Sequence alignment of WHV and HBV L proteins (wL and hL, respectively). The NCBI database accession numbers for WHV and HBV are AAA46770.1 and AAK58874.1, respectively. Alignment was via the T-Coffee program (24). Arrows are used to indicate the junctions of pre-S1, pre-S2, and S domains and also the boundaries of the major antigenic loop. Each protein is considered to have four TMD, as indicated by underlining. Further details are provided in the text and in Fig. 2. Identical (*), conserved (:), and semiconserved (.) amino acids are indicated.

Effect of wHDV and hHDV MOIs on infections of PHH and PWH. (A) PHH were infected with different MOIs of wHDV (open circles) or hHDV (shaded circles) in the presence of 5% PEG. (B) An experiment similar to that in panel A but using PWH. As described in the legend of in Fig. 3, total RNA was extracted at day 6 and assayed by qPCR, and the infectivity results were expressed as HDV RNA GE/average cell. The data are the average of duplicate infections, and the results were confirmed in two separate experiments.

Time course of wHDV and hHDV infections of PHH and PWH. Cultures of PHH (A and B) or PWH (C and D) were infected either with wHDV (open circles) or hHDV (shaded circles) at an MOI of 500 in the presence of 5% PEG. At 1, 3, 5, and 7 days after infection, total RNA was extracted and assayed by qPCR, as described in the legend of Fig. 3. The infectivity results are expressed as HDV RNA GE/average cell. The data are the average of duplicate infections.

Ability of soluble peptides to interfere with infections of PHH and PWH by wHDV and hHDV. Cultures of PHH (A and B) or PWH (C) were infected with either wHDV (open bars) or hHDV (shaded bars) at an MOI of 50 in the presence of 5% PEG. The six soluble peptides explained in Table 1 were tested for their ability at a concentration of 50 nM to inhibit the infections. As described in the legend of Fig. 3, total RNA was extracted at day 6 and assayed by qPCR. Here, the infectivity results are expressed as a percentage of the infectivity observed in the absence of peptide. The infections were performed in duplicate, and the results shown are the average of two separate experiments. We consider a reduction of infectivity to less than 25% of control values significant.

Ability of wHDV and hHDV to infect PHH and PWH. Both types of hepatocytes were infected at an MOI of 300 HDV GE/cell. (A) Infections were performed in the presence of 5% PEG, and at 6 days, cells with HDV replication were identified by immunostaining to detect newly synthesized δAg (red). Counterstaining was with DAPI (blue) and for either albumin in PHH or α-tubulin in PWH (green). (B) HDV infections were carried out either without (−) or with (+) 5% PEG. Cells were harvested at day 6 postinfection. Total RNA was assayed for HDV RNA by qPCR as previously described (16), and the infectivity results are expressed as HDV RNA GE/average cell. Graphs represent the average results of duplicate infections with wHDV (open bars) and hHDV (shaded bars). The results were confirmed in two separate experiments.

Predicted topologies for WHV and HBV L proteins (wL and hL, respectively). Protein folding of the sequences presented in Fig. 1, including prediction of the TMD, was performed using TMHMM2.0 software (19). Further details are provided in the text. The four dark cylinders, indicated as I to IV, represent the TMD inserted in the lipid membrane, indicated by the light shading. For the extracellular virus the membrane has inside-outside surfaces, while during intracellular assembly at an endoplasmic reticulum-Golgi membrane these are cytosol-lumen surfaces, respectively. The amino and carboxy termini are indicated by N and C, respectively. On the outside, or luminal side, of the membrane are pre-S1, pre-S2, and the major antigenic loop. On the inside, or cytosolic side, loops of 51 and 8 amino acids are indicated. The boundaries of the above-mentioned structural elements are indicated by position numbers of the corresponding amino acid residues. aa, amino acids.

Assembly and infectivity of the hepatitis delta virions coated with different intermolecular combinations of WHV and HBV envelope proteins. Pairwise combinations of plasmids expressing the L and S envelope proteins of either WHV (wL and wS) or HBV (hL and hS) were used in different transfections of Huh7 cells in order to achieve the assembly of hepatitis delta virions with different envelopes. The three sets of assembly experiments performed consisted of wL plus wS, wL plus hS, and hL plus wS. We used one plasmid to express L and another to express S protein; the mass amounts of each plasmid were varied, while the total mass was kept constant. The mass ratios are indicated at the bottom of the figure as the percentage of L-expressing construct, that is, 100 × L/(L+S). For each transfection the virion-containing medium was harvested for days 7 to 10 and assayed for HDV RNA by qPCR, with results as shown in panels A1 to A3. Aliquots of these media were also used for infection of PHH (B) and PWH (C). As described in the legend of Fig. 3, total RNA was extracted at day 6 and assayed by qPCR. However, here we divided these infectivity values (in HDV RNA GE/average cell) by the input MOI (also expressed as HDV GE/average cell). This ratio, which we have previously referred to as the specific infectivity (16), is a measure of the ability of the virus particles to infect susceptible cells, either PHH (B1 to B3) or PWH (C1 to C3). Rather than use duplicates of each assembly, we chose to consider additional combinations of L and S. Thus, in panel A1, the relatively low particle yield at 60% of L content is considered an experimental variation. The critical results in panels A1 to A3 and B1 to B3 were confirmed in a separate, less extensive repeat experiment.