the HDV-binding site (HDV-BS), including the major antigenic loop (MAL). The details
904
are described in Materials and Methods. The results of sequencing are expressed as translated amino
905
acid sequences. The alignments were obtained using the Muscle alignment software
906
(http://www.ebi.ac.uk/Tools/msa/muscle/). The residues identical to those in the reference sequences
907
(top lines) are shown as dashes, non-identical residues are shown as the single letter code. The
908
numbers of the recovered identical sequences are shown on the left hand side next to a particular
909
sequence. On the bottom of each alignment, the identical amino acids are marked as stars, conserved
910
- as colons, semi-conserved as periods. Panel A. For the first region, in agreement with the data
41
911
presented on Figs. 4 and 5, the complete sequences that begin at the N-terminus of the PreS1 were
912
obtained only for RNAs from Alexander cells. The displayed region covers the positions 1 to 211,
913
which includes the entire PreS1 and PreS2 domains and first 38 N-terminal residues of the S. The
914
first N-terminal residue of the PreS1 corresponds to the number one (the numbering is for HBV
915
genotype G (41)). The references sequence (accession number BAG50284.1, HBV genotype G) is
916
shown on the top. This sequence is the closest match (88% identity) to the most abundant sequence
917
recovered from Alexander cell (the second line from the top, the sequence is presented as a single
918
letter amino acid code). The most abundant sequence (marked with ρ) is identical to the sequence of
919
the PLCORF52. The other less abundant sequences are placed in the lines below. One of the least
920
abundant sequences (bottom line) marked with σ, and is identical to the PLCORF62. The sequences
921
spanning the area at and around of the HDV-binding site are presented in Panel B (for RNA from
922
PLC/PRF/5 cells) and Panel C (for RNA from Hep3B cells). The displayed region spans amino
923
acids from 95 to 209 of the small envelope protein (the numbering is for HBV genotype A (41), and
924
is given counting the first N-terminal residue of the S domain as the number one), which includes
925
the C terminus of the second transmembrane domain (C-TMII); the major antigenic loop (MAL); the
926
third transmembrane domain (TMIII); the small cytosolic loop, which is the HDV-binding site
927
(HDV-BS); and the N terminus of the fourth transmembrane domain (N-TMIV) (58). For the Panel
928
B, the reference sequence (shown on the top) is the closest match (99%) to the majority of the
929
sequences recovered from Alexander cells (accession number AAA45516.1, HBV genotype A,
930
subtype A1). A single sequence, marked with β had a downstream deletion, which should result in
931
the frame-shift and translation of the hybrid sequence that is different from the S sequence of HBV
932
downstream of the amino acid 209. For the Panel C, the reference sequence (on the top) is 100%
933
match to the majority of the sequences recovered from the Hep3B cells (CAC51288.1, HBV
42
934
genotype A, subtype A2). The least abundant sequence at the bottom of the alignment, marked with
935
β had an insertion T(113)TT and a unique deletion, which should result in translation of the non-
936
HBV sequences starting the position 161 and possible production of a hybrid protein. Four
937
premature stop codons (marked as Xs) were found in the non-HBV-part of this sequence.
938
Figure 1
A
109
Accumulation of HDV genomic RNA in the cells (Log10 (GE of HDV G RNA/g of total RNA))
108
107 106
105 104 103 3
B
108
5
7 9 11 13 Time post-transfection, days
15
17
Accumulation of HDV genomic RNA in the media (Log10 (GE of HDV G RNA/ml of media))
107
106 105 104
103 3
5
7 9 11 13 Time post-transfection, days
15
17
Fig 1. Replication of hepatitis delta virus (HDV) genome and assembly of HDV virions in cells transfected with the plasmid pSVLD3. Hep3B (Hep3B2.1-7) and PLC/PRF/5 (Alexander) cells were transfected with plasmid pSVLD3 to initiate HDV replication (44). As a control, Huh7 cells were transfected simultaneously with equal mass amounts of the vectors pSVLD3 and pNF-LMSD3 that expresses the L, M and S proteins of HBV (genotype D, variant D3, accession number V01460). During each transfection, each time point has been performed in duplicate. Panel A. Replication of HDV genomes. Total RNA was isolated from transfected cells at days 3, 5, 7, 9, 11, 13, 15 and 17 post-transfection and analyzed by HDV-specific qPCR (48). The vertical axis represents the levels of HDV genomic (G) RNA accumulation (Log10(GE of HDV G RNA/g of total RNA)) in Huh7 cells (open circles), Hep3B cells (black triangles) or in Alexander cells (open
rectangles). Panel B. Assembly of HDV virions. Total RNA was isolated from the media of the transfected cells at days 3, 5, 7, 9, 11, 13, 15 and 17 post-transfection and analyzed by qPCR for G RNA of HDV (48). The vertical axis represents the levels of HDV G RNA accumulation in the media (Log10(GE of HDV G RNA/ml of media)) of Huh7 cells (open circles), Hep3B cells (black triangles) or Alexander cells (open rectangles). In case of Hep3B and Alexander cells, each number represents an average value obtained by analysis of two independent transfections. In each qPCR assay, each RNA sample has been analyzed in triplicate. The standard errors of the mean are indicated in both panels.
Figure 2
Fig 2. Detection of primary human hepatocytes (PHH) infected with different types of HDV virions using immunofluorescence. At day nine post-infection, PHH were fixed, permeabilized and analyzed for HDV-infected cells by immunofluorescence, using rabbit polyclonal antibodies raised against recombinant small delta antigen (Ag) (48). Red staining is for the Ag accumulated in infected PHH. For detection of the cellular -tubulin (green staining), the mouse monoclonal antibodies (Fisher Scientific) were used. Blue staining (DAPI) represents nuclear DNA. The panels A-F represent PHH infected with: A – HDV assembled in Hep3B cells (HDV-Hep3B); B – HDV assembled in PLC/PRF/5 cells (HDV-PLC); C – HDV coated with the envelope proteins of HBV genotype B, variant B4 (HDV-B4); D – HDV coated with the envelope proteins of HBV genotype C, variant C5 (HDV-C5); E – HDV coated with the envelope proteins of HBV genotype F, variant
F1 (HDV-F1); and F – HDV coated with the envelope proteins of HBV genotype D, variant D1 (HDV-D1).
Figure 3 HDV Type HDV-D1
100.0
HDV-F1
57.3
HDV-C5
33.5
HDV-PLC
8.8
HDV-B4
6.4
HDV-Hep3B
1.4 0 10 20 30 40 50 60 70 80 90 100 110 Specific infectivity, %
Fig 3. Specific infectivity of different HDV types. The specific infectivity (SI) reflects the number of new HDV genomes per hepatocyte produced as a result of infection, which is normalized by the PreS1*-MOI. The PreS1*-MOI is the multiplicity of infection that represents the number of the PreS1*-HDVs per hepatocyte used in inoculum. The numbers of HDV genomes in inoculum and in total RNA harvested from infected primary human hepatocytes were determined using HDVspecific qPCR assay (48). The Figure represents the averaged data of four independent infection experiments (HDV-B4 was assayed in three infections out of four). The SI average number for HDV-D1 was the highest and was used as 100% value. All other SI numbers are presented as the percentage values relatively to the SI of HDV-D1. The SI, % values are shown as horizontal bars. The SI percentage scale is shown at the bottom. The name of HDV type is indicated at the left hand side next to the corresponding SI bar. The SI, % values are also indicated as numbers at the right hand side of each bar. The bar reflecting the SI, % for HDV-PLC is lightly shaded, while the corresponding bar for HDV-Hep3B has a dark shade. The standard errors of the mean are indicated.
Figure 4
A
M
FA BA E1A CA DA M
FA BA E1A CA DA 879 BA 814 E1A 506 CA
1500 1000 750 500 250
333 DA RT +
B
RT
F
B
PreC ~1832
E1
Core
1901
C
PreS1 2458
M
C
2854
FA BA E2A CA DA M
D
PreS2 3211 3221/1
R
470
752
S
155
FA BA E2A CA DA
1500 1000 750 500 250
D
A
889 FA 814 E2A 506 CA 333 DA RT +
RT
F
Core PreC 2057
1901 1815
E2
C
PreS1 2875
D
PreS2 3211 3221/1
A
R
470
752
S
155
Fig 4. Analysis of the presence of specific HBV regions on integrant-derived RNAs accumulated in Hep3B and PLC/PRF/5 cells. Total RNA from not transfected Alexander cells or Hep3B cells was extracted with TRI reagent, reverse transcribed using the primer R (shaded pentagon), and then assayed for the presence of the specific HBV sequences related to the production of the envelope proteins using six different PCR assays, which employed the pairs of primers FA, BA, E1A, E2A, CA and DA. The numbering for the primers' locations and for other indicated HBV-specific boundaries is based on the genomic organization of a typical genotype A of HBV (41). Primers A, B, and R were based on the sequences conserved among different HBV genotypes, A-I. Primers C, D, E1 (E1 was used only for the material from Alexander cells), E2 (E2
was used only for the material from Hep3B cells) and F were designed based on information (i) that we acquired by partial sequencing of the RNAs from Hep3B and Alexander cells and (ii) that was previously published regarding HBV integrants found in the above cell lines (30, 34-35, 51). The primer B is placed in the conserved 5-untranslated region (UTR) of the L mRNA. The occurrence of the PCR product BA should reveal the presence of the PreS1 domain and the most part of the open reading frame for the L protein on RNA regardless of the origin of HBV sequence, because the PCR assay BA is based on the sequences conserved among different HBV genotypes, A-I. Panel A represents the analysis of total RNA harvested from Alexander cells. Panel C represents the PCR analysis of total RNA harvested from Hep3B cells. On Panels A and C, the lanes marked as "M" are double-stranded DNA markers with selected molecular weights (in base pairs) indicated on the left hand side of the gel images. The PCR assays, when cDNA synthesis was performed (i) in the presence of the reverse transcriptase or (ii) in the absence of the reverse transcriptase (negative controls) are indicated at the bottom of the gel images. The types of the PCR assay that were conducted are indicated on the top of the images above the corresponding lanes. For example, BA indicates that the PCR using the B and A primers was conducted. Note that on Panel A, one of the PCR assays was E1A (specific for RNAs from Alexander cells), while on Panel C, it was replaced by E2A PCR (specific for RNAs from Hep3B cells). The sizes of the observed PCR products BA, E1A (or E2A), CA, DA and FA are indicated on the right hand side. Note that FA PCR product was not observed on Panel A, as expected (see diagram on Panel B). The results shown on Panel A indicate (i) the presence of the 5-UTR, sequences coding for the PreS1, PreS2 and for a considerable N-terminal part of the S domain on RNAs accumulated in Alexander cells; (ii) as well as the absence of the rearrangements in the assayed area on the corresponding integrated HBV DNA. The PCR products observed in Panel A are consistent with the diagram presented in Panel B. Panel B represents the locations and orientations of the primers relatively to non-rearranged HBV
integrant, which is the likely template for the synthesis of HBV RNAs producing the L, M and S envelope proteins in Alexander cells. The diagram is consistent with the description of one of the integrants found in Alexander cells, which has no alterations or rearrangements in the assayed area, bears the entire L open reading frame (51), and therefore likely facilitates the production of at least functional L and S proteins. The left hand side position of the integrant is shown as approximately at nucleotide 1832, which reflects the fact that the main substrate for integration is double-stranded linear (DSL) HBV DNA genome (16). On Panel C, the BA PCR product was not observed. The results shown on Panel C are consistent with (i) the absence of the 5-UTR and the sequences coding for the N-terminus of the PreS1 on RNAs expressed in Hep3B cells, and (ii) the deletion/rearrangement in the corresponding integrated DNA that resulted in the loss of (a) the PreS1 promoter, (b) 5-UTR, (c) N-terminus of the PreS1 and (d) a considerable region upstream of the PreS1 promoter, which led to the fusion of the remainder of the PreS1 to the inverted precore/core sequences. The occurrence of the FA PCR product strongly suggests that the corresponding RNA sequences were transcribed using unknown cellular (non-HBV) promoter. The PCR products observed in the Panel C are consistent with the diagram presented in Panel D. Panel D represents of the locations and orientations of the primers relatively to integrated HBV DNA, which is the likely template for the synthesis of HBV RNAs encoding the envelope proteins in Hep3B cells. The diagram is consistent with the description of the integrant found in Hep3B cells, which has a deletion/rearrangement that resulted in the loss of the PreS1 promoter, N-terminus of the PreS1, and fusion of the remainder of the PreS1 sequences to the inverted pre-core/core sequences. The positions of the pre-core and PreS1 sequences at the junction site are shown in the integrant as previously described (30).
Figure 5 Inverted pre-core 1819(1818) 1865-GAACAGTGGGACATGTACAAGAGATGATTAGGCAGAGGTGAAAAAGT 01 01 01 03 01
-------------------------------------------------------------C------------------------------------------------------------------------------------------------------------------------------------------------------------------------*************** *******************************
2877(2878) PreS1 TCGCAAAGGCATGGGGACGAATCTTTCTGTTCCCAATCCTCTG-2919 ------------------------------------C-----------------------------------------C-----------------------------------------C-----------------------------------------C-----------------------------------------C----C************************************ **** *
2920-GGATTCTTTCCCGATCATCAGTTGGACCCTGCATTCGGAGCCAACTCAAACAATCCAGATTGGGACTTCAACCCCGTCAAGGACGACTGG-3009 01 01 01 03 01
-------------------------------------------------------------------C-------A--------C-------------------------------------------------------------------------------A------G-C-------------------------------------------------------------------------------A--------C-------------------------------------------------------------------------------A--------C-------------------------------------------------------------------------------A--------C----******************************************************************* *******.******.* *****
3010-CCAGCAGCCAACCAAGTAGGAGTGGGAGCATTCGGGCCAAGGCTCACCCCTCCACACGGCGGTATTTTGGGGTGGAGCCCTCAGGCTCAG-3099 01 01 01 03 01
---------TG---G------------------------G--T--------------------G---------------------------------------G------------------------G--T--------------------G---------------------------------------G---------A--------------G--T--------------------G---------------------------------------G------------------------G--T--------------------G---------------------------------------G------------------------G--T--------------------G-------------------------*********:.***.*********.**************.** ********************.**************************
3100-GGCATATTGACCACAGTGTCAACAATTCCTCCTCCTGCCTCCACCAATCGGCAGTCAGGAAGGCAGCCTACTCCCATCTCTCCACCTCTA-3189 01 01 01 03 01
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------******************************************************************************************
3190-AGAGACAGTCATCCTCAGGCCATGCAGTGGAA-3221 01 01 01 03 01
----------------------C------------------------------C------------------------------C------------------------------C------------------------------C--------********************** *********
1-TTCCACTGCCTTCCACCAAACTCTGCAGGATCCCAGAGTCAGGGGTCTGTATCTTCCT-58 C------------------G--------------------------------T----C------------------G--------------------------------T----C------------------G--------------------------------T----C------------------G--------------------------------T----C------------------G--------------------------------T----******************.******************************** *****
59-GCTGGTGGCTCCAGTTCAGGAACAGTAAACCCTGCTCCGAATATTGCCTCTCACATCTCGTCAATCTCCGCGAGGACTGGGGACCCTGTG-148 01 01 01 03 01
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------C------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------******************************** *********************************************************
149-ACGAACATGGAGAACATCACATCAGGATTCCTAGGACCCCTGCTCGTGTTACAGGCGGGG-208 01 01 01 03 01
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------************************************************************
Fig 5. Sequences at and around of the inverted pre-core/truncated PreS1 junction site found on RNAs from Hep3B cells. The FA PCR product obtained by RT-PCR procedure (see the legend of Fig. 4) using the total RNA from Hep3B cells was cloned into the Topo-vector II, and the resulting plasmids were sequenced to reveal the sequences at and around of the inverted pre-
core/truncated PreS1 junction site. The alignment obtained using the Clustalw2 software (http://www.ebi.ac.uk/Tools/msa/clustalw2/) reflects the comparison of seven different sequences generated by the above cloning to Valenzuela's HBV sequence (accession number X02763, (57)). The reference nucleotide sequence (X02763) is shown on the top. The numbering is as for a typical HBV genotype A genome (41). The nucleotide positions are indicated at the top row. The nucleotides identical to the reference sequence are shown as dashes, non-identical nucleotides are shown as single letters. At the bottom, identical nucleotides are marked as the stars, purine to purine substitutions are marked by dots, and A to T change is indicated by a colon. The number of identical sequences obtained by the above cloning is indicated on the left hand side next to each sequence. The obtained sequences demonstrate the deletion and rearrangement, which resulted in the junction of the inverted pre-core/core sequences to the truncated PreS1 region. The Figure demonstrates the fragment of the inverted pre-core nucleotide sequences (positions 1865-1819(1818)) joined to the fragment of the truncated open reading frame for the L envelope protein (shown are the nucleotide positions 2877(2878)-3221/1-208). Note, that because the second T of the TT dinucleotide at the junction site could belong to either pre-core or to the PreS1, the adjoining positions are shown as 1819(1818) for the pre-core and 2877(2878) for the PreS1. The junction site that was mapped on the expressed RNA, pre-core 1819(1818)-PreS1 2877(2878) is not the exact match to the junction that was mapped previously on the DNA integrant and was pre-core 1815-PreS1 2875 (30).
Figure 6 A
PreS1 1-MGLSWTVPLEWGKNLSTSNPLGFLPDHQLDPAFRANTNNPDWDFNPKKDPWPEANKVGVGAYGPGFTPPHGGLLGWSPQSQGTLTTLPAD-90
07 03 01 01 01 01
MGLSWTVPLEWGKNQSTSNPLGFFPDHQLDPAFGANSNNPDWDLNSNKDHWPQANQVGVGAFGPGFTPPHGGLLGWSSQAQGTLHTVPAV --------------Q--------F---------G--S------L-SN--H--Q--Q-----F---------------S-A----H-V--V --------------Q--------F---------G--S------L-SN--H--Q--Q-----F---------------S-A----H-V--V --------------Q--------F---------G--S------L-SN--H--Q--Q-----F---------------S-A----H-V--V --------------Q--------F---------G--S------L-SN--H--Q--Q-----F---------------S-A----H-V--V --------------Q--------F---------G--S------L-SN--H--Q--Q-----F---------------S-A----H-V--V ************** ********:********* **:******:*.:** **:**:*****:***************.*:**** *:**
PreS1 PreS2 S 91-PPPASTNRQSGRQPTPISPPLRDSHPQA MQWNSTAFHQALQNPKVRGLYFPAGGSSSGIVNPVPTIASHISSIFSRIGDPAPN MENITS-180 07 03 01 01 01 01
PPPASTNRQTKRQPTPISPPLRDSHPQA ---------T--------------------------TK-------------------------TK-------------------------TK-------------------------T-----------------*********: *****************
MQWNSTAFHQALQHPRVRGLYFPAGGSSSGTVNPAQNIASHISSISSRTGDPAPN -L-----------D-R-----L--------T---AQN--------S--T------------------H-R--------------T---AQN--------S--T------------------H-R--------------T---AQN--------S--T------------------H-R--------------T---ARN--------S--T------------------D-R--------------T---AQN--------S--T-----* ***********.*:*****:******** ***. .******** ** ******
MENITS -------------------------******
S 181-GFLGPLLVLQAGFFLLTRILTIPQSLDSWWTS-211 07 03 01 01 01 01
GFLGPLLVLQAGFFLLTRILTIPQSLDSWWTS –L--------------------------------------------------T---------------P--------------------------------------------------------------------------------------*:**** ************** **********
B C-TMII Region of the major antigenic loop (MAL) TMIII 95-LVLLDYQ GMLPVCPLIPGSTTTSTGPCKTCTTPAQGNSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASVRFS WLSLLVPFVQW-182 12 01 01 01 01β
LVLLDYQ ------R ------------------******.
GMLPVCPLIPGSTTTSTGPCKTCTTPAQGNSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWGSVRFS ----------------------------------------------------------------G--------------------------------------------------------------------G--------------------------------------------------------------------G--------------------------------------------------------------------G----****************************************************************:*****
WLSLLVPFVQW ----------------------------------------***********
TMIII HDV-BS N-TMIV 183-FVGLSPTVWLS VIWMMWYW GPNLYNIL-209 12 01 01 01 01β
FVGLSPTVWLS ------------------R---------------------********.**
VIWMMWYW --------------------C-------****** *
GPNLYNIL ----------------------------********
C C-TMII Region of the major antigenic loop (MAL) TMIII 95-LVLLDYQ GMLPVCPLIPGS-TTTSTGPCKTCTTPAQGNSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASVRFS WLSLLVPFVQW-182 11 LVLLDYQ GMLPVCPLIPGS-TTTSTGPCKTCTTPAQGNSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASVRFS WLSLLVPFVQW 01β ------- ------------T----------------------------------------------NTYGSGPQSVSL GSVYXCHLFSG ******* ************ **********************************************: . .. :.. TMIII HDV-BS N-TMIV 183-FVGLSPTVWLS AIWMMWYW GPRLYSIV-209 11 FVGLSPTVWLS AIWMMWYW GPRLYSIV 01β SXGFPPLFGFQ LYGXCGIG GQDCTASX *:.* . :. * :
Fig 6. Examination of amino acid sequences encoded by HBV mRNAs transcribed from the integrated viral DNAs. Total RNA was extracted from Hep3B or Alexander cells and analyzed by RT-PCR, followed by cloning and sequencing in order to examine two regions. The first region spans the PreS1, PreS2 and the N-terminus of the S domain. The second region covers areas at and around of the HDV-binding site (HDV-BS), including the major antigenic loop (MAL). The details are described in Materials and Methods. The results of sequencing are expressed as translated amino acid sequences. The alignments were obtained using the Muscle alignment software (http://www.ebi.ac.uk/Tools/msa/muscle/). The residues identical to those in the reference sequences (top lines) are shown as dashes, non-identical residues are shown as the single letter code. The numbers of the recovered identical sequences are shown on the left hand side next to a particular sequence. On the bottom of each alignment, the identical amino acids are marked as stars, conserved - as colons, semi-conserved as periods. Panel A. For the first region, in agreement with the data presented on Figs. 4 and 5, the complete sequences that begin at the N-terminus of the PreS1 were obtained only for RNAs from Alexander cells. The displayed region covers the positions 1 to 211, which includes the entire PreS1 and PreS2 domains and first 38 N-terminal residues of the S. The first N-terminal residue of the PreS1 corresponds to the number one (the numbering is for HBV genotype G (41)). The references sequence (accession number BAG50284.1, HBV genotype G) is shown on the top. This sequence is the closest match (88% identity) to the most abundant sequence recovered from Alexander cell (the second line from the top, the sequence is presented as a single letter amino acid code). The most abundant sequence (marked with ) is identical to the sequence of the PLCORF52. The other less abundant sequences are placed in the lines below. One of the least abundant sequences (bottom line) marked with , and is identical to the PLCORF62. The sequences spanning the area at and around of the HDV-binding site are presented in Panel B (for RNA from PLC/PRF/5 cells) and Panel C (for RNA from Hep3B cells). The displayed region spans amino
acids from 95 to 209 of the small envelope protein (the numbering is for HBV genotype A (41), and is given counting the first N-terminal residue of the S domain as the number one), which includes the C terminus of the second transmembrane domain (C-TMII); the major antigenic loop (MAL); the third transmembrane domain (TMIII); the small cytosolic loop, which is the HDV-binding site (HDV-BS); and the N terminus of the fourth transmembrane domain (N-TMIV) (58). For the Panel B, the reference sequence (shown on the top) is the closest match (99%) to the majority of the sequences recovered from Alexander cells (accession number AAA45516.1, HBV genotype A, subtype A1). A single sequence, marked with β had a downstream deletion, which should result in the frame-shift and translation of the hybrid sequence that is different from the S sequence of HBV downstream of the amino acid 209. For the Panel C, the reference sequence (on the top) is 100% match to the majority of the sequences recovered from the Hep3B cells (CAC51288.1, HBV genotype A, subtype A2). The least abundant sequence at the bottom of the alignment, marked with β had an insertion T(113)TT and a unique deletion, which should result in translation of the nonHBV sequences starting the position 161 and possible production of a hybrid protein. Four premature stop codons (marked as Xs) were found in the non-HBV-part of this sequence.