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. 2016 Dec 6;7(6):e01998-16.
doi: 10.1128/mBio.01998-16.

Biliary Secretion of Quasi-Enveloped Human Hepatitis A Virus

Asuka Hirai-Yuki  1   2 , Lucinda Hensley  1 , Jason K Whitmire  1   3 , Stanley M Lemon  4   2   5
Affiliations

Biliary Secretion of Quasi-Enveloped Human Hepatitis A Virus

Asuka Hirai-Yuki et al. mBio. .

Abstract

Hepatitis A virus (HAV) is an unusual picornavirus that is released from cells cloaked in host-derived membranes. These quasi-enveloped virions (eHAV) are the only particle type circulating in blood during infection, whereas only nonenveloped virions are shed in feces. The reason for this is uncertain. Hepatocytes, the only cell type known to support HAV replication in vivo, are highly polarized epithelial cells with basolateral membranes facing onto hepatic (blood) sinusoids and apical membranes abutting biliary canaliculi from which bile is secreted to the gut. To assess whether eHAV and nonenveloped virus egress from cells via vectorially distinct pathways, we studied infected polarized cultures of Caco-2 and HepG2-N6 cells. Most (>99%) progeny virions were released apically from Caco-2 cells, whereas basolateral (64%) versus apical (36%) release was more balanced with HepG2-N6 cells. Both apically and basolaterally released virions were predominantly enveloped, with no suggestion of differential vectorial release of eHAV versus naked virions. Basolateral to apical transcytosis of either particle type was minimal (<0.02%/h) in HepG2-N6 cells, arguing against this as a mechanism for differences in membrane envelopment of serum versus fecal virus. High concentrations of human bile acids converted eHAV to nonenveloped virions, whereas virus present in bile from HAV-infected Ifnar1-/- Ifngr1-/- and Mavs-/- mice banded over a range of densities extending from that of eHAV to that of nonenveloped virions. We conclude that nonenveloped virions shed in feces are derived from eHAV released across the canalicular membrane and stripped of membranes by the detergent action of bile acids within the proximal biliary canaliculus.

Importance: HAV is a hepatotropic, fecally/orally transmitted picornavirus that can cause severe hepatitis in humans. Recent work reveals that it has an unusual life cycle. Virus is found in cell culture supernatant fluids in two mature, infectious forms: one wrapped in membranes (quasi-enveloped) and another that is nonenveloped. Membrane-wrapped virions circulate in blood during acute infection and are resistant to neutralizing antibodies, likely facilitating HAV dissemination within the liver. On the other hand, virus shed in feces is nonenveloped and highly stable, facilitating epidemic spread and transmission to naive hosts. Factors controlling the biogenesis of these two distinct forms of the virus in infected humans are not understood. Here we characterize vectorial release of quasi-enveloped virions from polarized epithelial cell cultures and provide evidence that bile acids strip membranes from eHAV following its secretion into the biliary tract. These results enhance our understanding of the life cycle of this unusual picornavirus.

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Figures

FIG 1
FIG 1
Microanatomy of the hepatocyte and three possible explanations for the presence of different forms of HAV in blood (quasi-enveloped) and feces (naked, nonenveloped). (A) Simplified diagram showing two hepatocytes in relation to the space of Disse and hepatic sinusoid that is bathed in blood and to the biliary canaliculus that encircles hepatocytes in a belt-like fashion and into which virus and bile are secreted across the hepatocyte apical membrane. Bile flows from the canaliculus to larger bile ductules and ultimately to the lumen of the gastrointestinal tract. (B to D) Competing hypotheses that could explain differences in membrane association and quasi-envelopment of virus in different compartments. See introductory text for details.
FIG 2
FIG 2
HAV infection of polarized Caco-2 cell monolayers. (A) Polarity was assessed by determining the intracellular localization of DPP4 (CD26) and TJP1 (ZO-1). Cells were seeded into chamber slides and cultured for 10 days before being fixed and stained with antibodies specific for DPP4 (red) and TJP1 (green). Nuclei were counterstained with DAPI (blue). Images of xy and xz sections were collected by confocal microscopy. z sections were compiled by taking 0.41-μm steps through each x-y section; “a” and “b” indicate approximate positions of apical and basolateral membranes, respectively. The scale bars represent 20 μm. (B) The integrity of monolayer cell cultures grown on Transwell membranes was assessed by measuring paracelluar diffusion of FITC-dextran prior to (day 0) and 7 days after virus infection. Results shown represent the mean percentage ± range of FITC-dextran delivered to the apical chamber that was detected in the basolateral chamber 60 min later (see Materials and Methods). (C) Vectorial release of quasi-enveloped HAV from polarized Caco-2 cells. After 14 days of culture on Transwell inserts, cells were infected by inoculating the apical surface with HAV. Total apical and basolateral fluids were collected at 24-h intervals, and HAV RNA was quantified by RT-qPCR. (D) The buoyant density of virus particles present in the apical (top) and basolateral (bottom) compartments of Transwell cultures on day 6 of the infection was determined by centrifugation in isopycnic iodixanol gradients followed by HAV-specific RT-qPCR.
FIG 3
FIG 3
HAV infection of polarized HepG2-N6 cell monolayers. (A) Polarity was assessed by determining the intracellular localization of DPP4 and TJP1. Cells were cultured for 17 days before being fixed and stained with specific antibodies. See the legend to Fig. 1A for additional details. (B) Paracellular diffusion of FITC-dextran in monolayer cultures of HepG2-N6 cells. See the legend to Fig. 2B for details. (C) Vectorial release of virus from HepG2-N6 cells. Cells were infected at the basolateral surface after 18 days culture on Transwell membranes. Total apical and basolateral fluids were subsequently collected at 24-h intervals. HAV RNA was quantified by RT-qPCR. (D) Buoyant densities of virions present in the apical (top) and basolateral (bottom) compartments of Transwell cultures 4 days after infection. (E) Basolateral to apical compartment transcytosis of nonenveloped (“naked”) and enveloped eHAV virions by HepG2-N6 cells. Gradient-purified HAV and eHAV (1 × 108 GE) were added to the basolateral compartment of polarized cultures of HepG2-N6 cells maintained at either 36°C or 4°C. Virus released into the apical chamber at hourly intervals was quantified by HAV-specific RT-qPCR. Results are shown as the mean percentage ± range of virus added to the basolateral chamber that underwent transcytosis in duplicate cultures during the period indicated. BLD, below the level of detection.
FIG 4
FIG 4
Stability of quasi-enveloped eHAV in human bile acids. (A) Quasi-enveloped eHAV virions were incubated with chenodeoxycholic acid (CDCA; 24 mM), taurocholic acid (TCA; 93 mM or 930 mM), or DMSO (10%) at 37°C for 2 h. The virus was then centrifuged to equilibrium in isopycnic iodixanol gradients. The buoyant density of virions was determined by measuring the quantity of HAV RNA in fractions 6 to 19 by RT-qPCR. The distribution of virus in the DMSO-treated sample matched that of virus incubated in PBS alone (not shown). The shaded zone indicates the expected density of nonenveloped HAV virions. (B) HAV capsid antigen ELISA of gradient-purified eHAV following treatment with bile acids. Virus samples (approximately 2 × 107 GE) were subjected to diafiltration to remove bile acids and then tested for the presence of detectable (exposed) capsid antigen by ELISA (see Materials and Methods). (C) Infectivity of eHAV following treatment with bile acids. Samples were subjected to diafiltration to remove bile acids and then diluted and inoculated onto FRhK-4 cells for IR-FIFA. The content of the 10−2 dilution of the CDCA-treated sample was 3.8 × 104 GE/ml, that of the 10−2 dilution of the TCA-treated sample was 3.1 × 104 GE/ml, that of the 10−2 dilution of the DMSO-treated sample was 2.3 × 104 GE/ml, and that of the 10−2 dilution of the PBS-treated sample was 3.6 × 104 GE/ml.
FIG 5
FIG 5
Buoyant densities of virions in serum and bile from infected Mavs−/− mice. (A) Supernatant fluids from infected AML12 cells were concentrated by filtration and subjected to iodixanol density gradient centrifugation. The buoyant density of virions was determined by measuring the quantity of HAV RNA in fractions by RT-qPCR. (B) Buoyant densities of virus circulating in blood of Mavs−/− mice 14 to 28 days after infection (top) and in bile from the gallbladder of Mavs−/− mice 7 days after infection (middle). Also included for reference are the previously published density of virus in feces of an infected DKO mouse (9) and previously published densities of virus in chimpanzee samples (red) (5) (bottom).
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