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

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antigens (HBsAgs) can be produced either by HBV replication, or from integrated HBV DNA

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regardless of the replication. The functional properties of the integrant-generated HBsAgs were

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examined using two human HCC-derived cell lines Hep3B and PLC/PRF/5 that contain HBV

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integrants, but do not produce HBV virions and have no signs of HBV replication. Both cell lines

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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

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frame (ORF) for the large (L) envelope protein that is essential for infectivity was present on HBV

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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

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at least some of HBV DNA sequences naturally integrated during infection can produce functional

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small and large envelope proteins capable of the formation of infectious HDV virions. Our data

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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

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integrants.

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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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INTRODUCTION

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Hepatitis delta virus (HDV) is a significant human pathogen with approximately 20 million chronic

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carriers worldwide. HDV is a natural subviral agent of human hepatitis B virus (HBV) that requires

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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

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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

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alpha-interferon is beneficial only for a subset of HDV carriers. There are no treatments in clinical

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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

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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

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available.

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The envelope proteins can be produced from integrated HBV DNA independently of hepadnavirus

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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

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this study, we examined the hypothesis that HDV can persist in the absence of HBV replication (or

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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.

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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

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surface antigens (HBsAgs) remain to be fully evaluated (16, 29-31, 34-35).

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We examined two human cell lines derived from HBV-induced HCCs, Hep3B and PLC/PRF/5

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(Alexander cells) that bear HBV integrated DNA, but do not produce HBV virions and display no

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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,

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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

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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

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collection of HBV sequences of genotypes A-I. The HBV variants D1, B4 and C5 were acquired

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during the chronic stage of HBV infection. For the variants D3 and F1, the information about their

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origin was not available ((37-40) and Abe K, personal communication). The HBV sequences were

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inserted into XhoI/XbaI sites of the pSVL vector (Pharmacia). Each HBV insert begins at the natural

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start codon of the L, contains the entire L coding sequence along with 3'-untranslated region that

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bears the post-transcriptional regulatory element (PRE) and ends at about one hundred nucleotides

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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

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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.

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Immunoprecipitation (IP). The immunoprecipitaion experiments were conducted using the method

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that was described previously and employs Pansorbin (Calbiochem) (45). The rabbit polyclonal

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"anti-matrix" antibodies were generated by GenScript. They were raised against the synthetic

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peptide that spans the amino acid residues 91-

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IPPPASTNRQSGRQPTPISPPLRDSHPQAMQWNSTAFH-128 of the PreS region of the L

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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/

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and mobyle.pasteur.fr/cgi-bin/portal.py?#forms::antigenic), the above peptide harbors a single

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antigenic determinant TPISPPLRDS, which is located within the PreS1 domain. Thus, it was

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anticipated that the IP with the "anti-matrix" antibodies would target specifically only the PreS1-

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containing HDV particles. The virions that contain the PreS1 domain of the L envelope protein

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(which interacts with HBV receptor (45)) on the outer surfaces are considered as potentially

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infectious. In a separate study, we examined the specificity of the "anti-matrix" antibodies. Two

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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-

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D1). The second type (HDV-D1 L-minus) was prepared using the L-minus version of the above

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mentioned vector, which was modified to express the M and S, but not the L envelope protein. The

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"anti-matrix" antibodies were compared to the S26 mouse monoclonal antibodies that recognizes

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QDPR sequence in the PreS2 domain (47), and therefore was able to bind the L and M proteins.

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Using HDV-D1 L-minus virions, it was found that the "anti-matrix" antibodies (as compared to the

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S26 antibody) were unable to immunoprecipitate about 78% of the L-M+S+ virions. This result

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suggested that the "anti-matrix" antibodies recognized very poorly the M protein, which was

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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

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the "anti-matrix" antibodies was at least 83%, while the rest of precipitated virions (about 17% or

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less) may consist of the particles that bore the PreS2 sequences in the context of the M protein,

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while the L was absent (i.e., L-M+S+ virions) (data not shown). Because the PreS1-HDVs

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represented the predominant majority of the virions that were immunoprecipitated by the "anti-

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matrix" antibodies, the contribution of the virions that contain the M, but not the L protein can be

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considered as not significant. The virions that were immunoprecipitated by the "anti-matrix"

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antibodies were designated as the PreS1*-HDVs, because these antibodies demonstrated significant

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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,

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were predominantly the PreS1-HDVs with an addition of not significant amounts of the particles

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that bear the M, but not the L protein. The PreS1*-HDVs served, therefore as acceptable

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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.

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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.

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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

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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

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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.

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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

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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

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among the three cell lines.

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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

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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

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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

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concentrated about 100-fold using polyethylene glycol (48). The results of comparison of the

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concentrated virus stocks are summarized in Table 1. The HDV-F1 displayed the highest total

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concentration of HDV virions. The total concentration of HDV-D1 virions was approximately five-

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fold lower. For the HDV-Hep3B and HDV-C5, the total HDV levels were 11.7% and 5.7%,

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respectively, as compared to that of HDV-F1. The concentrations of HDV-B4 and HDV-PLC were

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1.1% and 2.1%, respectively. Therefore, the total HDV yields facilitated by HBsAgs expressed in

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Hep3B and Alexander cells were within the range observed for the envelope proteins of natural

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HBV variants. The fraction of the PreS1-containing and therefore potentially infectious virions

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(because the N-terminus of the PreS1 domain harbors the receptor-binding region (50)) was

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analyzed using the immunoprecipitation (IP) with the "anti-matrix" antibodies against the peptide

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harboring conserved HBV matrix domain (3, 46, 50). As described in Materials and Methods, the

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"anti-matrix" antibodies displayed strong preferential, but not exclusive specificity to the PreS1. In a

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separate study, it was determined that the virions immunoprecipitated using the "anti-matrix"

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antibodies were predominantly (greater than 80%) the PreS1-containing HDVs along with not a

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significant fraction (less than 20%) of the L-M+S+ virions (not shown). The HDV virions pulled

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down with the "anti-matrix" antibodies were designated as the PreS1*-HDVs, indicating that

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predominant majority of the precipitated virions were the PreS1-HDVs. The PreS1*-HDVs

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quantification therefore served as acceptable approximation for the content of the potentially

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infectious HDV virions. The results of the IP-based quantification of PreS1*-HDVs are presented in

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Table 1. The highest fractions of the PreS1*-HDVs were found in HDV-D1 and HDV-F1, 32.1%

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and 27.3%, respectively. For HDV-B4 and HDV-C5, the percentages of PreS1*-HDVs were 16.1%

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and 12.4%, respectively. The lowest yields of PreS1*-HDVs were measured in HDV-Hep3B (0.5%)

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and HDV-PLC (1.0%). The percentages of the PreS1*-HDVs measured for HBV-Hep3B and HDV-

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PLC were not significantly higher than the cut-off value of the sensitivity of the IP procedure that

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was determined using HDV coated with the S envelope protein only (i.e., 0.2%).

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Infectivity of HDV virions coated with the envelope proteins produced from integrated HBV

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DNA. Infectivity of HDV-Hep3B and HDV-PLC was compared to that of HDV-B4, HDV-C5,

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HDV-D1 and HDV-F1 using in vitro infection of primary human hepatocytes (PHH). Four

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independent infections were conducted using four different PHH lots. For the experiments detecting

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HDV-positive hepatocytes using the immunofluorescence (IF), the multiplicity of infection (MOI)

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was in the range of 25-100 total HDV virions per hepatocyte. HDV-infected cells were detected

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using rabbit polyclonal antibodies against recombinant small delta antigen (48). The representative

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IF images are shown in Figure 2. No HDV-positive cells were observed during all infections among

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PHH inoculated with HDV-Hep3B virions (Fig. 2A). Therefore, HDV-Hep3B was considered non-

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infectious. Unlike HDV-Hep3B, HDV-PLC (Fig. 2B) was infectious on all occasions. The HDV-

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PLC was able to infect between 0.43% and 1.96 % of PHH (MOI 25-50). Similarly, HDV-positive

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PHH were observed during all infections for HDV-B4, HDV-C5, HDV-F1 and HDV-D1 (Figs. 2C,

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2D, 2E and 2F, respectively). For HDV-D1, the percentage of infected PHH was in the range of

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16.60% to 28.25% (MOI 100). For HDV-F1, the fraction of infected hepatocytes was 3.71 - 23.41%

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(MOI 100). For HDV-B4 (MOI 30) and HDV-C5 (MOI 50-100), the observed fractions of infected

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cells were 0.52 - 6.18% and 1.94 - 18.99%, respectively. In agreement with previous observations

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(48), two typical patterns of the delta antigen (δAg) subcellular localization in infected PHH were

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observed (Fig. 2). The major pattern was nucleoplasmic staining, and the minor one included an

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additional staining in cytoplasm. Next, the extent of HDV replication in infected PHH was measured

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by qPCR that specifically assayed a number of HDV genomic RNA copies in isolated total cellular

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RNA (48). Based on qPCR data, the specific infectivity (SI) values were calculated. The SI reflects

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the number of HDV genomes produced as a result of infection per average hepatocyte, which is

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normalized by the PreS1*-MOI (a number of the PreS1*-HDVs per hepatocyte used in inoculum).

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The average SI determined for HDV-D1 was the highest and was used as 100% value. All other SI

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numbers were expressed relatively to the SI of HDV-D1 (Fig. 3). The HDV-F1 and HDV-C5 had

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intermediate SI levels of 57.3% and 33.5% respectively, while HDV-B4 displayed a relatively low

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number of 6.4%. For HDV-PLC, the average SI value observed was 8.8%. As expected, HDV-

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Hep3B had the lowest SI number of 1.4%. Overall, the generated data demonstrated that HDV-PLC

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was infectious, while Hep3B appeared to be non-infectious.

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To examine further the infectivity of HDV-PLC, the entire open reading frame (ORF) for the L

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envelope protein was PCR-amplified using total RNA from not transfected Alexander cells and

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cloned into the vector that enables the expression of the L, M and S proteins after the transfection.

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Two different ORF sequences were used to produce HDV-PLCORF52 and HDV-PLCORF62 virus

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stocks using the above described co-transfection strategy of Huh7 cells. The total amounts of HDV

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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

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of HDV-F1 (see Table 1 for comparison), and are similar to the total yield of HDV as facilitated by

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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

REFERENCES

557

1. Taylor J. 2000. Hepatitis delta virus. Virology. 344:71-76.

558

2. Smedile A, Rizzetto M. 2011. HDV: thirty years later. Dig. Liver. Dis. 43 Suppl 1:S15-18.

559

3. Seeger C, Mason WS. 2000. Hepatitis B virus biology. Microbiol. Mol. Biol. Rev. 64:51-

560 561 562 563 564

68. 4. Lupberger J, Hildt E. 2007. Hepatitis B virus-induced oncogenesis. World J. Gastroenterol.

13:74-81. 5. Koytak ES, Yurdaydin C, Glenn JS. 2007. Hepatitis D. Curr. Treat. Options.

Gastroenterol. 10:456-463.

565

6. Verme G, Brunetto MR, Oliveri F, Baldi M, Forzani B, Piantino P, Ponzetto A, Bonio

566

F. 1991. Role of hepatitis delta virus infection in hepatocellular carcinoma. Dig. Dis. Sci.

567

36:1134-1136.

568

7. Fattovich G, Giustina G, Christensen E, Pantalena M, Zagni I, Realdi G, Schalm SW.

569

2000. Influence of hepatitis delta virus infection on morbidity and mortality in compensated

570

cirrhosis type B. The European Concerted Action on Viral Hepatitis (Eurohep). Gut. 46:420-

571

426.

26

572 573

8. Michielsen PP, Francque SM, van Dongen JL. 2005. Viral hepatitis and hepatocellular

carcinoma. World J. Surg. Oncol. 3:1-18.

574

9. Romeo R, Del Ninno E, Rumi M, Russo A, Sangiovanni A, de Franchis R, Ronchi G,

575

Colombo M. 2009. A 28-year study of the course of hepatitis Delta infection: a risk factor

576

for cirrhosis and hepatocellular carcinoma. Gastroenterology. 136:1629-1638.

577 578

10. Fattovich G, Stroffolini T, Zagni I, Donato F. 2004. Hepatocellular carcinoma in cirrhosis:

incidence and risk factors. Gastroenterology. 127:S35-50.

579

11. Vassilopulos D, Hadziyannis SJ. 2006. Clinical features of hepatitis delta virus. p. 76-80.

580

In: Handa H, Yamaguchi Y (eds). Hepatitis delta virus. Medical Intelligence Unit. 1st ed.

581

Landes Bioscience and Springer Science+Business Media, Georgetown, TX, New York, NY,

582

USA.

583

12. Niro GA, Ciancio A, Gaeta GB, Smedile A, Marrone A, Olivero A, Stanzione M, David

584

E, Brancaccio G, Fontana R, Perri F, Andriulli A, Rizetto M. 2006. Pegylated interferon

585

alpha-2b as monotherapy or in combination with ribavirin in chronic hepatitis delta.

586

Hepatology. 44:713-720.

587

13. Casey J, Cote PJ, Toshkov IA, Chu CK, Gerin JL, Hornbuckle WE, Tennant BC,

588

Korba BE. 2005. Clevudine inhibits hepatitis delta virus viremia: a pilot study of

589

chronically infected woodchucks. Antimicrob. Agents Chemother. 49:4396-4399.

590 591

14. Mason WS, Litwin S, Jilbert AR. 2008. Immune selection during chronic hepadnavirus

infection. Hepatol. Intl. 2:3-16.

592

15. Xu C, Yamamoto T, Zhou T, Aldrich CE, Frank K, Cullen JM, Jilbert AR, Mason WS.

593

2007. The liver of woodchucks chronically infected with the woodchuck hepatitis virus

27

594

contains foci of virus core antigen-negative hepatocytes with both altered and normal

595

morphology. Virology. 359:283-294.

596

16. Mason WS, Liu C, Aldrich CE, Litwin S, Yeh MM. 2010. Clonal expansion of normal-

597

appearing human hepatocytes during chronic hepatitis B virus infection. J. Virol. 84:8308-

598

8315.

599

17. Gowans EJ, Baroudy BM, Negro F, Ponzetto A, Purcell RH, Gerin JL. 1988. Evidence

600

for replication of hepatitis delta virus RNA in hepatocyte nuclei after in vivo infection.

601

Virology. 167:274-278.

602

18. Wu JC. 2006. Functional and clinical significance of hepatitis D virus genotype II infection.

603

p. 176-183-131. In: Casey JL (ed). Hepatitis delta virus. Current Topics in Microbiology and

604

Immunology. 1st ed. vol 307. Springer-Verlag, Berlin, Heidelberg, Germany.

605

19. Govindarajan S., Fields HA, Humphrey CD, Margolis HS. 1986. Pathologic and

606

ultrastructural changes of acute and chronic delta hepatitis in an experimentally infected

607

chimpanzee. Am. J. Pathol. 122:315-322.

608

20. Tavanez JP, Cunha C, Silva CM, David E, Monjardino J, Carmo-Fonseca M. 2002.

609

Hepatitis delta virus ribonucleoproteins shuttle between the nucleus and the cytoplasm.

610

RNA. 8:637-646.

611

21. Pollicino T, Raffa G, Santantonio T, Gaeta GB, Iannello G, Alibrandi A, Squadrito G,

612

Calvi C, Colucci G, Levrero M, Raimondo G. 2011. Replicative and transcriptional

613

activities of hepatitis B virus in patients coinfected hepatitis B and hepatitis delta viruses. J.

614

Virol. 85:432-439.

28

615

22. Wu JC, Chen PJ, Kuo MY, Lee SD, Chen DS, Ting LP. 1991. Production of hepatitis

616

delta virus and suppression of helper hepatitis B virus in a human hepatoma cell line. J.

617

Virol. 65:1099-1104.

618 619

23. Sureau C, Taylor J, Chao M, Eichberg JW, Lanford RE. 1989. Cloned hepatitis delta

virus cDNA is infectious in the chimpanzee. J. Virol. 63:4292-4297.

620

24. Jardi R, Rodriquez F, Buti M, Costa X, Cotrina M, Galimany R, Esteban R, Guardia J.

621

2001. Role of hepatitis B, C and D viruses in dual and triple infection: influence of viral

622

genotypes and hepatitis B precore and basal core promoter mutations on viral replicative

623

interference. Hepatology. 34:404-410.

624

25. Krogsgaard K, Wantzin P, Aldershvile J, Kryger P, Andersson P, Nielsen JO. 1986.

625

Hepatitis B virus DNA in hepatitis B surface antigen-positive blood donors: relation to the

626

hepatitis B e system and outcome in recipients. J. Infect. Dis. 153: 298-303.

627

26. Wang Y, Wu MC, Sham JS, Tai LS, Fang Y, Wu WQ, Xie D, Guan XY. 2002. Different

628

expression of hepatitis B surface antigen between hepatocellular carcinoma and its

629

surrounding liver tissue, studied using a tissue microarray. J. Pathol. 197:610-616.

630 631

27. Mason WS, Litwin S, Xu C, Jilbert AR. 2007. Hepatocytes turnover in transient and

chronic hepadnavirus infection. J. Viral. Hepat. 14 Suppl 1:22-28.

632

28. Von Loringhoven AF, Koch S, Hofschneider PH, Koshy R. 1985. Co-transcribed 3′ host

633

sequences augment expression of integrated hepatitis B virus DNA. EMBO J. 4:249-255.

634

29. Shaul Y, Ziemer M, Garcia PD, Crawford R, Hsu H, Valenzuela P, Rutter WJ. 1984.

635

Cloning and analysis of integrated hepatitis virus sequences from a human hepatoma cell

636

line. J. Virol. 51:776-787.

29

637 638 639 640

30. Su TS, Hwang WL, Yauk YK. 1998. Characterization of hepatitis B virus integrant that

results in chromosomal rearrangement. DNA Cell. Biol. 17:415-425. 31. Twist EM, Clark HF, Aden DP, Knowles BB, Plotkin SA. 1981. Integration pattern of

hepatitis B virus DNA sequences in human hepatoma cell lines. J. Virol. 37:239-243.

641

32. Takada S, Gotoh Y, Hayashi S, Yoshida M, Koike K. 1990. Structural rearrangement of

642

integrated hepatitis B virus DNA as well as cellular flanking DNA is present in chronically

643

infected hepatic tissues. J. Virol. 64:822-888.

644

33. Tokino T, Fukushige S, Nakamura T, Nagaya T, Murotsu T, Shiga K, Aoki N,

645

Matsubara K. 1987. Chromosomal translocation and inverted duplication associated with

646

integrated hepatitis B virus in hepatocellular carcinomas. J. Virol. 61:3848–3854.

647 648 649 650

34. Ou J, Rutter WJ. 1985. Hybrid hepatitis B virus-host transcripts in a human hepatoma cell.

Proc. Natl. Acad. Sci. USA. 82:83-87. 35. Ziemer M, Garcia P, Shaul Y, Rutter WJ. 1985. Sequence of hepatitis B virus DNA

incorporated into genome of a human hepatoma cell line. J. Virol. 53:885-892.

651

36. Barin F, Goudeau A, Brechot C, Romet-Lemonne J, Sureau C, Lesage G. 1983. Further

652

studies on production and characterization of HBsAg derived from a human hepatoma cell

653

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.

Envelope proteins derived from naturally integrated hepatitis B virus DNA support assembly and release of infectious hepatitis delta virus particles.

A natural subviral agent of human hepatitis B virus (HBV), hepatitis delta virus (HDV), requires only the envelope proteins from HBV in order to maint...
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