Abstract
A novel hepatitis B virus surface antigen mutant harboring a deletion of most of the major antigenic loop region was competent for self-assembly and secretion. Although the mutant protein was competent for interaction with and incorporation of free large hepatitis delta antigen, it was partially defective in hepatitis delta virus RNP incorporation.
Hepatitis delta virus (HDV) is an RNA agent which exists as a subviral satellite of hepatitis B virus (HBV) (9). HDV's 1,679-nucleotide genome contains a single open reading frame and encodes two proteins, the small and large delta antigens (HDAg-S and HDAg-L) (13). HDAg-S is expressed throughout infection and is required for HDV RNA replication. HDAg-L expression occurs later during infection, following a host enzyme-mediated RNA editing event which abolishes the HDAg-S stop codon, thereby extending the HDAg open reading frame by 19 codons. HDAg-L is a farnesylated protein which potently inhibits HDV RNA replication (11) and is required for the assembly of HDV virions (2).
HDV's envelope is provided by HBV, which encodes three envelope proteins (or surface antigens), HBsAg-S, -M, and -L. Expression of HBsAg-M and -L, which are amino-terminal extensions of HBsAg-S, is driven by promoters located upstream of -S. HBsAg-S is a hemiglycosylated protein which self-assembles in the endoplasmic reticulum (ER) membrane even in the absence of additional viral proteins or nucleic acid (4). Self-assembly of HBsAg-S is highly efficient, and noninfectious or “empty” HBsAg-S particles compose the vast majority of secreted viral particles during clinical infection (4). Coexpression of HDAg-L and HBsAg-S in tissue culture results in incorporation of HDAg-L into these empty particles (12). Assembly of infectious HBV virions has been shown to depend upon two different topologies assumed by HBsAg-L. Disposal of the pre-HBsAg-S region to the cytoplasm facilitates interaction with HBV core particles, while disposal to the ER lumen places the pre-HBsAg-S region on the particle's surface, enabling interaction with the host receptor (1, 7). In contrast to HBV assembly, HDV assembly does not require HBsAg-L, though HBsAg-L remains a requirement for HDV infectivity, and therefore both viruses are thought to use a common receptor on host hepatocytes (7, 10).
Previous studies have shown that sequences in the cytoplasmic domain of HBsAg-S, located between residues 28 and 80, as well as sequences in the carboxyl-terminal region of HBsAg-S, are important for HDV virion assembly (5, 6). Here, we address the role of residues 107 to 147 of the antigenic loop region in HDV assembly. Though this region is predicted to reside in the ER lumen and therefore on the outside of assembled virions, one can speculate that the hemiglycosylated phenotype of HBsAg-S might reflect an alternate membrane topology in which the antigenic loop is disposed to the cytoplasmic rather than the lumenal face of the ER membrane in some subunits. It is also notable that in contrast to HBV and woodchuck hepatitis virus (WHV), duck HBV (DHBV) is unable to act as a helper virus for HDV (3, 8). Despite its overall similarity to HBsAg-S and WHVsAg-S, DHBsAg-S lacks most of the structurally and functionally important antigenic loop region found in HBsAg and WHVsAg (Fig 2). We tested the possibility that the contrasting abilities of mammalian and avian hepadnaviruses to act as helpers in HDV assembly suggest a role for the antigenic loop region in HDV assembly.
FIG. 2.
Construction of a novel HBsAg-S mutant, HBsAgΔLoop. (A) Schematic of the HBsAg-S mutant HBsAgΔLoop. Cysteine residues at positions 107 and 147 were mutated to glycine, and all sequence between these two positions was removed. (B) Alignment of HBsAg-S with HBsAgΔLoop and DHBsAg-S.
DHBsAg-S cannot facilitate assembly of HDAg-L into virus-like particles.
We tested the envelope protein of DHBV, which naturally lacks sequence homologous to the antigenic loop region found in HBsAg-S, for its ability to facilitate assembly of HDAg-L-containing particles. HuH7 cells were transfected with expression constructs for DHBsAg-S, HBsAg-S, or the vector backbone of these constructs, together with an HDAg-L expression vector. Four days posttransfection, both the lysate and supernatant fractions were collected and analyzed by immunoblot analysis for secretion of DHBsAg-S and HDAg-L into the culture supernatant. As shown in Fig. 1, lane 1, while DHBsAg-S is secreted efficiently from HuH7 cells, it fails to facilitate secretion of HDAg-L, indicating that DHBsAg-S cannot act as a helper in the assembly of HDAg-L-containing particles. This may be due either to the absence of sequence corresponding to the antigenic loop region or to other differences between the DHBsAg and HBsAg proteins.
FIG. 1.
Immunoblot analysis of HDAg-L virus-like particle assembly in the presence of DHBsAg-S. (A and B) Immunoblot analysis (with mouse monoclonal anti-DHBsAg and 125I-labeled anti-mouse immunoglobulin G [IgG]) of lysate and supernatant fractions, respectively, from cells transfected with DHBsAg-S or HBsAg-S and HDAg-L, as indicated; (C and D) immunoblot analysis (with rabbit anti-HDAg and 125I-labeled protein A) of the samples shown in panels A and B.
HBsAgΔLoop facilitates incorporation of HDAg-L into virus-like particles.
In order to test more directly the role of the antigenic loop region in HDV assembly, we constructed a mutant HBsAg-S that lacks most of this region. Using PCR mutagenesis, the cysteine residues at positions 107 and 147 of HBsAg-S were mutated to glycine, and residues 108 to 146, representing most of the antigenic loop region and including all of the antigenic loop's cysteine residues, were deleted. An alignment comparing the resulting protein, HBsAgΔLoop, with HBsAg-S and DHBsAg-S is shown in Fig. 2. Because conformational epitopes in the antigenic loop region represent the major antigenic determinants of HBsAg-S (4), HBsAgΔLoop was undetectable by anti-HBsAg-S antibodies. We therefore constructed a plasmid for expression of HBsAgΔLoop with a carboxyl-terminal influenza virus hemagglutinin (HA) tag. We also constructed a vector expressing similarly tagged wild-type HBsAg-S in order to determine whether any impairment of function resulted from this modification (Fig. 3A, lanes 3 and 9).
FIG. 3.
Western analysis of HDAg-L virus-like particle assembly in the presence of HBsAgΔLoop. (A) Western analysis (with rabbit anti-HDAg and 125I-labeled protein A) of either cell lysate or pelleted culture supernatant (as indicated) from HuH7 cells transfected with HDAg-expressing plasmids and with plasmids for the expression of both untagged and C-terminally HA-tagged HBsAg-S and HBsAgΔLoop; (B) Western analysis (with mouse monoclonal anti-HA and 125I-labeled anti-mouse IgG) of culture supernatants from samples described in panel A. Panels A and B show the results of a typical experiment. The relative amount of HDAg-L incorporation per molecule of surface antigen was calculated by dividing the delta antigen signal by the surface antigen signal and was normalized using the value obtained for wild-type HBsAg-S. gp, glycosylated protein; p, nonglycosylated protein.
Figure 3A, lanes 8 and 10, shows that HDAg-L was secreted from HuH7 cells in the presence of HBsAgΔLoop. As shown in the anti-HA immunoblot in Fig. 3B, the overall amount of HBsAgΔLoop secreted into the culture supernatant (Fig. 3B, lane 2) was less than the overall amount of HBsAg-S secreted (Fig. 3B, lanes 1 and 3). However, neither HBsAg-S nor HBsAgΔLoop was found to be retained intracellularly at appreciable levels (data not shown), indicating that the reduced levels of HBsAgΔLoop present in the culture supernatant reflected either reduced expression or reduced stability of this protein compared to that of wild-type HBsAg-S. Quantitation of these immunoblots showed that the ratios of secreted HDAg-L signal to HA signal were similar in the two samples (Fig. 3B), indicating that the antigenic loop region of HBsAg-S is not required for the assembly of HDAg-L into virus-like particles and thus confirming the idea that sequences outside of the antigenic loop region are sufficient to mediate the interaction of HBsAg-S with HDAg-L. Additionally, HBsAgΔLoop did not form disulfide-linked homodimers (data not shown); this confirms that the protein's six remaining cysteines are incapable of mediating such interactions, at least in this context.
HBsAgΔLoop mediates the assembly of particles containing HDV RNPs.
Given the ability of HBsAgΔLoop to facilitate the assembly of HDAg-L-containing particles, we asked whether this protein could facilitate the assembly of HDV RNPs into virions. Though intracellular levels of accumulation of HDAg (Fig. 4) and HDV (data not shown) RNAs were similar for all samples, incorporation of RNPs into particles was more efficient in the presence of HBsAg-S (lane 1) than in the presence of HBsAgΔLoop (lane 2). The quantitation in Fig. 4A to D revealed that although specific incorporation of HDAg (total secreted HDAg signal/HA signal) (Fig. 4E) and HDV genomic RNA (Fig. 4F) was approximately fourfold less in the presence of HBsAgΔLoop than in the presence of wild-type HBsAg-S, the overall proportion of HDAg-L incorporated in HBsAgΔLoop particles was approximately 20% greater than that seen with HBsAg-S. This suggests that while particles composed of the mutant protein were somewhat hindered in their ability to incorporate RNPs, incorporation of free HDAg-L occurred as efficiently as it did in the case of the HDAg-L particles shown in Fig. 3. We conclude that although the antigenic loop region is not required for interaction with HDAg-L, it is still needed in order to achieve wild-type levels of RNP incorporation. We speculate that the lower efficiency of HDV RNP incorporation might reflect a subtle difference in the mutant particle's geometry, perhaps resulting in a reduction of volume within the particle.
FIG. 4.
Western and Northern analyses of HDV virion assembly in the presence of HBsAgΔLoop. (A and B) Western analysis (with rabbit anti-HDAg and 125I-labeled protein A) of cell lysates (A) and pelleted supernatants (B) from HuH7 cells transfected with expression plasmids encoding an antigenomic-sense HDV replicon and the envelope proteins indicated; (C) Western blot of the samples from panel B, using mouse monoclonal anti-HA and 125I-labeled anti-mouse IgG to detect the tagged envelope proteins; (D) Northern blot of the samples from panel B, using an anti-32P[UTP]-labeled probe for genomic-sense HDV RNA; (E) histogram showing relative amounts of delta antigen incorporation per molecule of surface antigen; (F) histogram showing relative amounts of HDV genomic RNA incorporation per molecule of surface antigen; (G) histogram showing the ratio of HDAg-L secretion to total HDAg secretion. Values in histograms are averages of results from two experiments.
Acknowledgments
We thank J. Coffin, R. Isberg, and N. Rosenberg (Tufts University) for helpful discussions. We thank J. Pugh (American Red Cross Holland Laboratory for the Biomedical Sciences, Rockville, Md.) for providing the anti-DHBsAg antibody.
This work was supported by NIH grant R01-AI40472 and by the Raymond and Beverly Sackler Research Foundation.
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