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

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.

Two Potentially Important Elements of the Hepatitis B Virus Large Envelope Protein Are Dispensable for the Infectivity of Hepatitis Delta Virus

“…We and others have realized that HDV offers some unique advantages as part of a strategy for understanding HBV attachment and entry (2,9,16,31,33). Unlike HBV assembly, HDV assembly does not require a pre-S1 or pre-S2 domain.…”

“…From these data, we could determine the initial virus titer (genome equivalents [GE]/ml of medium), the input multiplicity of infection (MOI) (GE per cell, with 200,000 hepatocytes per well of a collagen-coated, 48-well plate), and the HDV infectivity, which is the accumulation of HDV RNA (GE per cell) following infection. All infections were carried out at a multiplicity of Ͻ300 GE/cell, that is, in what has been previously determined to be the linear response range (16). Finally, we determined the ratio of the output of GE per cell accumulated in the infected hepatocytes to the input multiplicity of GE per cell.…”

“…c The indicated values of HDV assembly, infectivity, and specific infectivity were based on the average of independent, duplicate, quantitative real-time PCR assays of GE, as previously described (16).…”

“…Thus, as described here, we have been able to address certain requirements for infectivity without compromising assembly. The strategy is based on studies described in detail elsewhere (16).…”

“…Particles assembled with only HBV S protein were used as a negative control for infectivity. The assembly, infectivity, and specific infectivity of HDV were assayed as described in Table 1 and as previously described (16), and all are based on the average of independent, duplicate, quantitative real-time PCR assays of GE. Based on this and a separate experiment, we consider the range of specific infectivity values for wt and TLM Ϫ to be not significantly different.…”