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Journal of Virology, April 2007, p. 3608-3617, Vol. 81, No. 7
0022-538X/07/$08.00+0     doi:10.1128/JVI.02277-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Assembly of Hepatitis Delta Virus: Particle Characterization, Including the Ability To Infect Primary Human Hepatocytes{triangledown}

Severin Gudima,1 Yiping He,1 Anja Meier,1 Jinhong Chang,1 Rongji Chen,1 Michal Jarnik,1 Emmanuelle Nicolas,1 Volker Bruss,2 and John Taylor1*

Fox Chase Cancer Center, Philadelphia, Pennsylvania,1 Department of Virology, University of Göttingen, Göttingen, Germany2

Received 17 October 2006/ Accepted 8 January 2007


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ABSTRACT
 
Efficient assembly of hepatitis delta virus (HDV) was achieved by cotransfection of Huh7 cells with two plasmids: one to provide expression of the large, middle, and small envelope proteins of hepatitis B virus (HBV), the natural helper of HDV, and another to initiate replication of the HDV RNA genome. HDV released into the media was assayed for HDV RNA and HBV envelope proteins and characterized by rate-zonal sedimentation, immunoaffinity purification, electron microscopy, and the ability to infect primary human hepatocytes. Among the novel findings were that (i) immunostaining for delta antigen 6 days after infection with 300 genome equivalents (GE) per cell showed only 1% of cells as infected, but this was increased to 16% when 5% polyethylene glycol was present during infection; (ii) uninfected cells did not differ from infected cells in terms of albumin accumulation or the presence of E-cadherin at cell junctions; and (iii) sensitive quantitative real-time PCR assays detected HDV replication even when the multiplicity of infection was 0.2 GE/cell. In the future, this HDV assembly and infection system can be further developed to better understand the mechanisms shared by HBV and HDV for attachment and entry into host cells.


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INTRODUCTION
 
Hepatitis B virus (HBV) is the natural helper virus of hepatitis delta virus (HDV). In an infection of the liver, some hepatocytes can be infected by both viruses so that HBV replication can provide the envelope (HBsAg) proteins for the assembly, release, and spread of virions containing the HDV RNA genome (6).

Consider first the HBV assembly process as diverted by HDV. The HBV and the other mammalian hepadnaviruses encode three HBsAg proteins. They have a common C terminus and are known by their sizes as large, middle and small (L, M, and S). Relative to S, the M protein has a unique N-terminal domain referred to as preS2. Similarly, relative to M, L has a domain called preS1. As reviewed elsewhere, L, M, and S undergo a complex series of specific posttranslational modifications and intermolecular associations, ultimately leading to the release of particles (15, 17). If the genome of HBV is replicating in such a cell, there can be formation of the viral nucleocapsids, some of which can be enveloped by HBsAg, leading to the release of infectious particles. Similarly, if the HDV genome is present, then in the additional presence of the delta ({delta}Ag) proteins, there can also be assembly and release of infectious HDV (33).

While M is necessary for neither assembly nor infectivity (15, 35), the stoichiometry of L to S is a critical variable in these assembly processes, whether for HBV or HDV (33). The L protein is essential for both the assembly and the infectivity of HBV and the infectivity of HDV (34), and yet in the absence of S, L is unable even to be released from the cell (39). For HDV but not for HBV, the S protein is sufficient for assembly, but the particles are noninfectious (34).

Superimposed on this complex pathway of infectious-particle assembly is a gross inefficiency. Typically, a 1,000- to 1,000,000-fold excess of empty particles that do not contain an HBV genome is produced (15). From electron microscopy, the empty particles are seen to be either roughly spherical particles of 25-nm diameters or filaments of about 22-nm diameters but of variable lengths. The infectious HBV particle is about 42 nm in diameter (12, 13). The HDV particles are considered to be somewhat smaller, about 36 nm (30, 34).

In an experimental in vitro situation, HDV assembly can be achieved by cotransfecting cells with two plasmids, one to provide HBsAg and another to initiate the replication of the HDV RNA genome (31). Such particles were subsequently demonstrated to be infectious in an experimental animal (29). Largely from the work of Sureau and colleagues, it has become clear that HDV particles share some features with HBV in terms of assembly and especially in terms of the ability to attach to and enter host cells (23, 33). Others have also begun to use this approach to obtain virus particles with HDV genomes and various forms of hepadnavirus envelope proteins (1, 2, 14).

The present study was undertaken to provide a detailed analysis of the requirements of HDV in vitro assembly and infectivity. This analysis included the following variables. (i) As mentioned above, HBV S is sufficient to achieve efficient assembly of HDV, but such particles are noninfectious, and it is only when L is also present that the particles are infectious (34). Therefore, we undertook to explore how varying the amount of L in the envelope would influence the abilities of the virus particles to initiate infection. (ii) Since HBsAg proteins are expressed only transiently, we assayed whether at later times after transfection there would be inefficient particle assembly. (iii) The replication of the HDV RNA strictly depends upon the presence of the HDV-encoded small delta protein ({delta}Ag-S) (24). During HDV replication, there occurs site-specific editing by adenosine deaminase acting on RNA (7), allowing the production of an altered HDV mRNA. This mRNA leads to the translation of the large delta protein ({delta}Ag-L), which does not support HDV replication but is essential for the envelopment by HBV-encoded proteins L, M, and S (9). The fraction of genomes that have undergone this necessary editing increases steadily with time after replication has been initiated (38). However, while some specific editing has to occur in order that {delta}Ag-L might be produced, the edited HDV RNAs are no longer able to initiate HDV replication by infection, since they cannot provide the essential {delta}Ag-S. Thus, RNA editing is both a positive and a negative factor in obtaining infectious HDV. In addition to this site-specific editing, we and others have shown that additional nucleotide changes occur, many of which can be accounted for as editing at other sites (8, 10, 20, 29). Therefore, we undertook to determine whether all these changes would progressively decrease the infectivities of the released particles.

This study addresses these and other variables affecting the assembly of infectious HDV. The findings are important, since balancing of variables is not unique to in vitro assembly, and it can be readily imagined that similar problems arise during viral assembly in the liver, whether co- or superinfected with HDV. In addition, an understanding of in vitro HDV assembly will allow us to modify the components of the HDV envelope and clarify the process by which HDV and HBV are able to attach to and infect susceptible cells.


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MATERIALS AND METHODS
 
Antibodies. For immunoblots and immunostaining, {delta}Ag was detected using a rabbit polyclonal antibody (31) and HBsAg by a commercial rabbit polyclonal antibody (Fitzgerald Industries). The latter was confirmed by immunoblot analysis to be largely specific for HBV S by a comparison with an antibody raised against purified S (a gift from Camille Sureau). For immunoaffinity chromatography, we used S-26, a mouse monoclonal to HBV preS2 epitope QDPRVR, corresponding to positions 132 to 137 on genotype A, serotype adw2 (a gift from Vadim Bichko) (32). For immunostaining, we also used fluorescein isothiocyanate-conjugated goat polyclonal antibody to human albumin (Antibodies Incorporated), a mouse monoclonal to human E-cadherin (EMD Biosciences), and fluorescently labeled secondary antibodies (Jackson Immunoresearch Laboratories). For immunoblots, secondary antibodies labeled with infrared dyes were obtained from LI-COR.

Plasmids. The plasmids used in this study have been described previously: pSVL(D3) initiates HDV genome replication (11). pSVBX24H and pSVB45H express the HBV genotype A, serotype adw2 small envelope and all three envelope proteins, respectively (5). pCMV(LM–S–) expresses only the L protein (a gift from T. C. Benedict Yen).

Transfection. Virus assembly was performed with the human liver cell line Huh7 (28). All transfections with plasmid DNA combinations were performed using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. A typical combination for the transfection of a 10-cm dish of Huh7 cells was 2.5 µg pSVL(D3) and 10 µg of an HBV envelope-expressing plasmid.

Primary human hepatocytes and infection. Primary human hepatocytes, plated as confluent monolayers on rat tail collagen, were obtained commercially (Admet, Cambrex, and CellzDirect), typically in a 48-well configuration with 200,000 hepatocytes per well. They were maintained in Hepatostim medium supplemented with 0.01 µg/ml epidermal growth factor, receptor grade, both from BD Biosciences. Most infections were performed in the presence of 5% polyethylene glycol 8000 (PEG) (Sigma), with the media being changed after 6 h.

Virus. HDV virus-like particles were harvested from the media containing transfected Huh7 cells and immediately clarified by centrifugation (10 krpm, 30 min, 4°C in the HB-4 rotor of a Sorvall RC-5B). In some cases, this medium was stored at –80°C. Particles present in clarified media were precipitated by the addition of PEG to 10%, with stirring for 2 h at 4°C, followed by centrifugation (10 krpm, 30 min, 4°C). The pellet was resuspended in prechilled TAN buffer (50 mM Tris-HCl [pH 8.0]-100 mM NaCl), using 1/100 of the original volume, after which aliquots were stored at –80°C.

HBV was produced using the stable cell line HepAD38 (25), kindly provided by Christoph Seeger. The particles released between days 4 and 7 after induction by tetracycline removal were concentrated as described above. Woodchuck hepatitis virus (WHV) was obtained from sera of chronically infected woodchucks, kindly provided by William Mason.

Immunoaffinity purification of virus-like particles. The preS2-specific mouse monoclonal antibody S-26 was first complexed for 16 h at 4°C with protein A-agarose beads (Sigma). This was then incubated for 16 h at 4°C with PEG-concentrated virus and then washed four times with cold NT2 buffer (50 mM Tris-HCl [pH 7.5]-150 mM NaCl-1 mM MgCl2). The virus was competitively eluted by adding to the buffer 400 µg/ml of the peptide LQDPRVRG. This elution step was repeated four times. For samples to be examined by electron microscopy, the elutions were performed with only 10 µl. Throughout the purification, aliquots of 2 µl were removed for RNA extraction and the monitoring of recoveries by quantitative real-time PCR (qPCR).

Immunoblots. Protein samples were resuspended in Laemmli buffer containing 5% dithiothreitol and heated for 10 min at 95°C prior to electrophoresis on precast 12% polyacrylamide gels (Duramide; Cambrex). As size markers, we used Rainbow Markers (Amersham) and MagicMark (Invitrogen). After electrotransfer to nitrocellulose membranes, proteins were detected with specific antibodies followed by secondary antibodies conjugated with infrared fluorescent dyes (LI-COR). Detection and quantitation were performed with an Odyssey apparatus (LI-COR). Unlike enzyme-based chemiluminescence, these dye-based assays give a linear response over a >4,000-fold range (LI-COR and data not shown).

ELISA. HBV surface antigen (HBsAg) was detected using an enzyme-linked immunosorbent assay (ELISA) kit, ET-1-MAK-2 Plus, according to the manufacturer's instructions (DiaSorin).

RNA extraction, Northern analyses, and real-time PCR. All extractions used TRI reagent (Molecular Research Center) according to the manufacturer's instructions. RNA concentrations were measured using an ND-1000 spectrophotometer (Nanodrop).

Samples for Northern analyses were initially glyoxalated prior to electrophoresis on gels of 1.5% agarose. Electrotransfer and hybridization with 32P-labeled RNA probes were as previously described (24). Radiation was detected and quantitated with a bio-imager (Fujifilm BAS-2500) and ImageQuant software, respectively.

Prior to qPCR assays, the RNA samples were subjected to digestion with RQ1 RNase-free DNase (Sigma) and then reextracted with TRI reagent. This was done to remove HDV-specific plasmid DNA sequences carried over from the original transfection of Huh7 cells. For HDV qPCR assays, we used the following primers, with their locations indicated using the HDV genome positions reported by Kuo et al. (24): forward primer, 312-GGACCCCTTCAGCGAACA-329; and reverse primer, 393-CCTAGCATCTCCTCCTATCGCTAT-360. The TaqMan probe was 332-AGGCGCTTCGAGCGGTAGGAGTAAGA-357. The assays were normalized relative to a series of 10-fold dilutions of a genomic HDV RNA standard that had been transcribed in vitro and then gel purified. We deduce that 1 pg HDV RNA standard is equal to 1 million molecules. For cell RNA samples, we assume 25 pg RNA per one cell (20) and thus deduce the number of HDV genome equivalents (GE) per average cell. The qPCR assays for HBV and WHV follow the reports of others (16, 27).

Immunostaining. Immunostaining was generally as described previously (21), with the following minor modifications. Cells on collagen in 48-well plates were fixed with 4% paraformaldehyde for 15 to 30 min at room temperature, washed twice, and then permeabilized with N-octyl-glucopyranoside (EMD Biosciences). For detection of delta antigens, rabbit polyclonal antibody (1:1,000 dilution) was used. E-cadherin and human albumin were analyzed with various dilutions of the above-mentioned commercial antibodies. DNA was stained with 1 µg/ml DAPI (4',6'-diamidino-2-phenylindole) (Sigma). Prepared samples were analyzed using an inverted Nikon TE2000-U microscope with 40x or 20x objectives and specific filter blocks, equipped with a Cascade 650 monochrome camera (Photometrics), and utilizing MetaVue software (Universal Imaging). Images were further processed using Canvas 9.0 and Photoshop 7.0 software.

Electron microscopy. For electron microscopy studies, HDV was harvested from transfected cells between days 7 and 10, concentrated using PEG, and then purified by immunoaffinity chromatography as described above. Negative staining for transmission electron microscopy was performed as described previously (22). In brief, the viral suspension was adsorbed on a freshly glow-discharged collodion/carbon-coated electron microscopical grid for 4 min. The grid was briefly washed with phosphate-buffered saline (PBS) and the attached particles fixed with 2% glutaraldehyde in PBS for 2 min. After three PBS and six double-distilled-water washes, the grid was stained with 2% aqueous solution of uranyl acetate and air dried. The specimens were examined using a FEI Tecnai 12 electron microscope under 80-kV acceleration. The images were recorded on an AMT 2kx2k digital camera.

Rate-zonal sedimentation. Several sources of virus were used for Fig. 3. For Fig. 3A and B, the HDV was from media harvested between days 6 to 9 and 6 to 12, respectively, after transfection with pSVL(D3) and pSVB45H. For Fig. 3C, the harvest was between days 7 and 10 after transfection with pSVL(D3) and pSVBX24H. For Fig. 3D, the harvest was between days 0 and 6 after transfection with pSVB45H. The PEG-concentrated virus was sonicated and treated in the presence of 5 mM magnesium acetate-5 mM Tris-HCl (pH 8.0) with 0.1 mg/ml DNase I for 1 h at 37°C, after which EDTA was added to a 5 mM final concentration. The samples were dialyzed against STE (150 mM NaCl-10 mM Tris-HCl [pH 7.5]-1 mM EDTA) to remove residual PEG and then clarified by brief centrifugation prior to being layered onto a 10 to 30% (wt/wt) sucrose gradient in STE buffer. Centrifugation was performed as indicated in the text, using a Beckman SW41 rotor at 4°C. Fractions were collected from above and then monitored for refractive index. Aliquots were extracted with TRI reagent for RNA used in a subsequent qPCR assay for HDV RNA. Other aliquots were first digested with Pronase and sodium dodecyl sulfate and then extracted with TRI reagent to obtain the DNA used in qPCR assays for HBV and WHV DNA.


Figure 3
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  		FIG. 3. Rate-zonal sedimentation of assembled HDV. For panel A, a mixture of HDV, HBV, and WHV particles was subjected to sedimentation (Beckman SW41 rotor, 40 krpm, 2 h at 4°C) on a gradient of 10 to 30% sucrose. Fractions were collected and the viral nucleic acids quantitated with three specific qPCR assays. For panels B to D, the sedimentation time was increased to 4 h and the fractions were assayed for HBsAg by ELISA. For panel B, the HDV was assembled as for panel A. For panel C, the HDV RNA was assembled using only HBsAg S. For panel D, the subviral particles were assembled with all HBsAg proteins, but HDV was absent. The assay results, in arbitrary units, are indicated as follows: HDV RNA (shaded circles), WHV DNA (shaded diamonds), HBV DNA (open squares), and HBsAg (shaded squares).


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RESULTS
 
Time course of assembly. In order to achieve assembly of HDV particles, Huh7 cells were transfected with two plasmids. One expressed HBsAg. The other expressed a trimer of HDV genomic RNA and was sufficient to initiate the replication of the HDV genome, producing {delta}Ag-S and {delta}Ag-L. Figure 1A to C shows the presence of intracellular HBsAg, {delta}Ag, and HDV genomic RNA, respectively, at various times after transfection.


Figure 1
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  		FIG. 1. Intracellular accumulation of HBsAg and HDV proteins and RNA as a function of time after transfection of Huh7 cells. As described in the text, the transfection was performed with one plasmid to express the HBsAg and a second to initiate the replication of HDV. Panel A shows the accumulation of HBsAg, as detected by immunoblot analysis. Panel B shows a similar analysis for {delta}Ag. Panel C shows an analysis by qPCR to deduce the number of HDV RNA genomes (GE) per average cell. The immunoblot data in panels A and B were evaluated to deduce the indicated relative amounts of the viral proteins. MW, molecular mass.

Figure 1A shows the detection by immunoblot of two forms of L and of S, consistent with the effects of N-linked glycosylation within the S domain (33). The M species were not detected relative to L and S. The accumulation of L and S decreased with time, consistent with this being a transient transfection. From quantitation, as indicated, the percentage of L relative to that of L plus S remained constant with time.

The immunoblot in Fig. 1B shows the intracellular accumulation up to day 15 for {delta}Ag-S and {delta}Ag-L. Note that the amount of {delta}Ag-L (essential for assembly of particles) was initially undetectable and then progressively increased relative to that of {delta}Ag-S (essential for the replication of the HDV genome).

The qPCR data in Fig. 1C show that most of the accumulation of HDV genomic RNA was achieved by about 6 days after transfection.

Using the media from the same transfected cultures, we assayed for the presence of particles containing the HBsAg and HDV RNA genomic RNA. These data, shown in Fig. 2A and B, refer to particles released in a series of 3-day harvests. Figure 2A shows that the release of HBsAg was highest in the harvest taken at 3 days and then progressively decreased. Figure 2B shows that the release of HDV RNA containing particles was undetectable at 3 days, increased at 6, 9, and 12 days, and then decreased by day 15. It should also be noted that the percentage of L relative to that of L plus S for particles was less than the intracellular values (Fig. 1A) and that it decreased with time of harvest.


Figure 2
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  		FIG. 2. Release of HBsAg and HDV RNA as a function of time after transfection of Huh7 cells. Transfection was performed as described for Fig. 1. Panel A shows an immunoblot analysis of the HBsAg released during 3-day intervals at the times indicated. Panel B shows quantitation by qPCR of HDV GE/ml of medium. MW, molecular mass.

A plausible interpretation of these data is as follows. At 3 days, the largest amount of the HBsAg protein was translated and released into the media. However, insignificant amounts of HDV GE were assembled at this time. At 6 days, there was no further accumulation of HBsAg, but the amount of released GE increased and was maximal at day 12. This is consistent with an increase in the amount of edited HDV RNA, as evidenced by the production of more {delta}Ag-L, a component essential for the assembly of HDV RNA-containing particles.

From the data in Fig. 1 and 2, we deduce that in the period of days 9 to 12, when HDV release was maximal, 1,200 GE were released per average cell. (This corresponds to the release of one RNA-containing particle per cell per 4 min.) In addition, since the average cell contained 12,000 GE, we deduce that 10% of the accumulated HDV genomic RNA was assembled and released in 3 days.

Rate-zonal sedimentation analysis. As an initial step toward characterizing the released particles, we used rate-zonal sedimentation. As shown in Fig. 3A, we observed a narrow distribution of HDV RNA-containing particles centered in fraction 9, which we interpret as evidence of significant size homogeneity. Furthermore, as internal standards for this gradient, we used both HBV and WHV, in each case measuring the location of the DNA-containing particles. Both sources of hepadnaviruses sedimented to fraction 14. The sedimentation value of the HDV particles was thus about 67% of that for the HBV and WHV particles. This result is similar to an earlier report by Rizzetto et al. (30), although in that study, the HDV was in vivo assembled and was detected by {delta}Ag rather than by RNA, and the HBV was detected by core antigen rather than by DNA.

We next doubled the sedimentation time in order to assay the location of the HDV RNA relative to that of HBsAg, as detected by ELISA. As shown in Fig. 3B, for the majority of HBsAg, the sedimentation was only 50% relative to that of the HDV RNA, consistent with most of the HBsAg being in particles substantially smaller than the RNA-containing particles. Some HBsAg was in species that sedimented as far as the HDV RNA, although there was no discrete peak associated with the RNA.

In order to better understand the sedimentation pattern, we examined two additional sources of assembled particles. Figure 3C shows the sedimentation of particles assembled using only the S protein of HBV together with HDV. This was done with the expectation that in the absence of L, there might be relatively less production of filamentous structures, known to be heterogenous in length (18), all the while realizing that the HDV RNA would be assembled into noninfectious particles (34). The absence of L apparently had no significant effect on the sizes of the RNA-containing particles, since they had a sedimentation value similar to those in Fig. 3B. Again, most of the HBsAg sedimented more slowly than this. As expected, there was less HBsAg sedimenting as far as the RNA-containing particles, and yet, there was still no separate peak for HBsAg associated with that for the HDV RNA.

The second additional source of particles was assembled as for Fig. 3B but in the absence of HDV. As shown in Fig. 3D, the distribution of HBsAg was virtually identical to that in Fig. 3B. Therefore, we must interpret that even though we can resolve HDV RNA-containing particles as a band with a quite homogeneous sedimentation value (Fig. 3B and C), there is at the same mobility an excess of subviral HBV particles lacking HDV RNA. That is, there is produced an excess of particles with the same sedimentation value as HDV but lacking HDV RNA.

Immunoaffinity purification. Since the HBV L protein and its unique preS domain are essential in infectious HDV particles, we reasoned that by immunoaffinity selection, we might determine the fraction of the assembled particles that not only contain preS domains but also expose them on their surface. To do this, we used S-26, a well-characterized mouse monoclonal antibody that recognizes a linear epitope in the preS2 domain (32). This antibody was first coupled to protein A-agarose and then incubated with virus coated with HBV L, M, and S protein (LMS virus). Bound virus was specifically eluted with the peptide corresponding to the known epitope. A quantitation of the binding and elution of the HDV RNA-containing particles was made by qPCR. As a negative control, a parallel study was performed with HDV RNA-containing particles coated only with S (S virus).

From the results summarized in Table 1, we found that 77% (100 – 23) of the LMS-coated RNA-containing particles was bound. Furthermore, 50% was subsequently eluted from the S-26 antibody, in contrast to 0.1% of the S particles. We thus deduce that the binding and elution achieved a 500-fold purification of LMS relative to that of S particles. We also conclude that most of the RNA-containing LMS virus contains at least some preS2 exposed on the surface. However, such studies cannot distinguish whether all or just some of the preS2 is exposed. This qualification is relevant in that others have claimed that only a fraction of the preS1 and preS2 domains of HBV particles have been translocated to the outside of the virion (26).


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  		TABLE 1. Immuno-affinity purification of HDV RNA-containing particlesa

Electron microscopy evaluation. The above-described immunoaffinity procedure was performed at a preparative level, to provide sufficient purified and concentrated virus to allow examination by electron microscopy, with the results as presented in Fig. 4A. Three main particle forms were detected: filamentous structures of relatively constant diameters but variable lengths, along with roughly spherical particles that fell into two main size classes (Fig. 4B). A quantitation of the particle measurements is given in Table 2.


Figure 4
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  		FIG. 4. Electron microscope examination of affinity-purified particles. These were purified as described for Table 1. Panel A shows an example of the eluted particles as examined by electron microscopy, with magnification as indicated. Many such fields were examined, the particles quantitated, and their dimensions measured. Table 2 provides a summary of the quantitation. Panel B shows a histogram of these dimensions for only the spherical particles.


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  		TABLE 2. Dimensions of particles purified by immunoaffinity chromatography

In a previous study of in vivo-assembled HDV (30), the HDV RNA-containing particles were roughly spherical particles of 36-nm diameters. In Table 2, it can be seen that 29% of our in vitro-assembled particles fit this description, with a mean diameter of 36 ± 4 nm. In a separate purification and electron microscope study, only 14% of particles fit this description (data not shown). Nevertheless, as considered further in Discussion, there remain important caveats.

Ability to infect primary human hepatocytes. We then asked whether the particles assembled using L, M, and S were able to initiate infection of primary human hepatocytes. Infection was at a multiplicity of 300 GE/cell. After 6 days, total cell RNA was extracted and assayed for HDV genomic RNA both by Northern analyses and by qPCR. From the results summarized in Table 3, it can be seen that genomic RNA was detected in infected hepatocytes and the amount was more than that in the initial inoculum.


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  		TABLE 3. HDV replication following infection of primary human hepatocytes with LMS- and S-coated particles

Following the experience of others (1), we also tested infection in the presence of PEG. As shown, 5% PEG provided a major increase (typically 10- to 30-fold) in the amount of HDV RNA accumulated.

As a negative control for these studies, we used S virus. Such particles, when applied at the same multiplicity of 300 GE/cell, were unable to give detectable replication, with or without PEG.

It should be noted that quantitation by Northern analyses gave results lower than those obtained by qPCR. We speculate that this is because the Northern blot assays for full-length HDV RNAs, while the qPCR also detects species that are less than full-length. The qPCR was much more sensitive and could detect HDV RNAs even after cells were exposed to S particles. However, the amounts detected were typically 500 times less than those obtained with LMS particles. Also, the amounts detected were typically 5 times less than the amounts of virus to which the cells were initially exposed. As others have suggested, this detected RNA may thus be a residual of the inoculum (1).

We also used immunostaining to characterize the HDV infection. Figures 5A and B show detection of {delta}Ag in cells at 6 days after infection in the absence and presence, respectively, of 5% PEG. We observed that PEG increased the fraction of positive cells from 1 to 16%. Also shown in Fig. 5A and B is staining for albumin, considered a marker for mature, differentiated hepatocytes. Virtually all cells, as detected by DAPI staining of chromosomal DNA, also stained for albumin. It is thus not obvious why, with a multiplicity of 300 GE/cell and with the enhancement afforded by PEG, there was still infection of only 16% of cells.


Figure 5
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  		FIG. 5. Immunocytochemistry of primary human hepatocytes at 6 days after infection with assembled HDV. In panels A and B, the infections were initiated at a multiplicity of 300 GE/cell, in the absence or presence of 5% PEG. The images detect {delta}Ag (red), albumin (green), and DAPI staining (blue). For panels C and D, the infection was initiated as for panel B, and the images were collected at higher magnifications, with {delta}Ag (green), E-cadherin (red), and DAPI staining (blue). Images A and B were taken with a 20x objective, while C and D were taken with a 40x objective. All images were collected with a charge-coupled-device camera and superimposed using MetaVue software.

Immunostaining was also used to examine the infected cells at higher magnifications. In Fig. 5C and D, we detected not only {delta}Ag but also intercellular-junction protein E-cadherin. It can be seen that there are intercellular junctions in both infected and noninfected cells. Thus, neither the presence of intercellular junctions nor the expression of albumin provided us with insights as to why only a fraction of the hepatocytes were infected.

Two patterns of {delta}Ag staining were observed. The majority was of a general pattern of staining including both nucleus and cytoplasm, with some concentration within regions of the cytoplasm (Fig. 5C). Rarely did we detect a distribution that was predominantly nuclear (Fig. 5D).

Next, we examined the effect of multiplicity of infection. As shown in Fig. 6A, as the multiplicity was increased, a saturation was achieved, although with multiplicities higher than 500 GE/cell, we observed a small decrease in the level of HDV RNA accumulation. Conversely, as the input multiplicity was reduced, so also was the accumulation. Therefore, from this and other experiments, we consider that for multiplicities ranging from 300 down to 0.7 GE/cell, the observed amount of HDV RNA accumulation was directly proportional to the input (data not shown).


Figure 6
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  		FIG. 6. Effect of the time and multiplicity of infection on accumulation of intracellular genomic HDV RNA. For panel A, cells were infected at a multiplicity of 300 GE per cell and time points were taken out to 15 days. For panel B, cells were infected with a series of multiplicities, with harvesting at day 6. All infections were done in the presence of 5% PEG. Total RNA was extracted and HDV GE quantitated by qPCR. Panel C shows the specific infectivities of HDV released at various times after transfection of Huh7 cells. Specific infectivity is defined as the number of HDV GE detected per average infected cell divided by the input multiplicity of GE per average cell. The HDV used for the infections was harvested during 3-day periods, as indicated, and the amount of GE used as the inoculum was within the linear range, as determined in panel A.

The sensitivity of the HDV qPCR assay was so high that in some experiments following infection at an input multiplicity of about 0.2 GE/cell, we subsequently detected an accumulation that was 50 times greater than that in the inoculum (data not shown). Now, the earlier rate-zonal studies showed that the majority of the assembled particles were of homogenous sedimentation (Fig. 3A to C), consistent with the particles being mono-disperse. Similarly, with immunostaining, we observed that PEG during infection increased the fraction of {delta}Ag positive cells and not the accumulation per cell (Fig. 5A and B). This also supported an interpretation of mono-disperse virus.

Results for a time course experiment for HDV RNA accumulation are shown in Fig. 6B. Using infection at a multiplicity of 300 GE/cell and in the presence of 5% PEG, the maximum accumulation was reached within 6 days. At later times, the accumulation per cell dropped significantly. This could have been due to the progressive decline in the viability of the primary hepatocytes and/or the accumulation of changed HDV RNA genomes that are no longer replication competent.

Specific infectivities of assembled particles. As mentioned in the introduction, one of our aims was to determine whether as a consequence of nucleotide changes accumulated during HDV genome replication, especially changes due to RNA editing, there might be a reduction in the specific infectivities of HDV RNA genomes assembled late rather than early. Certainly, as shown in Fig. 1B, the detection of large amounts of {delta}Ag indicated that the fraction of edited RNA genomes was increasing with time.

Therefore, for harvests taken at a series of times after transfection, we measured specific infectivity, here defined as the ratio of HDV RNA accumulated per average cell as a consequence of infection divided by the input multiplicity of RNA genomes. The results are summarized in Fig. 6C. As an example, for the virus harvested from days 6 to 9, the specific infectivity was greater than 500. That is, the ability to infect primary hepatocytes was such that at 6 days after infection, the number of HDV RNA genomes produced by HDV replication was 500 times greater than that in the inoculum applied to infect these cells.

The data show that within the experimental errors, there was no drop in specific infectivity for later harvests relative to what was found for earlier harvests. That is, any drop was less than threefold. In other experiments, with harvests out to 21 days, the decreases in specific infectivity relative to what was found for earlier harvests were again no more than threefold (data not shown). We conclude that under these experimental conditions, we did not detect a time-dependent decrease in specific infectivity.

Effect of HBV L protein on assembly and infectivity. The above-mentioned studies confirm the results of others indicating that HBV S is sufficient for HDV assembly but not for infectivity, which depends upon the presence of HBV L (34). HBV assembly differs in that L and S are needed for both assembly and infectivity (17). Furthermore, expression of L alone is known to be cytotoxic (39). The following experiment was undertaken to determine the role of the relative amounts of L and S on both HDV assembly and infectivity. In this variation of the assembly procedure, we used separate plasmids to express L and S. A series of different ratios of these were cotransfected along with the plasmid to initiate HDV replication. The media were harvested from days 6 to 9, at which time the cells were also extracted. The media were then assayed for HDV RNA titer and tested for the ability to initiate infection in primary human hepatocytes. Thus, we were able to deduce the specific infectivity (as in Fig. 6C) for each sample.

The results are summarized in Table 4. Note that for each transfection, we determined by immunoblot analysis the percentage of L relative to that of L plus S in the transfected cells. The values ranged from 0 to 100%. Consider first the assembly efficiency. As the percentage of L was increased from zero, there was a threefold reduction in the assembly efficiency. Further increases in the percentage of L progressively led to more substantial reductions in assembly. At 100% L, no assembly could be detected, even though there was no obvious cell toxicity.


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  		TABLE 4. Requirement of HBV L in HDV assembly and infectivity

For the first four samples shown in Table 4, the assembly was sufficient to allow a determination by immunoblot analysis of the percentage of L relative to that of L plus S in the released particles. Note that increases from 0 to 57% were observed as the percentage of L in the cell was increased from 0 to 72%.

Aliquots of the media were also tested for their abilities to infect primary human hepatocytes. Such data are shown in Table 4, but what are also shown, and are more informative, are the specific infectivities (as in Fig. 6C). For the particles with 0% L, the specific infectivity was much less than 1, consistent with the particles being noninfectious. However, for all the other samples, as long as there was enough virus to detect, there was the ability to subsequently detect a genuine specific infectivity.

In summary, increasing the percentage of L in the transfected cells caused significant inhibition of particle release, but for those particles released, the specific infectivity was still significant. As long as some particles were released and they contained at least 7% L, they were infectious. Increasing the percentage of L gave no more than a 2.5-fold increase in the specific infectivity.


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DISCUSSION
 
We have described here the successful assembly of HDV RNA-containing particles from transfected Huh7 cells, along with evidence for their infectivity in susceptible primary human hepatocytes. While some aspects of our study confirm previous studies (1, 14, 34-37), we also provide significant extensions, along with novel findings, as discussed below.

(i) A detailed characterization of the assembly process was made. In the transfected cells, the S protein was most abundant, with somewhat lower levels of L and no detectable level of M (Fig. 1A). In contrast, the particles released into the medium had a 2-fold-lower relative amount of L (Fig. 2A). This was true at all harvest times, either early, at 0 to 3 days, when HDV RNA was not assembled, or at later times, 6 to 15 days, when the HDV was assembled. To specifically test the hypothesis that this reduction in the amount of assembled L was consistent with L protein interfering with the assembly mechanism, we considered assembly under conditions where we could control the L/S ratio within cells. We thus observed that as the amount of L relative to that of S was increased, the ability to achieve assembly of RNA-containing particles was progressively reduced to background levels (Table 4).

(ii) We showed that the released particles could be affinity purified using a monoclonal antibody to the preS2 domain. In a preparative analysis, 77% of the HDV RNA-containing particles bound (Table 1). For a separate analytical analysis, the binding was >90% (data not shown). Thus, the majority of the virus particles contained sufficient molecules of preS region exposed on the surface to interact with the antibody. Both the unbound virus and that which was eluted were infectious, but the specific infectivity of the unbound virus was 6.5 times less than that of the eluted virus (data not shown). We consider that many possible factors could contribute to the lower specific infectivity of the <10% of unbound virus.

(iii) The ability to carry out preparative affinity purification allowed us to examine the HDV particles by electron microscopy. We observed an abundance of the small spheres and filaments that are typically associated with natural HBV infections and considered to be empty particles (15). In addition, we were also able to detect larger spheres with a mean diameter of 36 nm. In an earlier study using serum from an infected chimpanzee, Rizzetto and coworkers detected a similar particle that had the same sedimentation value as particles containing the delta antigen (3, 4, 30). However, they noted that for some animals infected with HBV but not HDV, they could detect similar particles (30).

(iv) Like Rizzetto and coworkers (3, 4, 30), we also used rate-zonal sedimentation and were able to detect a specific sedimentation behavior for HDV (Fig. 3A). Our study has an advantage in that we could assay the RNA genome and not just {delta}Ag. Also, as an internal control, we were able to assay both HBV and WHV, thus demonstrating that they both have significantly larger sedimentation values (Fig. 3A). We also assayed for HBsAg particles and found that these were heterogenous with the majority, having a sedimentation value less than that of HDV (Fig. 3B). A minor fraction had a higher sedimentation value, overlapping with that of the HDV RNA-containing particles. And when particles were assembled using HBsAg but in the absence of HDV, we again detected particles in this region (Fig. 3D). Thus, like Bonino and coworkers for in vivo-assembled HDV (3), we conclude that in vitro-assembled HDV particles may have a discrete sedimentation value but that HBsAg particles of the same size that do not contain HDV RNA can be produced. Thus, the 36-nm particles detected by electron microscopy can be a mix of particles with and without the HDV RNA. For many other viruses, including HBV, the full and empty particles can be separated via differences in density; however, for HDV, where the genome is single stranded and significantly smaller than that of any other animal virus, there is not a sufficient density difference.

(v) As found for certain other studies (1, 14), we observed that HDV assembled in vitro could infect susceptible cells and that the extent of infection as assayed by the detection of HDV RNA could be significantly enhanced by the presence of 4 to 5% PEG (Table 3). However, we also used immunostaining to detect {delta}Ag and were able to show that PEG increased the fraction of hepatocytes infected from 1 to 16% (Fig. 5A and B). Thus, PEG increased the accumulation of progeny HDV RNA largely by increasing the fraction of cells infected rather than increasing the yield per infected cell.

(vi) These results raise the question of why, even with 5% PEG and a multiplicity of 300 GE/cell, we observed only 16% infection (Fig. 5B). That is, to what extent was the limited infection a consequence of the virus versus a consequence of the susceptibility of the cells? We found that the majority of primary hepatocytes, both infected and uninfected, seemed homogeneous, as judged by immunostaining for liver protein albumin (Fig. 5A and B) or by assaying for the cell-cell junction protein E-cadherin (Fig. 5A to D).

The alternative to faulting the hepatocytes for the limited extent of infection was to consider the infectivity of assembled virus. (i) We tested the specific infectivities of virus harvested during 3-day periods at a series of times after transfection of Huh7 cells. No significant differences (greater than threefold) were detected (Fig. 6C). (ii) To evaluate the effect of shorter harvest times on virus release and virus infectivity, we reduced the harvest time to 18 h and even 6 h. We observed that the amount of GE accumulated per unit time was unchanged and that the specific infectivities for these harvests were not significantly different (data not shown). Such data support the interpretation that virus released into medium was not significantly inactivated as the consequence of a 3-day harvest period. (iii) For most of our studies, virus was assembled in vitro from transfected Huh7 cells, and yet virus assembled in vitro from COS7, a line of monkey kidney cells that had a comparable specific infectivity (data not shown). (iv) We also tested virus assembled in vivo from HDV replicating in WHV-infected woodchucks. Several independent sources were tested and found to infect both primary human hepatocytes and primary woodchuck hepatocytes (kindly provided by William Mason), and yet the specific infectivities were not in excess of those obtained for the in vitro-assembled HDV (data not shown). It may be relevant to note certain data from Glebe and coworkers (19). They used HBV from infected patients to infect tupaia primary hepatocytes and observed, as we did, that with even a multiplicity of 10,000 particles per cell, only 20% of cells could be infected.

In summary, we show clearly that not all hepatocytes could be infected, but we have not been able to determine to what extent this was due to limitations of the virus and/or of the cultured hepatocytes.

(vii) It is clear from this study and earlier studies (34) that in vitro-assembled HDV particles are infectious only if the L protein of HBV is also present, and yet it has been recognized that the minimum amount required to confer infectivity is unknown (33). In unique experiments, we reduced the fraction of available L relative to that of L plus S and measured both the particle release and the corresponding specific infectivity. Reducing this from >57% to 7% increased the particle release but did not decrease the specific infectivity (Table 4). Recent structural studies indicate that an infectious HBV particle contains about 240 molecules of L plus S (13, 18), while a 25-nm empty particle contains 48 (18). The value for the HDV particle must be somewhere in between, so 7% L corresponds to somewhere between 3 and 17 molecules per average particle. Thus, we assert that this number, even though ascribed per average particle, is sufficient for maximal specific infectivity of HDV on susceptible cells. As experimental evidence for this, we reduced the fraction of L a further threefold, and the specific infectivity decreased to a value not distinguishable from that of noninfectious S-only particles (data not shown).

We further predict that at least 17 molecules of L per average particle are needed for maximal HBV infectivity. This demonstrates a unique advantage for our system since a comparable experiment for HBV would be very difficult to achieve since regions within preS1 are needed not only for infectivity but also for nucleocapsid assembly.

In summary, the studies described here provide novel information about the HDV particles that can be assembled in vitro, including their abilities to infect susceptible cells. We consider this experimental system to have many future applications. Some will be linked to HDV, for purposes such as to better determine the requirements for assembly of genomic rather than antigenomic RNA. However, important contributions will come from exploiting the unique advantage of this HDV system for addressing unsolved questions relating to the attachment and entry of both HDV and HBV.


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ACKNOWLEDGMENTS
 
J.M.T. was supported by grants AI-058269 and CA-06927 from the NIH and by an appropriation from the Commonwealth of Pennsylvania.

Glenn Rall, Richard Katz, and William Mason gave valuable comments on the manuscript. Thanks go to Vadim Bichko, Irina Shchaveleva, T. C. Benedict Yen, Camille Sureau, Chi Tarn, Donald Ganem, and Hans Netter for essential materials and/or encouragement. The fluorescence imaging studies were carried out in the Fox Chase Imaging Facility. The electron microscopy was aided by the Fox Chase Electron Microscopy Facility. The qPCR was performed in the Biochemistry and Biotechnology Facility.


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FOOTNOTES
 
* Corresponding author. Mailing address: Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111-2497. Phone: (215) 728-2436. Fax: (215) 728-3105. E-mail: john.taylor{at}fccc.edu. Back

{triangledown} Published ahead of print on 17 January 2007. Back


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Journal of Virology, April 2007, p. 3608-3617, Vol. 81, No. 7
0022-538X/07/$08.00+0     doi:10.1128/JVI.02277-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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