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.

Hepatitis delta virus (HDV) genome replication requires the virus-encoded small delta protein (␦Ag).During replication, nucleotide sequence changes accumulate on the HDV RNA, leading to the translation of ␦Ag species that are nonfunctional or even inhibitory. A replication system was devised where all ␦Ag was conditionally provided from a separate and unchanging source. A line of human embryonic kidney cells was stably transfected with a single copy of cDNA encoding small ␦Ag, with expression under tetracycline (TET) control. Next, HDV genome replication was initiated in these cells by transfection with a mutated RNA unable to express ␦Ag. Thus, replication of this RNA was under control of the TET-inducible ␦Ag. In the absence of TET, there was sufficient ␦Ag to allow a low level of HDV replication that could be maintained for at least 1 year. When TET was added, both ␦Ag and genomic RNA increased dramatically within 2 days. With clones of such cells, designated 293-HDV, the burst of HDV RNA replication interfered with cell cycling. Within 2 days, there was a fivefold enhancement of G 1 /G 0 cells relative to both S and G 2 /M cells, and by 6 days, there was extensive cell detachment and death. These findings and those of other studies that are under way demonstrate the potential applications of this experimental system.

Assembly of hepatitis delta virus (HDV) in infected human hepatocytes involves association of the 1,679-nucleotide single-stranded genomic RNA (␦RNA) with multiple copies of both small and large forms of the delta protein (␦Ag) to form a ribonucleoprotein particle which in turn interacts with envelope proteins of the natural helper virus, hepatitis B virus. Subsequently, for initiation of a new round of replication, the amount of small ␦Ag within the assembled HDV particle is both necessary and sufficient. Quantitative assays were used in order to better understand just how much ␦Ag is needed. The molar ratio of ␦Ag species to genomic ␦RNA in assembled HDV particles was approximately 200. Next, this ratio was determined for cells under several different experimental situations in which HDV genome replication was occurring. These included replication in woodchuck liver and also in mouse liver and skeletal muscle, as well as replication in stably and transiently transfected cultured human hepatoblastoma cells. Surprisingly, in almost all these situations the molar ratios were comparable to that observed for HDV particles. This was true for different times after the initiation of replication and was independent of whether or not virus assembly was occurring. Cell fractionation combined with quantitative assays was used to test whether the majority of ␦Ag and ␦RNA were colocalized during HDV replication in transfected cells. The cytoplasmic fraction contained the majority of ␦Ag and genomic ␦RNA. Finally, the quality of ␦Ag and ␦RNA, especially at relatively late times after the initiation of replication, was examined by using reverse transcription-PCR, cloning, and sequencing through the entire ␦Ag open reading frame. When virus assembly and spread were not possible, 20% or less of the predicted ␦Ag would have been able to support HDV replication. In summary, an examination of the quantity, quality and intracellular distribution of ␦Ag and ␦RNA in several different experimental systems has provided a better understanding of the parameters associated with the initiation, maintenance, and ultimate decline of HDV genome replication.Human hepatitis delta virus (HDV) was discovered in 1977 (34) and was soon recognized as a satellite of hepatitis B virus (HBV) (35). The helper role of HBV is limited to providing the envelope proteins needed for HDV assembly and subsequent cycles of infection (20). In other words, all other aspects of the intracellular replication of the HDV genome can be studied independently of assembly and infection.

A natural subviral agent of human hepatitis B virus (HBV), hepatitis delta virus (HDV), requires only the envelope proteins from HBV in order to maintain persistent infection. HBV surface antigens (HBsAgs) can be produced either by HBV replication or from integrated HBV DNA regardless of replication. The functional properties of the integrant-generated HBsAgs were examined using two human hepatocellular carcinoma-derived cell lines, Hep3B and PLC/PRF/5, that contain HBV integrants but do not produce HBV virions and have no signs of HBV replication. Both cell lines were able to support HDV replication and assembly/egress of HDV virions. Neither of the cell lines was able to produce substantial amounts of the pre-S1-containing HDV particles. HDV virions assembled in PLC/PRF/5 cells were able to infect primary human hepatocytes, while Hep3B-derived HDV appeared to be noninfectious. These results correlate with the findings that the entire open reading frame (ORF) for the large (L) envelope protein that is essential for infectivity is present on HBV RNAs from PLC/PRF/5 cells, while an L protein ORF that was truncated and fused to inverted precore sequences was found using RNAs from Hep3B cells. This study demonstrates for the first time that at least some of the HBV DNA sequence naturally integrated during infection can produce functional small and large envelope proteins capable of the formation of infectious HDV virions. Our data indicate that in vivo chronic HDV infection can persist in the absence of HBV replication (or when HBV replication is profoundly suppressed) if functional envelope proteins are supplied from HBV integrants. IMPORTANCEThe study addresses the unique mechanism of HDV persistence in the absence of ongoing HBV replication, advances our understanding of HDV-HBV interactions, and supports the implementation of treatments directly targeting HDV for HDV/HBV-infected individuals. Hepatitis delta virus (HDV) is a significant human pathogen, with approximately 20 million people worldwide being chronic carriers. HDV is a natural subviral agent of human hepatitis B virus (HBV) that requires from its helper hepadnavirus only the envelope proteins in order to form virions and infect hepatocytes via the HBV receptor. In infected livers, HDV coexists with HBV. Chronic HBV infection remains a main risk factor for hepatocellular carcinoma (HCC) and is associated with more than half of all HCC cases (1-4). Concomitant HDV infection is able to inflict additional liver damage associated with accelerated liver disease, cirrhosis, liver failure, and HCC (5-11). Treatment with alpha interferon is beneficial for only a subset of HDV carriers. There are no treatments in clinical practice that directly target HDV, and practically none of the anti-HBV drugs blocks HDV infection (5,12,13). In livers chronically infected with HBV, as many as 90% of hepatocytes may appear to be free of HBV replication markers. HBV-infected individuals can support HDV infection regardless of the presence of HBV replication markers...

Hepatitis delta virus (HDV) is unique relative to all known animal viruses, especially in terms of its ability to redirect host RNA polymerase(s) to transcribe its 1,679-nucleotide (nt) circular RNA genome. During replication there accumulates not only more molecules of the genome but also its exact complement, the antigenome. In addition, there are relatively smaller amounts of an 800-nt RNA of antigenomic polarity that is polyadenylated and considered to act as mRNA for translation of the single and essential HDV protein, the delta antigen. Characterization of this mRNA could provide insights into the in vivo mechanism of HDV RNA-directed RNA transcription and processing. Previously, we showed that the 5 end of this RNA was located in the majority of species, at nt 1630. The present studies show that (i) at least some of this RNA, as extracted from the liver of an HDV-infected woodchuck, behaved as if it contained a 5-cap structure; (ii) in the infected liver there were additional polyadenylated antigenomic HDV RNA species with 5 ends located at least 202 nt and even 335 nt beyond the nt 1630 site, (iii) the 5 end at nt 1630 was not detected in transfected cells, following DNA-directed HDV RNA transcription, in the absence of genome replication, and (iv) nevertheless, using in vitro transcription with purified human RNA polymerase II holoenzyme and genomic RNA template, we did not detect initiation of template-dependent RNA synthesis; we observed only low levels of 3-end addition to the template. These new findings support the interpretation that the 5 end detected at nt 1630 during HDV replication represents a specific site for the initiation of an RNA-directed RNA synthesis, which is then modified by capping.The two major species of RNA that accumulate during hepatitis delta virus (HDV) replication are the unit-length genome and its exact complement, the antigenome (Fig. 1, panels 1 and 2) (2). These 1,679-nucleotide (nt) RNAs are detected in both circular and linear forms. In addition, there are relatively smaller amounts of an 800-nt polyadenylated RNA of antigenomic polarity (Fig. 1, panel 3). This putative mRNA contains the open reading frame for the small delta protein, a 195-amino-acid species that is essential for HDV genome replication (10). All other proteins needed for HDV genome replication are provided by the host cell. This means that somehow a host RNA polymerase that is normally DNA dependent has to be redirected to act on HDV RNA as template.One approach to clarify the mechanism of HDV genome replication with its unique RNA-directed RNA synthesis and processing has been to adopt the rationale that a detailed characterization of the mRNA species will provide valuable insights. Previous studies include (i) Northern analyses (2), (ii) primer extension (9), (iii) ribonuclease protection assays (27), and (iv) 5Ј rapid amplification of cDNA ends (5Ј-RACE) (6). By primer extension, a 5Ј end was first mapped to position 1631 Ϯ 1 of the 1,679-nt RNA sequence (9). A more recent study using 5Ј-RACE indi...