Support of the infectivity of hepatitis delta virus particles by the envelope proteins of different genotypes of hepatitis B virus.

JVI Accepts, published online ahead of print on 19 March 2014 J. Virol. doi:10.1128/JVI.00346-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Support of the infectivity of hepatitis delta virus particles by the envelope proteins of different

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genotypes of hepatitis B virus.

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Natalia Freitasa, Kenji Abeb, Celso Cunhac, Stephan Menned 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; Department of Pathology, National Institute of Infectious

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Diseases, Tokyo, Japanb; Medical Microbiology Unit, Center for Malaria and Tropical Diseases,

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Institute of Hygiene and Tropical Medicine, New University of Lisbon, Lisbon, Portugalc;

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Department of Microbiology and Immunology, Georgetown University Medical Center,

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Washington, DC, USAd.

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Running title: Infectivity of hepatitis delta virus particles

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#Address correspondence to Severin O. Gudima, [email protected].

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Word counts: Abstract ( 250 words); text ( 6315 words)

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ABSTRACT

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The study examined how the envelope proteins of 25 variants of hepatitis B virus (HBV) genotypes

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A-I support hepatitis delta virus (HDV) infectivity. The assembled virions bore the same HDV

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ribonucleoprotein and differed only by HBV variant-specific envelope proteins coating the particles.

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The total HDV yield varied in 122-fold range. A residue Y(374) in the HDV-binding site was

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identified as critical for HDV assembly. The virions that bind the antibodies, which recognize the

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region that includes HBV matrix domain and predominantly but not exclusively immunoprecipitate

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the PreS1-containing virions, were termed as PreS1*-HDVs. Using in vitro infection of primary

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human hepatocytes (PHH), we measured the specific infectivity (SI), which is a number of HDV

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genomes/cell produced by infection and normalized by the PreS1*-MOI, which is a multiplicity of

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infection that reflects a number of PreS1*-HDVs/cell used in inoculum. The SI values varied in

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160-fold range and indicated probable HBV genotype-specific trend in supporting HDV infectivity:

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D>B>E>A. Three variants of genotypes B, C and D supported the highest SI values. We also

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determined the normalized index of infected PHH (NI), which is the percentage of HDV-infected

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hepatocytes normalized by the PreS1*-MOI. Comparison of the SI and NI values revealed that while

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a particular HBV variant may facilitate infection of relatively significant fraction of PHH, it may not

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always result in a considerable number of genomes that initiated the replication after the entry. The

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potential implications of these findings are discussed in the context of the mechanism of

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attachment/entry of HBV and HDV.

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IMPORTANCE

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The study advances the understanding of the mechanisms of (i) the attachment and entry of HDV

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and HBV; and (ii) transmission of HDV infection/disease.

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INTRODUCTION

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Human hepatitis B virus (HBV) remains a significant pathogen with approximately 400 million

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chronic carriers around the world. Chronic HBV infection is a main risk factor for developing of

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hepatocellular carcinoma (HCC) (1-2). A recent study demonstrated that the number of HBV virions

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in inoculum per se determined the kinetics of HBV spread through the liver, timing and magnitude

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of the immune response, as well as the outcome of infection, i.e. whether infection became transient

53

or chronic (3). These results indicated that the infectivity of the virions and the rate of the virus

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spread throughout the liver may play a decisive role for the clinical outcome of the infection. A

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number of previous studies suggested that HBV-associated liver pathogenesis is genotype-specific

56

and reported differences between HBV genotypes in terms of severity of induced liver disease, HCC

57

incidence, responsiveness to antiviral therapy and infectivity of HBV virions. Some of those reports

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appeared to be controversial (4-15). The current study examined in more mechanistic detail how the

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envelope proteins of HBV contribute to the infectivity of the virions. As the experimental model, we

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used the virions of human hepatitis delta virus (HDV). HDV is a sub-viral agent of HBV, which in

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nature co-exists with its helper virus in infected livers and uses HBV envelope proteins to form

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HDV virions and enter hepatocytes through HBV-specific receptor, which interacts with the specific

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sequences in the PreS1 domain of the L envelope protein (16). We assembled in cell culture twenty

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five different types of HDV virions, all of which bore the same HDV ribonucleoprotein (RNP)

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inside and differed only by the envelope proteins coating the virions. The envelope proteins

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analyzed in this study represent twenty five different natural variants of HBV, which belong to nine

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HBV genotypes, A-I (14, 17). It was found that HBV envelope proteins apparently were able alone

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to determine the number of infected hepatocytes and the number of genomes that successfully

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initiated HDV replication after entering the susceptible cell. It became apparent that HBV envelope

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proteins are not only involved in the attachment and entry, but also likely facilitate at least one of the

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immediate post-entry steps. These novel findings advance the understanding of the mechanism of

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the virus entry and possibly trafficking and suggest additional virus life cycle step(s) that are likely

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regulated by HBV envelope proteins.

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MATERIALS AND METHODS

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LMS vectors. Using the sequences of HBV variants listed in the Table 1, twenty four corresponding

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LMS vectors were constructed to express the large (L), middle (M) and small (S) envelope proteins.

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Each construct bears a fragment of a genome of a particular HBV variant inserted into XhoI/XbaI

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sites of the pSVL vector (Pharmacia). The inserted fragment begins at the start codon of the L,

81

includes the entire L open reading frame and ends at about one hundred nucleotides downstream of

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the post-transcriptional regulatory element (PRE) (PRE spans the positions 1151-1684 of HBV

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genotype A (18-20)). In the resulting LMS vectors, the mRNA for the L envelope protein is

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transcribed from the SV40 late promoter, and the transcription of the M/S mRNA is driven by

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authentic HBV promoter. The variants A1, B3, C1 and C5 are natural M-minus mutants. To express

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the L, M and S of the variant A0, the plasmid pSVB45H (21) was used. The mutations in the LMS-

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C4 and LMS-B4 vectors were introduced using QuickChange II site-directed mutagenesis kit

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(Agilent Technologies) according to the instructions of the manufacturer. To introduce the mutation

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F(374)Y, which would change the sequence in the HDV-binding site (HDV-BS) (22) of the variant

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B4 from the 368-VIWMIWFW-375 to 368-VIWMIWYW-375 (the numbering was according to

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HBV genotype A (20), counting as a number one, the residue at the very N-terminus of the PreS1

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92

domain), the following two oligonucleotides were used: 734-

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GTTATATGGATGATATGGTATTGGGGGCCAAGTCTGTACA-773 and 773-

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TGTACAGACTTGGCCCCCAATACCATATCATCCATATAAC-734 (the changed coding triplet

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is underlined). To create the mutants C4m1 and C4m2, the LMS vector, encoding the envelope

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proteins of variant C4 was used and either a single mutation V(12)M (for C4m1) or the combination

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of mutations N(7)K+V(12)M (for C4m2) was introduced into the N-terminus of the PreS1. To make

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the change V(12)M, the following two oligonucleotides were utilized: 2865-

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CAATCCTCGACAAGGCATGGGGACGAATCTTTC-2897 and 2897-

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GAAAGATTCGTCCCCATGCCTTGTCGAGGATTG-2865. The primers, 2848-

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ATGGGAGGTTGGTCTTCCAAGCCTCGACAAGG-2869 and 2869-

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CCTTGTCGAGGCTTGGAAGACCAACCTCCCAT-2848 were used to introduce the mutation

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N(7)K. To make a combination of two mutations N(7)K+V(12)M, the change V(12)M was

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introduced first into the LMS-C4 vector, and the resulting construct was used then in order to insert

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an additional N(7)K change. To create the L-minus version of the LMS-D1 vector, the above

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mentioned QuickChange II site-directed mutagenesis kit was used as well. The mutation was

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introduced using the following two oligonucleotides: ggatcgatccctcgac-2848-

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ACGGGGCAGAATCTTTCC-2865 and 2865-GGAAAGATTCTGCCCCGT-2848-

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gtcgagggatcgatcc. The sequences of the vector are shown in the lowercase letters. The changed

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position (2849 in HBV sequence) is underlined. The initiation codon for the L protein was changed

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from AUG to ACG (ACG codes for Thr), which resulted in the amino acid substitution M(1)T at the

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very N-terminus of the PreS1 domain. The resulting construct therefore is the L-M+S+ vector,

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expressing only M and S envelope proteins (but not the L) of the variant D1.

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Transfection. To assemble HDV virions coated with the envelope proteins of a particular HBV

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variant, Huh7 cells (a gift from Camille Sureau) were co-transfected with a 1:1 mixture of plasmids,

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pSVLD3 (to initiate HDV replication) and the corresponding LMS vector, using Fugene HD reagent

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(Roche) (21, 23).

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In vitro infection of primary human hepatocytes (PHH). Prior to infection, HDV virions were

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concentrated about 100-fold using polyethylene glycol (PEG) (Sigma). The infection of plated PHH

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in the format of 48-well plates (purchased from Life Technologies) was conducted in the presence of

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5% PEG 8000 as previously described (21). Briefly, the PHH were maintained in the serum-free

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fully-defined Hepato-STIM culture medium that was supplemented with the epidermal growth

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factor (EGF) (BD Biosciences). The infection media that contained HDV and 5% PEG was

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incubated with PHH for 8 hours. After the incubation with PHH, the infection media was replaced

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with Hepato-STIM/EGF medium that contained neither virus nor PEG. For the measurements of the

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specific infectivity (SI), the multiplicity of infection (MOI) of 10-20 of total HDV genome

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equivalents (GE)/per average hepatocyte was employed. For the analysis of infected cells using the

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immunofluorescence procedure, the MOI of 30-100 of HDV GE/cell has been used. Infected

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hepatocytes were analyzed at day nine post-infection.

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Immunoprecipitation. The immunoprecipitations of HDV virions were performed using the

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previously described protocol (23). The rabbit polyclonal "anti-matrix" antibodies (GenScript) have

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been raised against the peptide spanning the positions 91-

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IPPPASTNRQSGRQPTPISPPLRDSHPQAMQWNSTAFH-128 of the PreS region (HBV serotype

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adw2, genotype A), which includes the conserved matrix domain (underlined) (23-24). Using

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specific software (mobyle.pasteur.fr/cgi-bin/portal.py?#forms::antigenic and bips.u-

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strasbg.fr/EMBOSS/), it was predicted that the above peptide bears a single antigenic site

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(TPISPPLRDS) located in the PreS1 domain. Therefore, the immunoprecipitation (IP) with the

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"anti-matrix" antibodies was expected to target exclusively PreS1-HDVs. Briefly, aliquots of the

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prepared HDV virus stocks were incubated first with "anti-matrix" antibodies at 4°C overnight and

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after that - with 100μl of Pansorbin suspension in phosphate buffer saline (PBS) (Calbiochem) for

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the next 2 hours on ice. Next, Pansorbin was sedimented by centrifugation at 13,000 rpm for 1 min.

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The resulting pellet was washed four times with ice-cold PBS containing 0.5% of Nonidet P-40

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(Fisher) , and then used for RNA isolation.

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HDV-specific real-time PCR (qPCR). Total RNA from Huh7 cells, Pansorbin-bound particles,

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PHH or virions was isolated with TRI reagent (Fisher) and treated with DNase (Life Technologies)

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prior to qPCR. The qPCR was conducted using the 7500 Real Time PCR instrument (Applied

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Biosystems) as described previously (21, 25). The forward primer, 312-

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GGACCCCTTCAGCGAACA-329; the reverse primer, 393-

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CCTAGCATCTCCTCCTATCGCTAT-360; and the TaqMan probe, 332-

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AGGCGCTTCGAGCGGTAGGAGTAAGA-357 were used (the numbering was according to Kuo

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et al. (26)). The reverse primer was also used for reverse transcription. The copy numbers were

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measured using a 10-fold dilution series of in vitro transcribed and gel-purified genomic (G) unit-

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length HDV RNA standard (range: 20-200,000 genome equivalents (GE) of HDV), and considering

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that 1 million of HDV RNA molecules equals to 1 picogram of G RNA standard (21, 25).

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Immunofluorescence (IF). The details of the procedure are described elsewhere (21, 27). Briefly,

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PHH were fixed with 4% paraformaldehyde, washed twice with PBS and permeabilized using 0.2%

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Triton X-100. Anti-delta antigen rabbit polyclonal and mouse monoclonal antibodies against human

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alpha-tubulin (Sigma) were used to stain PHH. Nuclear DNA was stained using DAPI (4ƍ, 6ƍ-

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diamidino-2-phenylindole). Stained PHH were analyzed using an inverted Nikon TE2000-U

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microscope with 20x objective. For each HDV type, approximately 400,000 hepatocytes were

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assayed per infection. About ten different fields of stained cells were photographed. Using staining

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for alpha-tubulin and for nuclear DNA, the total number of cells was quantified. Using staining for

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delta antigens, the number of HDV-infected hepatocytes was counted. The percentage of HDV-

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infected cells was calculated via dividing the number of delta antigen-positive cells by the total

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number of hepatocytes.

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RESULTS

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Sequences of HBV variants used to express the envelope proteins. The Table 1 summarizes

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twenty five HBV variants used in this study, which belong to nine HBV genotypes (A-I) and were

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acquired at different stages of HBV infection (14, 17, 28-36). Sequences of the variants C4 and D5

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(Table 1) are absent from NCBI database. Therefore, the entire amino acid sequences of the L

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proteins of these variants are presented in Figure 1. None of the sequences tested in this study

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contained easily identifiable alterations, such as deletions, insertions or rearrangements. The variants

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A1, B3, C1 and C5 are natural M-envelope protein minus mutants, and thus can facilitate only the

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expression of the L and S proteins. Twenty five constructs that express the envelope proteins of

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different HBV variants (LMS vectors) were used for assembly of twenty five different types of

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HDV virions. Each HDV type was assembled by co-transfection of Huh7 cells with the plasmid

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pSVLD3 that initiates HDV genome replication and a particular LMS vector that expresses the

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envelope proteins of a single HBV variant. HDV types were designated HDV-A1, HDV-B2, HDV-

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C3 etc. accordingly to HBV variant used (Table 2). Therefore, (i) each HDV type contained within

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the virion the same HDV RNP (HDV RNA genome and approximately 200 copies of delta antigen

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(δAg) (37)), and (ii) the only difference between the assembled types of HDV is an unique envelope

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that contains the envelope proteins of a particular HBV variant. Furthermore, the differences that are

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anticipated to be observed in assembly and/or infectivity of the above HDV types will reflect the

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differences in the functioning of the envelope proteins that belong to different HBV variants.

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Characterization of the concentrated HDV stocks. The results of the quantitative analysis of the

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concentrated different HDV virus stocks are summarized in the Table 2. The total yield of

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assembled HDVs refers to the sum of all categories of HDV virions regardless of the combination of

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the envelope proteins in the virion. All secreted virions contained the S envelope protein, while only

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a fraction of virions bore the L envelope protein on the their outer surface (Table 2 and (16)). The

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values of the total HDV yields varied in 122-fold range. The highest yield was observed for the

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variant A0 and the lowest - for B4. Fig. 2 represents the alignment of the HDV-binding site (HDV-

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BS) sequences for all HBV variants examined. The HDV-BS is a small 8 amino acid-long cytosolic

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loop that is located between the third and fourth transmembrane domains, and is therefore present in

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the L, M and S proteins (16, 22). In the HDV-BS, each variant has unchanged all three tryptophans

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that were shown to be important for HDV assembly (22). Clearly, either of highly conserved

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versions of the HDV-BS, AIWMMWYW or VIWMM(I)WYW may facilitate considerable HDV

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yield. Only B4 has an unique change Y(374)F resulting in the VIWMIWFW, which correlates with

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the lowest efficiency of HDV assembly (Table 2). As expected, the created mutant B4m1 bearing

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the reverse change F(374)Y supported an approximately 88-fold increase of total HDV yield, which

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is (i) a 72.0% value as compared to that of HDV-A0, and (ii) the highest value among the variants of

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genotype B studied (Table 2). The significance of the Y(374) residue per se for HDV assembly was

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not recognized before (22, 38). The previously produced mutant, in which five amino acids

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including the sequence 373-WYW-375 of the HDV-BS were replaced by the Lys-Leu sequence,

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displayed comparable to wild-type levels of synthesis and egress of the S protein and supported

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about 10-fold reduced level of HDV assembly (38). Based on that and taking into consideration that

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all three important for HDV assembly tryptophans were intact in HDV-BS of B4, we interpret that

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Y(374) is involved in the regulation of HDV RNP binding to HDV-BS, and the presence of the

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hydroxyl group on Y (in contrast to F) appears to be important for HDV assembly efficiency.

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However, the mechanism, by which the residue Y(374) influences the assembly of HDV is yet to be

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revealed in the follow-up studies. Total HDV yields (Table 2) reflect the efficiency of interactions of

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HDV RNP with HBV envelope proteins and efficiency of the egress of the formed HDV RNP-

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envelope protein complexes. Given that 24 out 25 variants have highly conserved versions of HDV-

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BS sequences, it became apparent that the differences in the total HDV yield (with the exception of

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HDV-B4) cannot be attributed to the binding of HDV RNP to HBV envelope proteins, and possibly

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could be influenced by the specific amino acid residues outside of the HDV-BS, which may affect

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the efficiency of the S protein secretion.

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The virions that contain on their outer side the PreS1 domain of the L, which bears HBV receptor-

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interacting site, are potentially infectious (16). To access the fraction of the virions that bear the

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PreS1 domain on the outer surfaces, we used rabbit polyclonal "anti-matrix" antibodies. Although, it

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was predicted as described in the Materials and Methods that "anti-matrix" antibodies are expected

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to exclusively interact with the PreS1 domain, previously it was not experimentally examined. We

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therefore tested the specificity of these antibodies. Based on the LMS vector for the D1 variant

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(LMS-D1), a modified version of this construct was created, in which the initiation codon for the L

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envelope protein was knocked-out (AUG was changed to ACG that codes for Thr). The resulting

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construct was designated therefore as the "L-" vector. The parental LMS-D1 vector and the newly-

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made L-M+S+ vector were used to assemble HDV virions. The assembled HDV virions were

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concentrated with PEG and then were subjected to the IP procedure using either the "anti-matrix"

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antibodies or the S26 mouse monoclonal antibody. The S26 monoclonal antibody is well-studied. It

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recognizes a very short sequence in the PreS2 domain, QDPR (positions 121-124 in HBV variant

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D1 sequence) (39). The S26 antibody therefore binds to the L and M envelope proteins. As a

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negative control, we used HDV virions assembled with the S protein only (HDV-S only virions),

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which do not bear any PreS sequences. The results are summarized in Figure 3. As expected, both

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kinds of antibodies bind HDV-S only virions extremely poorly. The S26 antibody precipitated only

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0.5% of HDV-S only virus particles. Similarly, the "anti-matrix" antibodies pulled down only 0.3%

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of HDV-S only virions. Furthermore, the S26 antibody pulled down 30.8% of all HDV-D1 L-minus

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virions. This result confirms that a fraction of the assembled virions contained the M protein along

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with the S (i.e., these virions were L-M+S+). The "anti-matrix" antibodies only precipitated 6.6% of

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HDV-D1 L- particles, which is only 21.6% (=(6.6/30.8)x100) of the virions that were

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immunoprecipitated by the S26 antibody. This result strongly suggests that the "anti-matrix"

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antibodies poorly recognize the PreS2 sequences, since they were unable to pull down 78.4%

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(=100-21.6) of L-M+S+ virions that were otherwise available for precipitation (as judged by the

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virions that were brought down by the S26 antibodies). Next, the HDV-D1 virions that were

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assembled using the vector that expresses the L, M and S proteins (LMS-D1 vector) were analyzed.

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The "anti-matrix" antibodies pulled down 39.6% of all HDV-D1 virions (Fig. 3). The S26 antibody,

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which recognizes its linear antigenic site on both, the L and M proteins, was able to precipitate

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34.1% of the same HDV-D1 virions. Considering these data and also taking into consideration the

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poor ability of the "anti-matrix" antibodies to recognize the M protein (Fig. 3), it became apparent

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that in the HDV-D1 stock (assembled using the LMS-D1 vector) the majority of the M was present

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in the context of the L+ particles, and there was no great excess of the L-M+S+ particles. The above

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experiments demonstrated that the maximal percentage of the HDV-D1 L-minus virions that can be

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precipitated by the "anti-matrix" antibodies was 6.6%. It can be reasonably assumed that in the

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context of the HDV-D1 virions assembled with unmodified LMS-D1 vector, which expresses the L,

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M and S proteins, the percentage of the L-M+S+ virions that can be precipitated with the "anti-

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matrix" antibodies (relatively to the total number of virions) cannot exceed the above 6.6%.

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Therefore, the fraction of the PreS1-containing virions among the HDV-D1 virions precipitated with

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the "anti-matrix" antibodies is expected to be greater or equal to 100-16.8 = 83.2%, where

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16.8%=(6.6/39.6)x100. In other words, out of total amount of the virions that were

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immunoprecipitated from the HDV-D1 stock using the "anti-matrix" antibodies, the percentage of

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the PreS1-HDVs is anticipated to be at least 83.2%. Therefore, the "anti-matrix" antibodies when

275

used in IP procedure were predominantly pulling down the L protein-containing (and thus

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potentially infectious) virions. However, the "anti-matrix" antibodies did not display the exclusive

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specificity for the PreS1 region. They demonstrated preferential specificity for the PreS1 sequences,

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but at the same time were able to react with the PreS2 sequences (of the M protein) to a small extent.

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Overall, we concluded that (i) the predominant majority of the virions immunoprecipitated by the

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"anti-matrix" antibodies are the PreS1-HDVs; and (ii) the contribution of the binding to the M

281

envelope protein during the IP with the "anti-matrix" antibodies can be considered as not significant.

282

Since the "anti-matrix" antibodies displayed predominant, but not exclusive specificity for the

283

PreS1-containing virions, the virions pulled down by the IP using the "anti-matrix"-antibodies were

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termed as the PreS1*-HDVs, which can be considered as a good acceptable approximation reflecting

285

the fraction of potentially infectious HDV virions. The asterisk is introduced to indicate that the

286

immunoprecipitated virions contain predominantly the PreS1-containing particles along with not

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significant amounts of the L-M+S+ virions.

288 289 290

The determined absolute amounts of the PreS1*-HDVs varied between different HDV types within

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the approximately 270-fold range (Table 2). The averaged percentage values of PreS1*-HDVs did

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not exceed 32% of the total number of secreted virions, strongly indicating that the majority of the

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assembled HDV virions do not contain the L envelope protein (16). The percentage of the PreS1*-

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HDVs facilitated by different HBV variants varied in approximately 3-fold range. The majority of

295

HBV variants supported the yield of PreS1*-HDVs within the range of 21-32%, while the highest

296

numbers of 31.9% and 31.8% were observed for HDV-B2 and HDV-D1, respectively. The

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envelope proteins of HBV genotypes D and E consistently yielded significant fractions of the

298

PreS1*-HDVs. The lowest percentages of PreS1*-HDVs were facilitated by B3 (11.0%), C4

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(11.3%) and C5 (11.8%) variants (Table 2). The "anti-matrix" antibodies used for the

300

immunoprecipitation were raised against the peptide that spans the positions 91-128 in the L protein

301

of HBV variant A0. This peptide includes the sequence of conserved HBV matrix domain (23-24).

302

Fig. 4 represents the alignment of the region spanned by the above peptide for the all sequences used

303

in this study. The predominant majority of amino acids that differ from the A0 sequence represent

304

either conserved or semi-conserved changes (Fig. 4). It became apparent that none of the observed

305

amino acid changes seems to interfere with IP procedure, since the presence of a particular change

306

or combination of changes did not correlate with determined percentage of the PreS1*-HDVs.

307

Overall, the generated data (Table 2 and Fig. 4) strongly suggest that the "anti-matrix" antibodies

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recognized similarly the region of the amino acid positions 91-128 (numbering is for genotype A

309

(20)) in the sequences of HBV variants that were examined. The following observations can serve

310

as examples of the several lines of supporting evidence for the above conclusion:

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(i) If there would exist serious differences between different HBV variants in terms of the

312

binding to the "anti-matrix" antibodies, then one can expect that at least the presence of some

313

variant-specific amino acid residues, which differ from the A0 sequence within the region of the

314

positions 91-128 (Fig. 4), will be consistently correlating with low percentage of the PreS1*-HDVs

315

measured (as compared to the A0). This would suggest the existence of defect(s) in the binding by

316

the "anti-matrix" antibodies. However, according to the Table 2 and Fig. 4, 19 out of 25 variants,

317

which represent HBV genotypes A to I, displayed the percentage of the PreS1*-HDVs within not a

318

wide range of values, between 21 and 32%.

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(ii) The HDV-A0 (A0 sequence is 100% identical to the peptide that was used to produce the

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"anti-matrix" antibodies) did not display the highest percentage of the PreS1*-HDVs (i.e., 28.0%).

321

Six other viruses are above the HDV-A0 in the Fig. 4, while displaying very close, but slightly

322

higher percentages of the PreS1*-HDVs: (1) HDV-B2 (31.9% of the PreS1*-HDVs; four amino

323

acids that are non-identical to the above peptide sequence of A0); (2) HDV-D1 (31.8%; five non-

324

identical residues); (3) HDV-I1 (31.0%; two non-identical residues); (4) HDV-E2 (31.0%; four non-

325

identical amino acids); (5) HDV-E3 (29.8%; four non-identical amino acids); and (6) HDV-D5

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(29.4%; five non-identical residues).

327

(iii) The variants, B2, B1 and B4 have identical sequence within the fragment spanning the

328

positions 91-128, which bears four residues that are not identical to the sequence of A0. However,

329

B2 facilitates 31.9% of the PreS1*-HDVs, while B1 - 22.7%, and B4 - 12.6%. These differences

330

cannot be explained by the differences in sequences between the above variants of genotype B and

331

A0 variant (within the region of the positions 91-128).

332

(iv) The F1 has six non-identical residues (as compared to A0) within the region between

333

pos. 91 and 128, and still displayed very similar percentage of PreS1*-HDVs - 27.3% (for A0, the

334

number is 28.0%).

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In addition, the IP procedure was optimized, so the "anti-matrix" antibodies were able to pull

336

down the sequences that belong to different HBV variants with practically equal efficiency, leading

337

to recovery of about 90% (and in some cases >90%) of PreS1*-HDVs (data not shown).

338

The above observations indicate that the amino acid residues, which define the antigenic

339

sites within the region that is recognized by the "anti-matrix" antibodies, likely remain identical or

340

sufficiently conserved among different HBV variants tested. Based on the data summarized in Table

16

341

2, no overall HBV genotype-specific trend in regard to either the total yield of HDV, or to the yield

342

of the PreS1*-HDVs was observed.

343 344 345

Infectivity of HDV virions coated with the envelope proteins of different HBV variants.

346

The infectivity of assembled HDVs was assayed using in vitro infection of primary human

347

hepatocytes (PHH). We examined numbers of infected PHH by staining for newly made delta

348

antigens using rabbit polyclonal antibodies raised against recombinant small δAg (21). We also

349

quantified the numbers of newly made HDV genomes produced as the result of infection of PHH by

350

assaying total RNA harvested from infected hepatocytes using qPCR (21, 25). Selected images of

351

PHH infected with different HDV types are presented in Figures 5-8. The origin of the envelope

352

proteins clearly makes a difference in terms of the numbers of HDV-infected cells. For example,

353

HDV-B2 and HDV-D1 virions consistently infected larger fractions of PHH (Figs. 5B and 5D),

354

while for HDV-A2 and HDV-C2, infected PHH were observed only during one out of three and 2/4

355

separate infections, respectively. For HDV-C4, no infected cells were observed in 3/3 infections

356

(Figs. 7B, 8B and data not shown). Therefore, HDV-C4 was considered as non-infectious virus.

357

Consistent with HDV replication in PHH, δAg most frequently was detected in the nucleoplasm

358

(Figs. 5-8, and (21, 27)). Some infected hepatocytes displayed a different, but also previously

359

observed pattern, when δAg was found also in the cytoplasm (Figs. 6C and 6D). Since the

360

assembled HDVs did not contain HBV and therefore a formation of new virions and HDV spread

361

did not take place during the infection, the appearance of δAg in cytoplasm is consistent with the

362

occurrence of replication-derived mutations in the δAg nuclear localization signal (21, 37, 40). To

363

describe quantitatively the infectivity of the different HDV types, we employed two parameters,

17

364

specific infectivity and normalized index of infected PHH. The specific infectivity (SI) is the

365

number of HDV genomes per hepatocyte produced by infection and normalized by the value of the

366

PreS1*-MOI, which is the multiplicity of infection that reflects a number of PreS1*-HDVs in

367

inoculum used per average hepatocyte. The normalized index of infected cells (NI) is the percentage

368

of infected PHH normalized by the PreS1*-MOI. The SI and NI numbers of HDV-C3 were used as

369

100% values. The SI and NI numbers of other viruses were normalized relatively to that of HDV-

370

C3. The results are summarized in Fig 9. Interestingly, the envelope proteins of different HBV

371

variants supported infectivity of the HDV within fairly wide 160-fold range of the SI(%) values. The

372

observed NI(%) values were within approximately 150-fold range. The most infectious viruses

373

HDV-C3, HDV-D1 and HDV-B2 demonstrated the highest SI values (81%-100%), and

374

considerably high NI numbers (40%-100%). Next six HDV types, HDV-F1, HDV-D3, HDV-D2,

375

HDV-D5, HDV-B3, and HDV-E3 also displayed relatively high infectivity (SI values within the

376

range of 40%-49%), while being not as highly infectious as above mentioned three HDV types. For

377

this group, NI values ranged between 15% and 39%. Next group of moderately infectious HDVs

378

included HDV-E2, HDV-I1, HDV-C5, HDV-E1 and HDV-B1, whose SI and NI numbers were in a

379

range of 27%-34% and 13%-49%, respectively. HDVs coated with envelope proteins of HBV

380

variants G1, B4, A1, A0 and H1 displayed SI values between 9% and 15%, and NI numbers

381

between 10% and 56%. The rest of HDV types demonstrated low infectivity with SI values below

382

8%. Among them were HDV-A3, HDV-C1 and HDV-A4 with SI values in the range of 3%-8%, and

383

NI values between 8% and 12%. HDV-A2, HDV-C2 and HDV-C4 displayed very low SI numbers

384

(1.6%, 0.6% and 0.1%, respectively). The observed very low SI values are consistent with the

385

results of the immunofluorescence experiments. As mentioned above, no infected PHH were

386

observed on all occasions for HDV-C4. For HDV-A2 and HDV-C2, infected PHH were observed in

18

387

1/3 and in 2/4 infections, respectively. Therefore, the ability of these virions coated with envelope

388

proteins of variants C2 or A2 to achieve the infection of PHH critically depended on the

389

susceptibility of tested hepatocytes, which varied between different PHH lots. All natural M-minus

390

mutants A1, B3, C1 and C5 supported production of infectious HDVs, as expected (41). The

391

envelope proteins of four genotype D variants supported production of virions with considerably

392

high infectivity (SI average is 59.9%), which was higher than SI average for four genotype B

393

variants (41.0%) and for three genotype E variants (34.8%). Although, the SI average for E variants

394

was not dramatically lower than that of the variants of the genotype B. All tested variants of

395

genotypes D, B and E supported formation of infectious HDVs. The variants of genotype A

396

facilitated assembly of HDVs with low infectivity (SI average is 6.9%). Therefore, based on the

397

average SI values (Fig. 9) for these HBV genotype-specific envelope proteins, the following trend

398

reflecting the support of HDV infectivity was observed: D(59.9%)>B (41.0%)> E(34.8%)>A(6.9%).

399

Within the group of genotype C variants, the SI values were diverse. The HDV-C3 had highest

400

infectivity, while HDV-C5 was moderately infectious. The HDV-C1 and HDV-C2 displayed low

401

infectivity, and HDV-C4 was apparently non-infectious. For genotypes F, G, H and I, only one

402

sequence per genotype was available. The HDV-F1, HDV-G1, HDV-H1 and HDV-I1 showed

403

diverse SI levels, between 9.8 and 49%.

404 405 406

Another interesting observation was made comparing the SI and NI values. The viruses in Fig. 9

407

were placed in the descending order of the SI values. It became apparent, that the NI values do not

408

always follow the same trend. Thus, there was no direct correlation between the SI and NI values for

409

a number of viruses tested. Several HDV types illustrating lack of such correlation were next

19

410

carefully considered, using only the SI and NI values with relatively low standard errors of the

411

mean. For example, HDV-B3 seemed to have the NI value that was unreasonably low as compared

412

to the corresponding SI value. In other words, HDV-B3 induced relatively high level of HDV

413

replication in infected PHH, while not infecting a considerable number of cells (as normalized by

414

PreS1*-MOI). The examples of an opposite tendency are HDV-F1, HDV-D3, HDV-I1, HDV-C5,

415

HDV-B1, HDV-G1, HDV-B4 and HDV-H1. They demonstrated the NI values that were higher than

416

what could be anticipated based on the corresponding SI values. These virions seemed to infect

417

sufficient numbers of PHH, which did not result in respectively high levels of HDV replication.

418

Apparently, the infection of considerable numbers of hepatocytes does not guarantee high levels of

419

HDV replication. The implications of these findings are further evaluated in Discussion section.

420 421 422

These interpretations are in tune with the results of testing the infectivity of the B4 mutant, bearing a

423

single change F(374)Y. As compared to HDV-B4, HDV coated with mutated B4 envelope proteins

424

(HDV-B4m1) displayed about 3-fold higher SI value (34.3% versus 12.2%), while exhibiting a

425

similar NI value (65.0% versus 55.9%) (Fig. 9). The data suggest that there was definitely an

426

increase in the number of HDV genomes that started HDV RNA replication after the successful

427

entry, while there was no significant increase in the number of infected cells. One possible

428

explanation is that the introduced mutation was important for coordinated mechanism of HDV RNP

429

disassembly from the envelope proteins during the post-entry intracellular trafficking and had no

430

effect on the entry per se.

431 432

20

433

Finally, we investigated why HDV-C4 appeared non-infectious. Only two unique substitutions,

434

M(12)V and K(7)N, were identified for the C4 variant in the critical for infectivity PreS1 domain

435

(Fig. 10A). While the significance of the former substitution was not obvious, the latter, according

436

to analysis using the software web.expasy.org/myristoylator/, could affect the functionality of the

437

myristoylation signal. Two new HDV types of virions were produced. The envelope of the HDV-

438

C4m1 contained the mutated version of the L protein of C4 variant, bearing V(12)M change. In the

439

HDV-C4m2, the L envelope protein of C4 variant contained two amino acid changes,

440

V(12)M+N(7)K. The assembled concentrated virus stocks, HDV-C4m1 and HDV-C4m2, did not

441

differ significantly from HDV-C4 in terms of the total yield of virions or the percentage of the

442

PreS1*-HDVs (Table 2). Both newly made HDV types were demonstrated to be infectious. The

443

corresponding images of the infected PHH are shown in Fig. 10B. A single mutation V(12)M led to

444

the observation of infected PHH (NI=5.8%), while no improvements of the SI were detected. The

445

double mutant appeared more infectious than HDV-C4m1 and HDV-C2 (Fig. 9). However, the NI

446

and SI values of the HDV-C4m2 were still considerably lower than those of more infectious HDV-

447

C3 and HDV-C5. The results suggest that while positions 7 and 12 were critical for infectivity, the

448

overall infectivity likely was additionally regulated by other amino acid residue(s) outside the PreS1

449

region.

450 451 452

DISCUSSION

453

Our study contributes to the understanding of the complex mechanisms that define the infectivity of

454

HBV and HDV virions. Since both viruses have indistinguishable envelopes (16), they share the

455

same receptor and the steps of the attachment and entry that are regulated by HBV envelope

21

456

proteins. HDV model allows us to study separately the contribution of HBV envelope proteins to

457

overall infectivity in absence of other HBV components.

458 459 460

This is the first report that demonstrates that HDV of genotype I (26, 42) is fully compatible with

461

nine HBV genotypes A-I in terms of assembly and infectivity. This finding suggests that although

462

HDV genotypes were not uniformly distributed around the world (43-44), it became apparent that at

463

least for HDV genotype I, there likely will be no restrictions in terms of support of the transmission

464

and developing of the infection by HBV of different genotypes. Our data therefore, not only

465

provides new insights in the understanding of HDV-HBV interactions, but has a certain clinical

466

value regarding the transmission of HDV disease and its relation to the geographical distribution of

467

HBV genotypes.

468 469 470

Out of twenty five HBV variants that were examined, the isolates A2, B1, B2, B4, C2, C5, D1, D2,

471

E1, E3, G1 and H1 correspond to the chronic stage of HBV infection, while A1, B3, C1 and I1 were

472

collected during the acute HBV infection ((17, 28-36) and Abe K., personal communication). The

473

information regarding the rest of the variants is not available. Small number of isolates from acute

474

stage of infection that were analyzed is insufficient in order to make the conclusions regarding the

475

extent of infectivity that was supported by the envelope proteins expressed during acute infection

476

versus that of the chronic phase of infection. Based on our data that the envelope proteins from the

477

variants collected during chronic infection facilitated production of HDV types with a wide variety

478

of infectivity parameters (both, NI and SI) (Fig. 9), including several most infectious HDV types, it

22

479

cannot be asserted that at the chronic stage of infection, HBV envelope proteins are responsible for

480

assembly of mainly virus particles with low infectivity that do not support efficient virus

481

transmission. Further studies, which will employ larger numbers of samples from acute and chronic

482

stages of HBV infection, and different HBV strains collected from the same host, are warranted in

483

order to better understand the mechanisms of (i) maintenance of chronic HBV infection, (ii) HDV-

484

HBV interactions and (iii) dependence of HDV persistence on the variety of different HBV

485

sequences that co-exist in a particular host.

486 487 488

It was observed that the envelope proteins of several HBV variants ensured relatively high numbers

489

of infected PHH and high levels of overall replication (variants C3, D1, B2). Other variants (for

490

example, F1, D3, I1, C5, B1, G1, B4 and H1) supported the levels of HDV replication (SI values)

491

that were unproportionally lower than what could be anticipated based on the corresponding NI

492

values (normalized numbers of infected PHH). In other words, infection of sufficient number of

493

PHH did not always assure overall high levels of HDV replication. Another variant, B3 facilitated

494

considerable level of replication and unexpectedly low NI value. Therefore, since all tested types of

495

HDV virions had the same HDV RNP inside and differed only by the variant-specific envelope

496

proteins coating the particle, it became apparent that HBV envelope proteins alone determined two

497

parameters (i) the number of infected cells and (ii) the number of HDV genomes, which

498

successfully started the replication after the entry. One interpretation is that HBV envelope proteins

499

facilitate not only the attachment and entry, but also regulate at least one post-entry step of the virus

500

life cycle (i.e., likely one or more events related to intracellular trafficking). Our data further suggest

501

that HBV and HDV likely employ endocytosis as the entry mechanism as was also suggested by

23

502

other labs (45-46). The entry via endocytosis explains how the envelope proteins determine the

503

number of viral genomes that successfully reach the replication site(s) and start the replication. It

504

needs to be explored further, whether the determinants of the envelope proteins that regulate the

505

number of infected cells and the number of genomes that will succeed in initiation of the replication,

506

are structurally and functionally separated.

507 508 509

Kinetics of the virus spread throughout the liver, as it was previously noted by Chisari’s lab, likely

510

determines the timing and magnitude of the host immune response, which in turn will determine

511

whether infection will become transient or chronic (3). The ability of HBV envelope proteins to

512

determine the number of infected cells and the fraction of genomes that will initiate replication will

513

likely have a considerable impact on defining the kinetics of virus spread throughout the liver. This

514

is likely true for both HDV and HBV, since both viruses share the same envelope proteins. Our data

515

suggest that the kinetics of the virus spread through the liver can be tightly linked to the overall

516

infectivity of the virions. Therefore, it is reasonable to consider the infectivity as a critical

517

determinant of the virus-induced pathogenesis, which is likely involved in determining the outcome

518

(transient or chronic) of either HBV or HDV infection. Considering the impact of infectivity on the

519

kinetics of virus spread, it is interesting to hypothesize that HDV even with a relatively low rate of

520

replication may achieve the state of persistent infection, when the spread of HDV is supported by

521

the envelope ensuring high number of infected hepatocytes.

522 523

24

524

The observed trend for HBV genotypes, D(59.9)>B (41.0)> E(34.8)>A(6.9) (based on the average

525

SI values, %), in regard of supporting HDV infectivity (Fig. 9) may advocate in favor of the follow-

526

up studies using larger numbers of tested HBV variants. The data generated also suggest that

527

genotyping of HBV could be helpful in anticipating the outcomes of HBV and HBV/HDV

528

infections.

529 530 531

Acknowledgements. SG and SM were supported by NIH grant NCI R01CA166213. SG was also

532

supported by NIH grants NIAID R21AI097647 and NCRR P20RR016443, and by the University of

533

Kansas Endowment Association.

534

We are grateful to Stefan Wieland, Frank Chisari, Hans Will, Volker Bruss, Yu-Mei Wen

535

and Stephan Schaefer, who provided us with constructs bearing sequences of different HBV

536

variants. We thank Vadim Bichko for his generous gift of the S26 monoclonal antibody. We

537

acknowledge help of Jessica Salisse, Megan Dudek and Louise Rodrigues. We thank Igor

538

Prudovsky for constructive comments.

539 540 541 542 543 544 545

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663

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664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681

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31

682

Table 1. HBV variants used in the current study.

683

Genotype of HBV

684

-------------------------------------------------------------------------------------------------------------------------

Accession number

Stage of HBV infection

A0

X02763

unknown

686

A1

AB126580

acutec

687

A2

AB205118

chronicc

688

A3

X51970

unknown

689

A4

M57663

unknown

685

690

A

Name of the varianta

------------------------------------------------------------------------------------------------------------------------B1

AB205121

chronicc

692

B2

AB205119

chronicc

693

B3

AB205122

acutec

694

B4

D00330

chronic (28)

691

695

B

------------------------------------------------------------------------------------------------------------------------C1

AB205125

acutec

697

C2

AB205124

chronicc

698

C3

AB033550

unknown (28)

699

C4

N/Ab

unknown

700

C5

AF411411

chronic (29)

696

701

C

------------------------------------------------------------------------------------------------------------------------D1

AB205127

chronicc (30)

703

D2

AB205126

chronicc

704

D3

V01460

unknown

702

D

32

D5

705 706

N/Ab

unknown

------------------------------------------------------------------------------------------------------------------------E1

AB205192

chronicc (31)

708

E2

AY739675

unknown (32)

709

E3

AB032431

chronic (33)

E

707

710

------------------------------------------------------------------------------------------------------------------------F

711 712

G

H

719 720

G1

AF405706

chronic (35)

H1

AB205010

chronicc (36)

------------------------------------------------------------------------------------------------------------------------I

717 718

unknown (34)

-------------------------------------------------------------------------------------------------------------------------

715 716

X69798

-------------------------------------------------------------------------------------------------------------------------

713 714

F1

I1

AB231908

acutec (17)

------------------------------------------------------------------------------------------------------------------------a

, The name of each variant reflects HBV genotype and a number of a particular variant among the

other variants of the same genotype in the collection of HBV sequences.

721 722

N/Ab, accession number is not available, since the sequences of the indicated above HBV variants

723

were not previously deposited into NCBI database. Therefore, the complete amino acid sequences of

724

the L envelope proteins of the variants C4 and D5 are presented in the Fig. 1.

725 726 727

c

, Abe K., personal communication.

33

728

Table 2. Characterization of the concentrated virus stocks of different HDV types.

729

Genotype of HBV

730 731

(GE/μl) (%b) (GE/μl) (%c) -------------------------------------------------------------------------------------------------------------------------

Name of HDV typea

Total yield

Total yield PreS1*-HDV PreS1*-HDV

HDV-A0

1.13x107

100.0

3.18x106

28.0

733

HDV-A1

5.27x106

46.5

1.38x106

26.3

734

HDV-A2

5.27x105

4.7

1.03x105

19.5

735

HDV-A3

6.23x105

5.5

1.36x105

21.8

736

HDV-A4

1.21x106

10.7

3.21x105

26.5

732

737

A

------------------------------------------------------------------------------------------------------------------------HDV-B1

2.64x106

23.3

5.97x105

22.7

739

HDV-B2

1.67x106

14.7

5.32x105

31.9

740

HDV-B3

1.38x106

12.2

1.52x105

11.0

741

HDV-B4

9.27x104

0.8

1.16x104

12.6

742

HDV-B4m1d

8.16x106

72.0

2.01x106

24.6

738

743

B

------------------------------------------------------------------------------------------------------------------------HDV-C1

3.19x106

28.2

8.69x105

27.2

745

HDV-C2

6.83x106

60.3

1.62x106

23.6

746

HDV-C3

7.85x105

6.9

1.06x105

13.4

747

HDV-C4

5.27x106

46.5

5.97x105

11.3

748

HDV-C5

4.98x105

4.4

5.86x104

11.8

749

HDV-C4m1d

1.04x107

91.9

1.44x106

13.8

750

HDV-C4m2d

3.46x106

30.5

4.72x105

13.7

744

751

C

-------------------------------------------------------------------------------------------------------------------------

34

HDV-D1

1.59x106

14.1

5.07x105

31.8

753

HDV-D2

8.83x105

7.8

1.96x105

22.1

754

HDV-D3

1.51x106

13.3

4.00x105

26.5

755

HDV-D5

2.53x106

22.3

7.43x105

29.4

D

752

756

------------------------------------------------------------------------------------------------------------------------HDV-E1

2.49x106

22.0

6.65x105

26.7

758

HDV-E2

5.30x105

4.7

1.64x105

31.0

759

HDV-E3

2.84x106

25.1

8.45x105

29.8

E

757

760

------------------------------------------------------------------------------------------------------------------------F

761 762

G

76.9

2.38x106

27.3

HDV-G1

7.53x105

6.7

1.71x105

22.7

------------------------------------------------------------------------------------------------------------------------H

765 766

8.71x106

-------------------------------------------------------------------------------------------------------------------------

763 764

HDV-F1

HDV-H1

7.58x106

67.0

1.89x106

24.9

------------------------------------------------------------------------------------------------------------------------I

767

HDV-I1

1.16x106

10.3

3.61x105

31.0

768

-------------------------------------------------------------------------------------------------------------------------

769

The robust procedure for HDV assembly was developed that (i) ensured that technical factors (such

770

as variations between transfections, etc.) have no significant contribution to the HDV yields

771

measured; and (ii) allowed us to achieve reproducible results, when independent assemblies were

772

performed.

773 774

a

, The name of each type of assembled HDV indicates a particular HBV variant, which envelope

35

775

proteins were used for the assembly of this HDV type. Total yield was quantified using qPCR (21)

776

and shows the number of HDV genome equivalents (GE) per one microliter of 100-fold

777

concentrated HDV virus stock. Total yield reflects a total number of HDV genome-containing virus

778

particles and refers to the sum of all categories of HDV virions regardless of the combination of the

779

envelope proteins in the virion. All secreted virions contained the S envelope protein, while only a

780

fraction of virions bore the L envelope protein on the their outer surface (16). The PreS1*-HDV

781

value represents the number of HDV virions that were immunoprecipitated using the "anti-matrix"

782

antibodies (which predominantly, but not exclusively precipitate the PreS1-containing HDVs (see

783

the text)) per one microliter of 100-fold concentrated HDV stock. As described in Materials and

784

Methods, after the immunoprecipitation with the "anti-matrix" antibodies, the total RNA was then

785

extracted from precipitated viral particles with TRI reagent and HDV genomes were quantified

786

using qPCR (21).

787 788 789

b

, The calculations of the percentages of the total yields of different HDV types were done relatively

to the total yield of HDV-A0 virions, which was the highest and therefore was used as 100% value.

790 791

c

, The percentage of the PreS1*-HDV reflects a portion of the PreS1*-HDVs in the total population

792

of the assembled and released HDV particles. The immunoprecipitation procedure was optimized in

793

order to achieve its maximal efficiency. The best IP efficiency was achieved using 3μl of the "anti-

794

matrix" antibodies with 10μl of the concentrated virus stock. Under these conditions the overall

795

efficiency of the immunoprecipitation was approximately 90%.

36

796

d

, B4m1 is a mutant of the variant B4, which bears the mutation F(374)Y in the HDV-binding site;

797

C4m1 and C4m2 are the mutants of the variant C4, which bear either a single V(12)M mutation or a

798

double change N(7)K+V(12)M, respectively, in the PreS1 domain of the L envelope protein.

799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820

37

821

FIGURE LEGENDS

822 823

Fig 1. Complete amino acid sequences of the large (L) envelope proteins of HBV variants C4

824

and D5. The sequences of HBV variants C4 and D5 (see Table 1) used in the current study were not

825

previously deposited into NCBI database, and therefore do not have accession numbers. The

826

complete amino acid sequences of the corresponding large (L) envelope proteins were deduced from

827

the nucleotide sequences and are presented in a single letter code. The L protein of the C4 contains

828

400 amino acids, while the L of D5 – 389. As compared to the C4, the L of D5 does not contain

829

eleven N-terminal amino acids of the PreS1 domain.

830 831 832

Fig 2. Comparison of the HDV-binding site sequences among different HBV variants. HDV

833

ribonucleoprotein (HDV RNA genome in complex with approximately 200 molecules of delta

834

antigen (37)) binds to the small cytosolic loop that is located in the S domain, which is present on

835

the L, M, and S envelope proteins of HBV. The amino acid sequence of this HDV-binding site

836

(HDV-BS) of the variant A0 is shown as the reference sequence above the alignment. The amino

837

acid position numbers are given as for the L protein of HBV genotype A. The alignment was

838

performed using the MUSCLE alignment software (www.ebi.ac.uk/Tools/msa/muscle/). The

839

variants of HBV (see Table 1) are indicated at the left side of the alignment. The relative values of

840

total yield of HDV virions (total population of released HDV virions that refers to the sum of all

841

categories of HDV virions regardless of the combination of the envelope proteins in the virion) are

842

shown as the percentages next to the name of each HBV variant. The absolute numbers of secreted

843

HDV virions were quantified using qPCR as described in Materials and Methods. The highest total

844

yield of HDV virions facilitated by envelope proteins of the variant A0 was used as 100% value.

38

845

The sequences are arranged (from the top to the bottom) in a descending order of the total HDV

846

assembly efficiency. The amino acids that are identical to the reference sequence (HDV-BS of A0)

847

are shown as dashes. The amino acids that are not identical to those of the reference sequence are

848

shown as a single letter amino acid code. At the bottom of the alignment, the identical amino acid

849

positions are indicated as stars, conservative residue – as a colon, and semi-conservative residue – as

850

a period. The HDV-BS sequence is very conserved among the variants of HBV. The HDV-BS of B4

851

bears an unique amino acid change Y(374)F.

852 853 854

Fig 3. Analysis of the specificity of the "anti-matrix" antibodies. Three types of HDV virions

855

were assayed. As a negative control, HDV particles coated with the S envelope protein only were

856

used (indicated as "S only"). The second type of virions was HDV-D1 L-minus (shown as "D1 L-

857

M+S+", indicating that the vector that expresses only the M and S proteins was used for the

858

assembly). This type of virions was assembled using the construct that was modified from the

859

original LMS-D1 vector that drives the expression of the L, M and S proteins of the variant D1. To

860

make the modified vector, the initiation codon for the L protein was changed from AUG to ACG

861

(ACG codes for Thr). The mutated vector was designated as the "L-" construct (i.e., L-M+S+). The

862

virions that were prepared using this L- construct, contained the M and S, but not the L protein. The

863

third type of virions was HDV-D1 (shown as "D1 L+M+S+", indicating that LMS-D1 vector that

864

expresses the L, M and S proteins was used for the assembly ). All assembled HDV stocks were

865

concentrated with PEG. The concentrated stocks were subjected to the immunoprecipitation with the

866

S26 or the "anti-matrix" antibodies. The S26 mouse monoclonal antibody (a gift from Vadim

867

Bichko) recognizes the short sequence QDPR in the PreS2 domain (39). The S26 binds to the L and

39

868

M envelope proteins. The "anti-matrix" antibodies (GenScript) are rabbit polyclonal antibodies that

869

were raised against the synthetic peptide that spans the positions 91-

870

IPPPASTNRQSGRQPTPISPPLRDSHPQAMQWNSTAFH-128 of the PreS region (HBV genotype

871

A(20)), which incorporates conserved HBV matrix domain (underlined) (23-24). Each IP used 10

872

μl of the concentrated virus stock and either 2 μl of the S26 antibody or 3 μl of the "anti-matrix"

873

antibodies, respectively. The immunoprecipitations performed using the S26 antibody are marked as

874

"S26" at the bottom of the Figure. The immunoprecipitations performed using the "anti-matrix"

875

antibodies are marked as "aM." The results of each of the immunoprecipitations are expressed as the

876

percentages of the total amount of HDV virions in the stock that was analyzed (the Y axis). The bars

877

representing the results of IP that used the S26 antibody are not shaded (open rectangles). The bars

878

that represent the results of the IP that used the "anti-matrix" antibodies have light gray shading.

879

Each bar represents the average of several independent immunoprecipitations. The precipitations of

880

(i) HDV-S only virions with the S26 antibody, (ii) HDV-D1 L-minus virions with the "anti-matrix"

881

antibodies and (iii) HDV-D1 virions with the "anti-matrix" antibodies were repeated two times each.

882

The other three immunoprecipitation experiments were repeated three times each. The standard

883

errors of the mean are indicated at the bars. The quantification of HDV genomes was done using

884

HDV-specific qPCR (21). During the real-time PCR procedure, each measurement was performed

885

as three independent qPCR reactions.

886 887 888

Fig 4. Comparison of the sequences at and around of the matrix domain among different HBV

889

variants. Rabbit polyclonal "anti-matrix" antibodies raised against the peptide (shown above the

890

alignment) that spans amino acid positions 91-128 of the L envelope protein (HBV variant A0 (see

40

891

Table 1)) were used for immunoprecipitation of HDV virions and quantification of the percentages

892

of the PreS1*-HDVs (see text). The sequence of the HBV matrix domain is underlined. The variants

893

of HBV are indicated at the left side of the alignment. The absolute numbers of HDV virions that

894

were immunoprecipitated with the "anti-matrix" antibodies were quantified using qPCR as described

895

in Materials and Methods. The percentages of the PreS1*-HDVs in the total population of secreted

896

HDV genome-containing virus particles are indicated next to the variant name. The sequences are

897

presented in a descending order (from the top to the bottom) of the percentage of the PreS1*-HDVs

898

with B2 shown at the top position, since it produces the highest percentage of PreS1*-HDVs

899

(31.9%). The amino acids that are identical to the reference sequence (variant A0) are shown as

900

dashes. The amino acids that are not identical to those of the reference sequence are shown as a

901

single letter amino acid code. At the bottom of the alignment, the identical amino acid positions are

902

indicated as stars, conservative residue – as a colon, and semi-conservative residue – as period. The

903

sequence of the matrix domain appeared to be conserved among different HBV variants as expected.

904 905 906

Fig 5. Detection of delta antigen-positive primary human hepatocytes (PHH) infected in vitro

907

with either HDV-A0, HDV-B2, HDV-C3, HDV-D1, HDV-E3 or HDV-F1. Shown are the images

908

of primary human hepatocytes (PHH) that were infected in vitro with either: HDV-A0 (panel A),

909

HDV-B2 (panel B), HDV-C3 (panel C), HDV-D1 (panel D), HDV-E3 (panel E) or HDV-F1 (panel

910

F). At day nine post-infection, hepatocytes were washed, fixed, permeabilized and then stained for

911

delta antigens (red staining), alpha-tubulin (green) and nuclear DNA (blue) as previously described

912

(21, 27). The infection and immunofluorescence procedure are described in Materials and Methods.

913 914

41

915

Fig 6. Detection of delta antigen-positive PHH infected in vitro with either HDV-G1, HDV-H1,

916

HDV-I1, HDV-D2, HDV-B1 or HDV-C5. Infection of PHH and immunofluorescence assay of

917

infected cells were conducted as described in Materials and Methods. The PHH were assayed at nine

918

days post-infection. Green staining is for alpha-tubulin, red – for newly synthesized as a result of

919

HDV infection delta antigens, and blue – is a DAPI staining for nuclear DNA. The images represent

920

PHH infected in vitro with either: HDV-G1 (panel A), HDV-H1 (panel B), HDV-I1 (panel C),

921

HDV-D2 (panel D), HDV-B1 (panel E) or HDV-C5 (panel F). On panels C and D, there are

922

hepatocytes that displayed staining for delta antigens (δAgs) not only in the nucleoplasm but also in

923

the cytoplasm. These are examples of a less frequent (as compared to nucleoplasmic staining)

924

pattern of δAgs’ subcellular localization in infected hepatocytes.

925 926 927

Fig 7. Detection of delta antigen-positive PHH infected in vitro with either HDV-A1, HDV-A2,

928

HDV-A3, HDV-A4, HDV-B3 or HDV-B4. Infection of PHH and immunofluorescence procedure

929

were conducted as described in Materials and Methods. The PHH were analyzed at day nine after

930

infection. Staining for delta antigens is in red, green staining is for alpha-tubulin, and blue – is a

931

DAPI staining for nuclear DNA. The images show PHH infected in vitro with either: HDV-A1

932

(panel A), HDV-A2 (panel B), HDV-A3 (panel C), HDV-A4 (panel D), HDV-B3 (panel E) or

933

HDV-B4 (panel F). Panel B shows no infected cells, since no cells positive for delta antigens were

934

observed during two out of three separate infections, when PHH were inoculated with HDV-A2.

935 936 937

Fig 8. Detection of delta antigen-positive PHH infected in vitro with either HDV-C1, HDV-C4,

42

938

HDV-D3, HDV-D5, HDV-E1 or HDV-E2. The details of the in vitro infection of PHH and

939

subsequent analysis of HDV-infected cells at day nine post-infection using immunoflurescence are

940

described in Materials and Methods. Newly synthesized delta antigens are stained in red, in green is

941

alpha-tubulin, in blue (DAPI) – nuclear DNA. The images represent PHH infected in vitro with

942

either: HDV-C1 (panel A), HDV-C4 (panel B), HDV-D3 (panel C), HDV-D5 (panel D), HDV-E1

943

(panel E) or HDV-E2 (panel F). There were observed no cells positive for delta antigens during

944

three out of three separate infections, when PHH were inoculated with HDV-C4 (panel B).

945 946 947

Fig 9. Infectivity parameters of different types of HDV virions that were coated with envelope

948

proteins of different HBV variants, which belong to nine genotypes of HBV (A-I). Two

949

parameters, the specific infectivity (SI) and normalized index of infected cells (NI), which define the

950

infectivity of different types of HDV virions were determined at day nine post-infection. The

951

specific infectivity (SI) is a number of HDV genomes per cell, which were produced and

952

accumulated during infection of PHH, normalized per each PreS1*-HDV virion (see text) used in

953

the inoculum per average hepatocyte. The normalized index (NI) of infected hepatocytes represents

954

a percentage of HDV-positive PHH normalized by the number of the PreS1*-HDVs per cell in

955

inoculum. For SI quantification measurements, the multiplicity of infection (MOI) of 10-20 of total

956

genome equivalents (GE) of HDV/hepatocyte was used. For immunofluorescence experiments, the

957

MOI of 30-100 GE of HDV/cell was used. The data presented in the Figure is based on the results

958

of: (i) two independent infections (that used two different lots of PHH) for the HDV-A3, HDV-B1,

959

HDV-B2, HDV-B3, HDV-D2, HDV-D3 and HDV-G1; (ii) three independent infections (that used

960

three different lots of PHH) for HDV-A1, HDV-A2, HDV-A4, HDV-C1, HDV-C3, HDV-C4,

43

961

HDV-D5, HDV-E1, HDV-E2, HDV-E3, HDV-H1, and HDV-I1; (iii) four independent infections

962

(that used four different lots of PHH) for HDV-A0, HDV-B4 and HDV-C2; (iv) five independent

963

infections (that used five different lots of PHH) for HDV-C5 and HDV-F1; and (v) eight

964

independent infections (that used eight different lots of PHH) for HDV-D1. The mutants of variant

965

C4 (C4m1 bearing the change V(12)M and the mutant C4m2 harboring two changes

966

N(7)K+V(12)M) were compared to their wild type counterpart (i.e., HDV-C4m1 and HDV-C4m2

967

versus HDV-C4) in one independent infection. The mutant of B4 (B4m1, bearing the mutation

968

Y(374)F) was compared to wild type B4 (i.e., HDV-B4m1 versus HDV-B4) in another independent

969

infection. Within each infection (a single individual lot of PHH), the SI value for each HDV type

970

was determined based on three independent repeats (three different wells of infected PHH), while

971

the immunofluorescence assay was conducted using two different wells of infected PHH (i.e., two

972

independent repeats). The Figure displays the averaged SI, % and NI, % values. The averaged SI

973

and NI values for HDV-C3 were used as 100% values. The NI bars have light gray shading, while SI

974

bars are not shaded (open rectangles). The standard errors of the mean are indicated at the bars. The

975

HDV type used as an inoculum is indicated as the abbreviation for HBV variant, which envelope

976

proteins were used for HDV assembly. Thus, for example, C3 stands for HDV virions assembled

977

with the envelope proteins of HBV genotype C, variant number 3 (i.e., HDV-C3). The data arranged

978

from the top to the bottom of the Figure, in a descending order of the SI, % values. An example,

979

when the calculated NI value appeared to be lower than it was anticipated, based on the

980

corresponding SI value (HDV-B3) is marked with an asterisk . Several examples, when NI values

981

were higher than it was anticipated, based on the corresponding SI values (HDV-F1, HDV-D3,

982

HDV-I1, HDV-C5, HDV-B1, HDV-G1, HDV-B4, and HDV-H1) are marked with two asterisks.

983 984

44

985

Fig 10. Analysis of two mutants of the variant C4. The in vitro infections of PHH with HDV-C4

986

resulted in absence of delta antigen-positive cells on three separate occasions (Fig. 8B). The PreS1

987

domain, and especially the region between positions 12 and 86 (this numbering is for genotypes that

988

have additional 11 amino acid extension at the N terminus of the PreS1, which results in 119 amino

989

acid long PreS1 domain) was demonstrated previously as being critical for HBV and HDV

990

infectivity (23). Panel A. Alignment of the PreS1 domains of the variants C1-C5 (indicated on the

991

left). The top row displays the sequence in a single letter code of the PreS1 domain of the C3

992

(positions 1-119). For the other HBV genotype C variants, non-identical amino acids are shown as

993

the corresponding single letters, and identical amino acids are shown within the alignment as dashes.

994

At the bottom of the aligned sequences, the stars correspond to the identical residues, the colons

995

mark conserved residues, and the periods stand for semi-conserved amino acids. In the PreS1, C4

996

has only two unique changes K(7)N and M(12)V (underlined) that were not found in any other

997

sequences tested in this study (Table 1). The third change I(84)L, which was found only in C4 and

998

not in other genotype C sequences, was also present in the variant B4 that facilitated production of

999

infectious HDV (Fig. 9). Two mutants of C4 were made, the C4m1 bearing a single reverse change

1000

V(12)M and the C4m2 harboring two reverse changes N(7)K+V(12)M (shown are at the bottom of

1001

the alignment). Panel B. Immunofluorescence analysis of PHH infected with either HDV-C4m1 (left

1002

image) or with HDV-C4m2 (right image). Infectivity of both mutants was assayed using in vitro

1003

infection of primary human hepatocytes (PHH). The infection procedure and immunofluorescence

1004

assay (at nine days post-infection) were performed as described in Materials and Methods. The red

1005

staining represents delta antigens, blue is a DAPI staining of nuclear DNA and alpha-tubulin is

1006

stained in green. Image on the right side displays staining for delta antigens not only in nuclei, but

45

1007

also in cytoplasm. The quantitative results of the infectivity tests (SI and NI values) were

1008

summarized in Fig. 9.

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

Virus entry mediated by hepatitis B virus envelope proteins.

The role of envelope proteins in hepatitis B virus assembly.

Hepatitis B e antigen and infectivity of hepatitis B virus.

Assembly of hepatitis delta virus particles.

The unexpected structures of hepatitis C virus envelope proteins.

Distribution of hepatitis delta virus genotypes in mashhad, northeast iran.

Autopresentation of hepatitis B virus envelope antigens by T cells.

Hepatitis delta virus genotype-1 alone cocirculates with hepatitis B virus genotypes A and D in Pakistan.

Small-form hepatitis B surface antigen is sufficient to help in the assembly of hepatitis delta virus-like particles.

Molecular interactions between hepatitis B virus and delta virus.

Genotypes and genetic variability of hepatitis B virus.

Distribution of hepatitis B virus genotypes in azerbaijani patients with chronic hepatitis B infection.

The hepatitis B virus.

Determinants of hepatitis B and delta virus host tropism.

Smouldering hepatitis B virus replication in patients with chronic liver disease and hepatitis delta virus superinfection.

The large form of hepatitis delta antigen is crucial for assembly of hepatitis delta virus.

Hepatitis delta virus: a peculiar virus.

Modification of the hepatitis B virus envelope protein glycosylation pattern interferes with secretion of viral particles, infectivity, and susceptibility to neutralizing antibodies.

Intrahepatic expression of pre-S1 and pre-S2 antigens in chronic hepatitis B virus infection in relation to hepatitis B virus replication and hepatitis delta virus superinfection.

Myristylation is involved in intracellular retention of hepatitis B virus envelope proteins.

Control of Hepatitis B Virus by Cytokines.

Interference of Apoptosis by Hepatitis B Virus.

Three envelope proteins of hepatitis B virus: large S, middle S, and major S proteins needed for the formation of Dane particles.