Attenuation Phenotype of a Cell Culture-Adapted Variant of Hepatitis A Virus (HM175/p16) in Susceptible New World Owl Monkeys

Taylor, Kerry L.; Murphy, Paula C.; Asher, Ludmila V.; LeDuc, James W.; Lemon, Stanley M.

doi:10.1093/infdis/168.3.592

Cited by 15 publications

(16 citation statements)

References 25 publications

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“…The virus that we used, HM175/p16, is adapted to growth in cell culture and relatively attenuated in its capacity to cause disease in primates and causes no detectable cytopathic effect in cell culture (20, 21). The FITC-dextran diffusion rate was not increased by infection over a period of 7 days (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Biliary Secretion of Quasi-Enveloped Human Hepatitis A Virus

Hirai-Yuki

Hensley

Whitmire

et al. 2016

mBio

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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.

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Section: Resultsmentioning

confidence: 99%

“…A noncytopathic, cell culture-adapted variant of the HM175 strain of HAV, HM175/p16 (20, 21), was grown in Huh-7.5 cells (42), which were derived from a human hepatoma. Supernatant fluids were clarified by low-speed centrifugation and frozen in aliquots as virus stock.…”

Section: Methodsmentioning

confidence: 99%

Biliary Secretion of Quasi-Enveloped Human Hepatitis A Virus

Hirai-Yuki

Hensley

Whitmire

et al. 2016

mBio

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“…Huh-7.5 human hepatoma cells (35) and FRhK-4 fetal rhesus kidney cells (36) were cultured in DMEM supplemented with 10% FBS, except as otherwise noted. HM175/p16 virus, a low-passage, noncytopathic, cell cultureadapted variant of the HM175 strain of HAV, has been described previously (37,38). An infectious molecular clone of its genome, pHM175p16.2 (GenBank accession no.…”

Section: Methodsmentioning

confidence: 99%

Protein composition of the hepatitis A virus quasi-envelope

McKnight

Xie

González‐López

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

124

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The Picornaviridae are a diverse family of RNA viruses including many pathogens of medical and veterinary importance. Classically considered "nonenveloped," recent studies show that some picornaviruses, notably hepatitis A virus (HAV; genus Hepatovirus) and some members of the Enterovirus genus, are released from cells nonlytically in membranous vesicles. To better understand the biogenesis of quasienveloped HAV (eHAV) virions, we conducted a quantitative proteomics analysis of eHAV purified from cell-culture supernatant fluids by isopycnic ultracentrifugation. Amino acid-coded mass tagging (AACT) with stable isotopes followed by tandem mass spectrometry sequencing and AACT quantitation of peptides provided unambiguous identification of proteins associated with eHAV versus unrelated extracellular vesicles with similar buoyant density. Multiple peptides were identified from HAV capsid proteins (53.7% coverage), but none from nonstructural proteins, indicating capsids are packaged as cargo into eHAV vesicles via a highly specific sorting process. Other eHAVassociated proteins (n = 105) were significantly enriched for components of the endolysosomal system (>60%, P < 0.001) and included many common exosome-associated proteins such as the tetraspanin CD9 and dipeptidyl peptidase 4 (DPP4) along with multiple endosomal sorting complex required for transport III (ESCRT-III)-associated proteins. Immunoprecipitation confirmed that DPP4 is displayed on the surface of eHAV produced in cell culture or present in sera from humans with acute hepatitis A. No LC3-related peptides were identified by mass spectrometry. RNAi depletion studies confirmed that ESCRT-III proteins, particularly CHMP2A, function in eHAV biogenesis. In addition to identifying surface markers of eHAV vesicles, the results support an exosome-like mechanism of eHAV egress involving endosomal budding of HAV capsids into multivesicular bodies.exosome | multivesicular body | ESCRT | extracellular vesicle | picornavirus T he Picornaviridae are a large and diverse family of positivestrand RNA viruses that include numerous pathogens (1). Classically considered "nonenveloped," these viruses package their single-stranded genomes within stable icosahedral protein capsids that are released from cells following cell lysis. However, recent work has revealed that several picornaviruses gain egress from cells in a nonlytic fashion within the lumen of extracellular vesicles shed from the cell. Most notably, hepatitis A virus (HAV; genus Hepatovirus), an important cause of enterically transmitted hepatitis in humans, replicates without cytopathic effect and is released from cells as membrane-wrapped, "quasienveloped" (eHAV) virions similar in size and density to exosomes (2). eHAV virions have been identified in sera collected from persons with acute hepatitis A, as well as in supernatant fluids of infected cell cultures. These virions are completely cloaked in host-derived membranes that protect the capsid from neutralizing antibody until the quasi-envelope is degraded wi...

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“…Generally, it is accompanied by more efficient replication and increased yields of virus. Several cell culture-adapted HAV variants are attenuated for their ability to induce liver injury in primates, although cell culture adaptation and attenuation are distinct phenotypes (29,40,42).The genomes of several cell culture-adapted HAV strains have been completely sequenced (7,18,24). One of these cell culture-adapted viruses, HM175/P16, differs from its wt parent, HM175/wt, by only 19 nucleotide (nt) substitutions scattered throughout the entire genome (24).…”

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confidence: 99%

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Functional Significance of the Interaction of Hepatitis A Virus RNA with Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH): Opposing Effects of GAPDH and Polypyrimidine Tract Binding Protein on Internal Ribosome Entry Site Function

Schultz

Lemon

2000

J Virol

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Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a cellular enzyme involved in glycolysis, binds specifically to several viral RNAs, but the functional significance of this interaction is uncertain. Both GAPDH and polypyrimidine tract binding protein (PTB) bind to overlapping sites in stem-loop IIIa of the internal ribosome entry site (IRES) of Hepatitis A virus (HAV), a picornavirus. Since the binding of GAPDH destabilizes the RNA secondary structure, we reasoned that GAPDH may suppress the ability of the IRES to direct cap-independent translation, making its effects antagonistic to the translation-enhancing activity of PTB (D. E. Schultz, C. C. Hardin, and S. M. Lemon, J. Biol. Chem. 271:14134-14142, 1996). To test this hypothesis, we constructed plasmids containing a dicistronic transcriptional unit in which the HAV IRES was placed between an upstream GAPDH-coding sequence and a downstream Renilla luciferase (RLuc) sequence. Transfection with this plasmid results in overexpression of GAPDH and in RLuc production as a measure of IRES activity. RLuc activity was compared with that from a control, null-expression plasmid that was identical except for a frameshift mutation within the 5 GAPDH coding sequence. In transfection experiments, GAPDH overexpression significantly suppressed HAV IRES activity in BSC-1 and FRhK-4 cells but not in Huh-7 cells, which have a significantly greater cytoplasmic abundance of PTB. GAPDH suppression of HAV translation was greater with the wild-type HAV IRES than with the IRES from a cell culture-adapted virus (HM175/P16) that has reproducibly higher basal translational activity in BSC-1 cells. Stem-loop IIIa RNA from the latter IRES had significantly lower affinity for GAPDH in filter binding experiments. Thus, the binding of GAPDH to the IRES of HAV suppresses cap-independent viral translation in vivo in African green monkey kidney cells. The enhanced replication capacity of cell culture-adapted HAV in such cells may be due in part to reduced affinity of the viral IRES for GAPDH.Hepatitis A virus (HAV) is a plus-strand RNA virus that is classified within the genus Hepatovirus of the family Picornaviridae. The genomic RNA of HAV contains a single long open reading frame encoding a polyprotein which is proteolytically processed by a virus-encoded proteinase (27). Like other picornavirus RNAs, it has a relatively lengthy 5Ј-terminal nontranslated RNA segment (5ЈNTR) and a shorter 3ЈNTR with a 3Ј terminal poly(A) tail. As in all picornaviruses, the 5ЈNTR of HAV forms a highly ordered RNA structural element (the internal ribosome entry site [IRES]) that is responsible for directing the cap-independent translation of the polyprotein (5,23,32). This occurs by a unique mechanism that involves internal entry of the ribosome on the RNA hundreds of bases downstream of the 5Ј end and is absolutely dependent on the secondary and probably the tertiary structure of the 5ЈNTR. Although HAV can be propagated in conventional cell culture systems, the replication of wild-type (wt) virus is slow and ...

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Attenuation Phenotype of a Cell Culture-Adapted Variant of Hepatitis A Virus (HM175/p16) in Susceptible New World Owl Monkeys

Cited by 15 publications

References 25 publications

Biliary Secretion of Quasi-Enveloped Human Hepatitis A Virus

Biliary Secretion of Quasi-Enveloped Human Hepatitis A Virus

Protein composition of the hepatitis A virus quasi-envelope

Functional Significance of the Interaction of Hepatitis A Virus RNA with Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH): Opposing Effects of GAPDH and Polypyrimidine Tract Binding Protein on Internal Ribosome Entry Site Function

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