Hepatitis delta virus infects the cells of hepadnavirus-induced hepatocellular carcinoma in woodchucks

Cunha

Menne

et al. 2014

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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...

Section: Discussionsupporting

confidence: 84%

Envelope Proteins Derived from Naturally Integrated Hepatitis B Virus DNA Support Assembly and Release of Infectious Hepatitis Delta Virus Particles

Cunha

Menne

et al. 2014

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“…In addition, using quantitative real-time PCR assays (qPCRs), we were able to demonstrate the presence of pregenome/precore RNA (pgRNA) and covalently closed circular DNA (cccDNA) of WHV in harvested samples of WHV-induced HCC tissues. However, the amounts of pgRNA and cccDNA in HCC tissues were reduced, often significantly, compared to those in corresponding nonmalignant liver tissues (31). In the current study, we also used three WHV carrier woodchucks with already formed WHV-induced HCCs.…”

mentioning

confidence: 95%

Superinfection with Woodchuck Hepatitis Virus Strain WHVNY of Livers Chronically Infected with Strain WHV7

Rodrigues

Kallakury

et al. 2015

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The determinants of the maintenance of chronic hepadnaviral infection are yet to be fully understood. A long-standing unresolved argument in the hepatitis B virus (HBV) research field suggests that during chronic hepadnaviral infection, cell-to-cell spread of hepadnavirus is at least very inefficient (if it occurs at all), virus superinfection is an unlikely event, and chronic hepadnavirus infection can be maintained exclusively via division of infected hepatocytes in the absence of virus spread. Superinfection exclusion was previously shown for duck HBV, but it was not demonstrated for HBV or HBV-related woodchuck hepatitis virus (WHV). Three woodchucks, which were chronically infected with the strain WHV7 and already developed WHV-induced hepatocellular carcinomas (HCCs), were superinfected with another WHV strain, WHVNY. Six weeks after the superinfection, the woodchucks were sacrificed and tissues of the livers and HCCs were examined. The WHVNY superinfection was demonstrated by using WHV strain-specific PCR assays and ( Worldwide, approximately four hundred million people are chronic carriers of human hepatitis B virus (HBV), which is a prototype hepadnavirus. Chronic HBV infection is a main risk factor of hepatocellular carcinoma (HCC), and it is associated with more than 50% of all known cases of HCC (1-14). During the early stage of hepadnaviral infection, virtually the entire liver becomes infected with a hepadnavirus within several weeks. However, during chronic infection, areas of apparently hepadnavirusfree hepatocytes were observed in HBV carrier humans and chimpanzees and also in woodchucks chronically infected with woodchuck hepatitis virus (WHV), which is closely related to HBV. The hepadnavirus-free hepatocytes tested negative for (i) the hepadnavirus core antigen (by immunostaining) and (ii) the viral DNA (by in situ hybridization) regardless of the ongoing viremia. In HBV carrier humans, up to 90% of all hepatocytes could appear to be HBV free. Some of the virus-free hepatocytes form foci of altered hepatocytes (FAH) with morphology similar to that of liver cancer cells and are considered premalignant. In addition, the cells of hepadnavirus-induced HCCs often were reported to be virus free as well (1,4,(15)(16)(17)(18)(19)(20)(21)(22)(23). In contrast, several previous studies reported the presence of hepadnaviral , making the issue still controversial. The observations of apparent hepadnavirus-free HCC cells and hepatocytes, along with the analysis of the kinetics of the emergence of drug-resistant HBV mutants, laid the foundation for a long-standing unresolved argument in the HBV field that during chronic infection, cell-to-cell spread of a hepadnavirus is at least very inefficient (if it occurs at all), and superinfection is an un-

“…We quantified infected PHH by staining for newly made delta antigens using rabbit polyclonal antibodies raised against recombinant small ␦Ag (21). We also quantified the newly made HDV genomes produced as the result of infection of PHH, by assaying total RNA harvested from infected hepatocytes using qPCR (21,25). Selected images of PHH infected with different HDV types are presented in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Total RNA from Huh7 cells, Pansorbin-bound particles, PHH, or virions was isolated with TRI reagent (Fisher) and treated with DNase (Life Technologies) prior to quantitative real-time PCR (qPCR). The qPCR was conducted using the 7500 real-time PCR instrument (Applied Biosystems) as described previously (21,25). The forward primer was 312-G GACCCCTTCAGCGAACA-329, the reverse primer was 393-CCTAGCATC TCCTCCTATCGCTAT-360, and the TaqMan probe was 332-AGGCGCTT CGAGCGGTAGGAGTAAGA-357 (the numbering is according to Kuo et al [26]).…”

Section: Lms Vectors Using the Sequences Of The Hbv Variants Listed Inmentioning

confidence: 99%

“…The reverse primer was also used for reverse transcription. The copy numbers were measured using a 10-fold dilution series of in vitro-transcribed and gel-purified genomic (G) unit-length HDV RNA standard (range, 20 to 200,000 genome equivalents [GE] of HDV) and considering 1 million HDV RNA molecules equal to 1 picogram of G RNA standard (21,25). IF.…”

Section: Lms Vectors Using the Sequences Of The Hbv Variants Listed Inmentioning

confidence: 99%

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Support of the Infectivity of Hepatitis Delta Virus Particles by the Envelope Proteins of Different Genotypes of Hepatitis B Virus

Abe

Cunha

et al. 2014

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This study examined how the envelope proteins of 25 variants of hepatitis B virus (HBV) genotypes 1, 2). A recent study demonstrated that the number of HBV virions in an inoculum per se determined the kinetics of HBV spread through the liver and the timing and magnitude of the immune response, as well as the outcome of infection, i.e., whether the infection became transient or chronic (3). These results indicated that the infectivity of the virions and the rate of virus spread throughout the liver may play a decisive role for the clinical outcome of an infection. A number of previous studies suggested that HBV-associated liver pathogenesis is genotype specific and reported differences between HBV genotypes in terms of the severity of induced liver disease, HCC incidence, responsiveness to antiviral therapy, and infectivity of HBV virions. Some of those reports appeared to be controversial (4-15). The current study examined in more mechanistic detail how the envelope proteins of HBV contribute to the infectivity of the virions. As the experimental model, we used virions of human hepatitis delta virus (HDV). HDV is a subviral agent of HBV that, in nature, coexists with its helper virus in infected livers and uses HBV envelope proteins to form HDV virions and enter hepatocytes through the HBV-specific receptor that interacts with specific sequences in the PreS1 domain of the L (large) envelope protein (16). We assembled in cell culture 25 different types of HDV virions, all of which bore the same HDV ribonucleoprotein (RNP) inside and differed only by the envelope proteins coating the virions. The envelope proteins analyzed in this study represent 25 different natural variants of HBV that belong to nine HBV genotypes, A to I (14, 17). It was found that HBV envelope proteins apparently were able alone to determine the number of infected hepatocytes and the number of genomes that successfully initiated HDV replication after entering a susceptible cell. It became apparent that HBV envelope proteins are not only involved in attachment and entry but also likely facilitate at least one of the immediate postentry steps. These novel findings advance the understanding of the mechanism of virus entry and, possibly, trafficking and suggest an additional virus life cycle step(s) that is likely regulated by HBV envelope proteins. MATERIALS AND METHODS LMS vectors.Using the sequences of the HBV variants listed in Table 1, 24 corresponding vectors were constructed to express the large (L), middle (M), and small (S) envelope proteins (LMS vectors). Each construct bears a fragment of the genome of a particular HBV variant inserted into XhoI/ XbaI sites of the pSVL vector (Pharmacia). The inserted fragment begins at the start codon of the L, includes the entire L open reading frame,