Parameters influencing the attachment of hepatitis A virus to a variety of continuous cell lines

Zajac, Allan; Amphlett, Elizabeth M.; Rowlands, David J.; Sangar, D. V.

doi:10.1099/0022-1317-72-7-1667

Cited by 28 publications

(23 citation statements)

References 31 publications

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confidence: 96%

“…The uncoating of HAV has been demonstrated to be a slow and asynchronous process (3,5,9,30). By contrast, the attachment to and penetration of cells by HAV appear to be as efficient as for other picornaviruses (3,8,29,32).Intact, antigenic particles from HAV-infected cells or cell culture supernatants have recently been demonstrated to be of three types: (i) virions, containing capsid proteins VP1, VP2, VP3, and possibly VP4 and viral RNA; (ii) provirions, or "immature" virions, containing VP1, VP0, VP3, and viral RNA; and (iii) procapsids, which are empty HAV particles with the same capsid composition as provirions but lacking RNA (2, 7, 23). Provirions have been implicated as direct precursors of mature picornavirus virions via the cleavage of VP0 (to VP2 and VP4) in an RNA-dependent manner (6).…”

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Uncoating Kinetics of Hepatitis A Virus Virions and Provirions

Bishop

Anderson

2000

J Virol

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When the growth kinetics of immature hepatitis A virus provirions and mature virions were monitored, distinct eclipse phases were noted for both types of particles. Strikingly, uncoating of virions occurred around 4 h postinfection, while uncoating of provirions occurred predominantly between 8 and 10 h postinfection. It is proposed that the heterogenous mixture of infectious hepatitis A virus particles (virions and provirions) typically present in inocula is responsible for the normally asynchronous nature of hepatitis A virus uncoating kinetics.Human hepatitis A virus (HAV), a member of the family Picornaviridae originally classified in the Enterovirus genus, is now the type member of the Hepatovirus genus (12, 31). The reclassification of HAV was based on a number of differences between HAV and other enteroviruses, including an extreme stability against elevated temperature and low pH (25, 26) and a slow and inefficient replicative cycle in cell culture (see references 27 and 31). The uncoating of HAV has been demonstrated to be a slow and asynchronous process (3,5,9,30). By contrast, the attachment to and penetration of cells by HAV appear to be as efficient as for other picornaviruses (3,8,29,32).Intact, antigenic particles from HAV-infected cells or cell culture supernatants have recently been demonstrated to be of three types: (i) virions, containing capsid proteins VP1, VP2, VP3, and possibly VP4 and viral RNA; (ii) provirions, or "immature" virions, containing VP1, VP0, VP3, and viral RNA; and (iii) procapsids, which are empty HAV particles with the same capsid composition as provirions but lacking RNA (2, 7, 23). Provirions have been implicated as direct precursors of mature picornavirus virions via the cleavage of VP0 (to VP2 and VP4) in an RNA-dependent manner (6).HAV provirions, while infectious, have a lower specific infectivity than mature virions (6, 7). We proposed that the mixture of particle types (i.e., of virions and provirions and of particles with intermediate levels of VP0 and VP2) in viral inocula was responsible for the asynchronous nature of the HAV uncoating process, on the basis that provirions might uncoat more slowly than virions. Here we report that using an HAV inoculum with a high virion content resulted in rapid as well as synchronous uncoating.Single-cycle growth kinetics of HAV and PV. Typical singlecycle growth kinetics of HAV and poliovirus (PV) are shown in Fig. 1. An African green monkey cell line, BS-C-1, was infected with HAV strain HM175A.2 (1, 9) or PV type 1 (Mahoney), and the accumulation of infectious virus within cells was determined after various incubation times at 37°C.In PV-infected cells, eclipse of the inoculated virus was evident at 2 h, reflecting rapid uncoating of the virus, followed by logarithmic accumulation of virus to 12 h. From 12 h, infectious virus accumulated slowly and much of the virus at this time was found in the supernatant. By contrast, eclipse of the HM175A.2 inoculum appeared to be incomplete, with much of the input virus being recovered...

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Uncoating Kinetics of Hepatitis A Virus Virions and Provirions

Bishop

Anderson

2000

J Virol

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“…Although cell lines originating from tissues other than liver, such as fibroblasts and kidney cells, are also susceptible to HAV infection (9, 11) and although HAV antigen and the putative receptor for HAV could be detected in different organs, such as kidney, spleen, and gastrointestinal tract (2, 6, 10, 18), no extrahepatic sites of HAV replication have been clearly identified. The data on the ubiquitous expression of a receptor for HAV and the ability of HAV to replicate in a number of nonliver cells in cell cultures, but obviously not in the organism, suggest that HAV may be targeted to the liver by a particular mechanism.Data from several laboratories showed that HAV virions are partially associated with immunoglobulin A (IgA) molecules (19,22) and other host organism-derived materials, such as fibronectin or ␣ 2 -macroglobulin (21,23,24,35,43). As viruses may find entry into host cells via receptors specific for molecules of the host organism ligated to the virion (13,16,20,26) and as the liver plays a central role in IgA metabolism by eliminating IgA as well as antigen-IgA complexes (4), we wondered if HAV-specific IgA ligated to HAV supports the targeting of HAV to the liver and is able to mediate the entry of HAV into hepatocytes via receptors specific for the IgA molecule and if such a carrier-mediated mechanism may result in viral infection.…”

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confidence: 99%

“…Data from several laboratories showed that HAV virions are partially associated with immunoglobulin A (IgA) molecules (19,22) and other host organism-derived materials, such as fibronectin or ␣ 2 -macroglobulin (21,23,24,35,43). As viruses may find entry into host cells via receptors specific for molecules of the host organism ligated to the virion (13,16,20,26) and as the liver plays a central role in IgA metabolism by eliminating IgA as well as antigen-IgA complexes (4), we wondered if HAV-specific IgA ligated to HAV supports the targeting of HAV to the liver and is able to mediate the entry of HAV into hepatocytes via receptors specific for the IgA molecule and if such a carrier-mediated mechanism may result in viral infection.…”

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Hepatitis A Virus-Specific Immunoglobulin A Mediates Infection of Hepatocytes with Hepatitis A Virus via the Asialoglycoprotein Receptor

Dotzauer

Gebhardt

Bieback

et al. 2000

J Virol

102

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The mechanisms underlying the hepatotropism of hepatitis A virus (HAV) and the relapsing courses of HAV infections are unknown. In this report, we show for a mouse hepatocyte model that HAV-specific immunoglobulin A (IgA) mediates infection of hepatocytes with HAV via the asialoglycoprotein receptor, which binds and internalizes IgA molecules. Proof of HAV infection was obtained by detection of HAV minus-strand RNA, which is indicative for virus replication, and quantification of infectious virions. We demonstrate that human hepatocytes also ingest HAV-anti-HAV IgA complexes by the same mechanism, resulting in infection of the cells, by using the HepG2 cell line and primary hepatocytes. The relevance of this surrogate receptor mechanism in HAV pathogenesis lies in the fact that HAV, IgA, and antigen-IgA complexes use the same pathway within the organism, leading from the gastrointestinal tract to the liver via blood and back to the gastrointestinal tract via bile fluid. Therefore, HAV-specific IgA antibodies produced by gastrointestinal mucosaassociated lymphoid tissue may serve as carrier and targeting molecules, enabling and supporting HAV infection of IgA receptor-positive hepatocytes and, in the case of relapsing courses, allowing reinfection of the liver in the presence of otherwise neutralizing antibodies, resulting in exacerbation of liver disease.Hepatitis A virus (HAV), a hepatotropic picornavirus (for a review, see reference 15), causes acute viral hepatitis in humans by an immunopathogenetic mechanism (41). The HAV infection is characterized by a short, self-limited disease and does not lead to chronic cases. However, after initial improvement in symptoms and liver test values, one or more relapses of the disease are described for up to 20% of patients (14,40). These relapses occur between 30 and 90 days after the primary episode, when high titers of neutralizing antibodies are already detectable (12). HAV is transmitted by the fecal-oral route, but the mechanism by which the virus first enters the bloodstream and reaches the liver as well as the pathogenetic mechanism leading to a relapsing disease remains unclear. Kaplan et al. (17) reported that a mucin-like class I integral membrane glycoprotein which was identified on African green monkey kidney cells acts as an attachment molecule for HAV. It was demonstrated that the human homolog is a binding receptor for HAV; it has been suggested that it is also a functional receptor (10). Although cell lines originating from tissues other than liver, such as fibroblasts and kidney cells, are also susceptible to HAV infection (9, 11) and although HAV antigen and the putative receptor for HAV could be detected in different organs, such as kidney, spleen, and gastrointestinal tract (2, 6, 10, 18), no extrahepatic sites of HAV replication have been clearly identified. The data on the ubiquitous expression of a receptor for HAV and the ability of HAV to replicate in a number of nonliver cells in cell cultures, but obviously not in the organism, suggest that HAV...

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“…per well) at 4 mC for 2 h. HAV used as the inoculum was sucrose density gradient-purified, radiolabelled, column-sieved, density gradient-purified again to remove free radioisotopes and resuspended in Dulbecco's modified MEM (Zajac et al, 1991). Cultures were washed with PBS three times and solubilized with 1 % SDS.…”

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Molecular cloning of the hepatitis A virus receptor from a simian cell line.

Ashida

Hamada

1997

Journal of General Virology

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Using a eukaryotic expression system in combination with a monoclonal antibody (MAb) capable of blocking hepatitis A virus (HAV) adsorption, a cDNA clone was selected from a library of S.la/Ve-1 cells, a cell line that is highly susceptible to the virus. Sequence analysis of the cDNA revealed a single open reading frame that encoded a protein consisting of 460 amino acids. The deduced primary structure of the protein included a signal sequence, a transmembrane domain, four sites for N-linked glycosylation, cysteine residues attributable to an immunoglobulin domain and threonine clusters characteristic of mucin-like protein. By employing a vaccinia virus expression vector, the cDNA was expressed in HeLa cells where it induced marked HAV attachment which was specifically blocked by the MAb. The cDNA obtained was thus assumed to encode a functional receptor for HAV.Hepatitis A virus (HAV) is the causative agent of hepatitis A, which infects via the faecal-oral route. The virus belongs to the family Picornaviridae, genus Hepatovirus and shares some physicochemical characteristics with members of the genus Enterovirus, but is distinct from these in its biological properties, showing slow growth in susceptible cells in culture and a lack of cytopathic effects (CPE) in infected cells (Lemon & Robertson, 1994). These unique biological properties of HAV have hampered precise and rapid assays of virus infectivity titres and have hindered analysis of the growth mechanisms of the virus at the molecular level, including identification of the cellular receptor, which is critical in understanding the pathogenicity of the virus. Previously, we have generated hybrid cell lines between marmoset liver and Vero cells that are highly susceptible not only to cell culture-adapted strains but also to wild strains of HAV (Ashida et al., 1989). This prompted us to attempt to identify the cellular receptor for HAV by employing one of the hybrid cell lines. Here we describe the isolation of cDNA encoding a membrane-associated glycoprotein which is a plausible candidate for the receptor. During the course of this study, Kaplan et al. (1996) reported similar results with an African green monkey kidney cell line (GL37), but there are several variations in the primary structure of these two cloned cDNAs.To identify the HAV receptor, we used cells which were highly susceptible to the virus as immunogens to raise monoclonal antibodies (MAb) capable of blocking virus adsorption. A cDNA library was then made from the susceptible cells and cDNA from the library encoding an MAb-reactive product was selected. The cells used were S.la\Ve-1 cells, a hybrid between marmoset liver cells and Vero cells. The HAV strain used as the challenge virus was cell culture-adapted strain T.T. (Kojima et al., 1981).To prepare MAb against S.la\Ve-1 cells, the membrane fraction of 2i10( S.la\Ve-1 cells was mixed with an equal amount of complete Freund's adjuvant and used for immunization of BALB\c mice. At 6 weeks post-inoculation, mice received a booster injection wi...

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Parameters influencing the attachment of hepatitis A virus to a variety of continuous cell lines

Cited by 28 publications

References 31 publications

Uncoating Kinetics of Hepatitis A Virus Virions and Provirions

Uncoating Kinetics of Hepatitis A Virus Virions and Provirions

Hepatitis A Virus-Specific Immunoglobulin A Mediates Infection of Hepatocytes with Hepatitis A Virus via the Asialoglycoprotein Receptor

Molecular cloning of the hepatitis A virus receptor from a simian cell line.

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