Infection of Polarized Cultures of Human Intestinal Epithelial Cells with Hepatitis A Virus: Vectorial Release of Progeny Virions through Apical Cellular Membranes

Abstract

Although hepatitis A virus (HAV) is typically transmitted by the fecal-oral route, little is known of its interactions with cells of the gastrointestinal tract.We studied the replication of HAV in polarized cultures of Caco-2 cells, a human cell line which retains many differentiated functions of small intestinal epithelial cells. Virus uptake was 30-to 40-fold more efficient when the inoculum was placed on the apical rather than the basolateral surface of these cells, suggesting a greater abundance of the cellular receptor for HAV on the apical surface. Infection proceeded without cytopathic effect and did not influence transepithelial resistance or the diffusion of inulin across cell monolayers. Nonetheless, there was extensive release of progeny virus, which occurred almost exclusively into apical supernatant fluids (36.4% ؎ 12.5% of the total virus yield compared with 0.23% ؎ 0.13% release into basolateral fluids). Brefeldin A caused a profound inhibition of HAV replication, but also selectively reduced apical release of virus. These results indicate that polarized human epithelial cell cultures undergo vectorial infection with HAV and that virus release is largely restricted to the apical membrane. Virus release occurs in the absence of cytopathic effect and may involve cellular vesicular transport mechanisms. Human hepatitis A virus (HAV) is a nonenveloped viruswith a single-stranded, 7.5-kb positive-sense RNA genome (17,18). It is classified as the type species of the genus Hepatovirus within the family Picornaviridae and is a common cause of both sporadic and epidemic acute hepatitis in humans (17,19). The transmission of HAV is generally due to the ingestion of material contaminated with feces containing HAV. However, the pathological sequence of events that begins with entry of the virus via the gastrointestinal tract and ultimately results in hepatitis is not well understood. A primary, extrahepatic site of replication for this highly hepatotropic agent has long been postulated, but has proven difficult to demonstrate. Early experiments involving immunohistologic evaluation of intestinal tissue from infected nonhuman primates provided no evidence for the presence of virus within the gastrointestinal mucosa. Both Mathiesen et al. (25) and Krawczynski et al. (16) were unable to identify viral antigen in the gut of enterically infected primates. However, more recent studies, possibly with better immunologic reagents, have resulted in the demonstration of HAV antigen within cells of the small intestine. Karayiannis et al. (14) found specific HAV antigen within the cytoplasm of ϳ3% of cells in duodenal biopsies from two of three tamarins (Saguinus labiatus) infected intravenously with a tamarinadapted HAV variant. Similarly, Asher et al. (2) demonstrated the presence of HAV antigen in the cytoplasm of epithelial cells lining small intestinal crypts within 3 days of the oral inoculation of New World owl monkeys (Aotus trivirgatus) with virus. In these animals, virus was present in the gut prior to its d...

Functional Analysis of Picornavirus 2B Proteins: Effects on Calcium Homeostasis and Intracellular Protein Trafficking

“…Moreover, it did not inhibit protein trafficking through the secretory pathway. The latter finding is consistent with the hypothesis that HAV uses the secretory pathway to leave the cell, as virus release of the cell culture-adapted HAV strain from polarized epithelial cells was shown to be sensitive to trafficking-inhibiting drugs (6). Analysis of the subcellular localization of HAV 2B demonstrated that it localized predominantly in the ER and was absent from the Golgi complex.…”

Hepatocytes Traffic and Export Hepatitis B Virus Basolaterally by Polarity-Dependent Mechanisms

“…Epstein-Barr virus, for example, is exported nonvectorially in mucosal epithelial cells (30), correlating with its local and systemic infection, while rotavirus infects intestinal cells apically and is released via the same domain into the gastrointestinal lumen, largely limiting its serious clinical effects to the gastrointestinal tract (9). Recently, we showed that hepatitis A virus (HAV) was exported from the basolateral domain of polarized hepatocytes (26), contrary to the expected biliary export of the virus and in contrast to what was seen previously in similar studies using enterocyte-derived cells (3), demonstrating the importance of using relevant hepatocytederived cell lines to study interactions for hepatotropic viruses.…”

Effects of Picornavirus 3A Proteins on Protein Transport and GBF1-Dependent COP-I Recruitment

“…However, the mechanisms by which the replication complexes are formed differ among the different picornaviruses, as shown by different sensitivities towards the inhibitory action of the drug brefeldin A (BFA). Enterovirus, rhinovirus, and HAV replication is blocked by BFA, whereas BFA has little effect on replication of EMCV and FMDV (6,22,29,39). For enteroviruses, it has been suggested that these rearranged membranes are derived from the secretory pathway through the action of the viral 2BC protein (5), possibly in conjunction with the 3A protein (44).…”