Abstract
Chronic HDV infections cause the most severe form of viral hepatitis. HDV requires HBV envelope proteins for hepatocyte entry, particle assembly and release. Eight HDV and 8 HBV genotypes have been identified. However, there are limited data on the replication competence of different genotypes and the effect that different HBV envelopes have on virion assembly and infectivity. We subcloned complementary DNAs (cDNAs) of all HDV and HBV genotypes and systematically studied HDV replication, assembly and infectivity using northern blot, western blot, reverse-transcription quantitative PCR, and in-cell ELISA. The 8 HDV cDNA clones initiated HDV replication with noticeable differences regarding replication efficacy. The 8 HBV-HBsAg-encoding constructs all supported secretion of subviral particles, however variations in envelope protein stoichiometry and secretion efficacy were observed. Co-transfection of all HDV/HBV combinations supported particle assembly, however, the respective pseudo-typed HDVs differed with respect to assembly kinetics. The most productive combinations did not correlate with the natural geographic distribution, arguing against an evolutionary adaptation of HDV ribonucleoprotein complexes to HBV envelopes. All HDVs elicited robust and comparable innate immune responses. HBV envelope-dependent differences in the activity of the EMA-approved entry inhibitor bulevirtide were observed, however efficient inhibition could be achieved at therapeutically applied doses. Lonafarnib also showed pan-genotypic activity. HDVs from different genotypes replicate with variable efficacies. Variations in HDV genomes and HBV envelope proteins are both major determinants of HDV egress and entry efficacy, and consequently assembly inhibition by lonafarnib or entry inhibition by bulevirtide. These differences possibly influence HDV pathogenicity, immune responses and the efficacy of novel drug regimens. HDV requires the envelope protein of HBV for assembly and to infect human cells. We investigated the ability of different HDV genotypes to infect cells and replicate. We also assessed the effect that envelope proteins from different HBV genotypes had on HDV infectivity and replication. Herein, we confirmed that genotypic differences in HDV and HBV envelope proteins are major determinants of HDV assembly, de novo cell entry and consequently the efficacy of novel antivirals.
Highlights
About 5–10% of chronic HBV-infected patients are coinfected with HDV
To differentiate between vector borne production of hepatitis D antigen (HDAg) and replication-related antigen expression, we implemented a replication deficient HDV complementary DNA (cDNA) named as HDV-def (Table S1)
To verify the establishment of HDV replicons, HuH7 cells were transfected with the respective cDNAs and RNA was analyzed by northern blot using Dig-labeled PCR-probes of an equimolar mixture of the 8 genotypes (Fig. S1B and S1C, Table S2)
Summary
About 5–10% of chronic HBV-infected patients are coinfected with HDV. Compared to chronic HBV monoinfection, chronic hepatitis D (CHD) manifests as the most severe form of viral hepatitis, accelerating progression to cirrhosis, hepatic decompensation and hepatocellular carcinoma.[1]. The respective HBVs distribute differently (Genotype A: Sub-Saharan Africa and Asia; genotype B and C: Asia; genotype D: Asia, Africa and Europe; genotype E: Sub-Saharan Africa; genotypes F, G and H: Latin America).[14] genotype-dependent differences in the stoichiometry of the 3 HBV envelope proteins were described.[15] synthetic preS1-lipopeptides derived from diverse genotypes differ in their ability to interfere with the binding of HBV to the sodium taurocholate cotransporting polypeptide (NTCP) receptor.[16] differently enveloped HDV might vary regarding infection efficacy, spreading and pathogenesis in patients It poses the question of whether the naturally occurring combinations are favored and may have undergone evolutionary co-adaptation. The generated tool set established here facilitates further investigations aiming to understand the genotype-dependent differences of HBV/HDVrelated pathogenesis and drug responses
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