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Principal Investigator: Stanley M. Lemon
Organization: UNIV OF NORTH CAROLINA CHAPEL HILL
Fiscal Year: 2024
Award: $518,380
Funding agency: National Institute of Allergy and Infectious Diseases
PROJECT ABSTRACT
Hepatitis A virus (HAV) is an unusual picornavirus (genus Hepatovirus) that is released without lysis from infected
hepatocytes within small extracellular vesicles (EVs) resembling exosomes. These membrane-cloaked virions,
or `quasi-enveloped' HAV (eHAV), lack virus-encoded glycoprotein peplomers on their surface, yet they are
infectious and the only form of the virus found circulating in the blood of persons with acute hepatitis A. Numerous
other `non-enveloped' viruses have been found to be released from cells as quasi-enveloped virions in EVs of
varying size, making studies of eHAV relevant to a broad range of viral pathogens. This application proposes to
investigate two aspects of lipid metabolism found to be essential for productive hepatovirus infection in a
genome-wide forward genetic screen, and will test the following hypotheses: (i) that GD3 and possibly other
gangliosides play a crucial role in cellular entry and the initiation of infection by both naked HAV and quasi-
enveloped eHAV, by binding to and possibly triggering uncoating of the capsid within late endosomes or
endolysosomes, and (ii) that HAV-infected cells and quasi-enveloped eHAV virions are highly enriched in
sphingolipids with very long-chain fatty acid (VLCFA, C≥22) tails, and that VLCFA synthesis is required for
efficient production and release of infectious eHAV. In Aim 1, exceptional lipidomics expertise will be brought to
bear on the question of which ganglioside species are necessary and sufficient for HAV and eHAV to enter cells
and initiate infection, and will define the carbohydrate headgroups and acyl tail structures that are optimal for
restoring the capacity of virus to infect CRISPR-derived ceramide glucosyltransferase (UGCG) knockout cells.
The proposed experiments will also determine the impact of UGCG knockout on endocytosis and intracellular
trafficking of HAV and eHAV, and the subcellular localization of exogenous gangliosides that restore the capacity
of virus to enter these cells and initiate infection. Aim 2 will identify which ganglioside classes bind specifically to
the naked HAV capsid, and whether ganglioside binding destabilizes its structure, lessening its thermostability
at neutral or acidic pH, as a surrogate measure of uncoating. Aim 3 will characterize the role played by VLCFA
in the hepatovirus lifecycle, and determine the degree to which VLCFA are enriched in infected cells, and in
eHAV virions versus non-viral exosomes, and whether VLCFA are required for the production and cellular
release of infectious eHAV, or for assembly of replication organelles involved in the synthesis of viral RNA.
Additional experiments will determine whether and how HAV infection upregulates VLCFA synthetic flux, and
whether this is essential for productive infection. By studying the role of lipids in hepatovirus replication, the
proposed research will address an unexplored and neglected facet of the pathobiology of this important human
pathogen.
Terms: <Acute Hepatitis><Address><Antibodies><Asialogangliosides><Assay><Binding><Bioassay><Biological Assay><Biology><Blood><Blood Reticuloendothelial System><CRISPR><CRISPR editing screen><CRISPR screen><CRISPR-based screen><CRISPR/Cas system><CRISPR/Cas9 screen><Capsid><Capsid Proteins><Carbohydrates><Carbon><Cell Body><Cells><Ceramide glucosyltransferase><Cessation of life><Clustered Regularly Interspaced Short Palindromic Repeats><Coat Proteins><Complex><Cytolysis><Data><Death><Disease Outbreaks><Endocytosis><Enteral><Enteric><Enzyme Gene><Enzymes><Family Picornaviridae><Fatty Acids><Gangliosides><Gene Transcription><Genes><Genetic><Genetic Screening><Genetic Transcription><GlcCer synthase><Glucocerebrosides><Glucosyl Ceramides><Glucosylceramides><Glycoproteins><Glycosphingolipids><Hepatic Cells><Hepatic Parenchymal Cell><Hepatitis A><Hepatitis A Virus><Hepatocyte><Hepatovirus><Host Factor><Host Factor Protein><Human><Infection><Infection prevention><Infectious Hepatitis Virus><Integration Host Factors><Investigators><Knock-out><Knockout><Laboratories><Life Cycle><Life Cycle Stages><Lipids><Liver Cells><Lysis><Maps><Measures><Membrane><Modern Man><Molecular Interaction><N-Acetylneuraminic Acids><Non-Polyadenylated RNA><Organelles><Outbreaks><Persons><Picornaviridae><Picornaviruses><Play><Prevent infection><Production><Productivity><Proteins><RNA><RNA Expression><RNA Gene Products><Replication Unit><Replicon><Reproducibility><Research><Research Personnel><Researchers><Ribonucleic Acid><Role><Sialic Acids><Sialoglycosphingolipids><Site><Sphingoglycolipids><Sphingolipids><Structure><Surface><Tail><Testing><Transcription><Transfection><UDP-glucose-N-acylsphingosine glucosyltransferase><UDP-glucose-ceramide glucosyltransferase><UDPglucose-ceramide glucosyltransferase><United States><Very Long Chain Fatty Acid><Viral><Viral Coat Proteins><Viral Diseases><Viral Outer Coat Protein><Viral hepatitis><Virion><Virus><Virus Diseases><Virus Particle><Virus Replication><ceramide UDPG glucosyltransferase><ceramide synthase><cerebroside synthase><clustered regularly interspaced short palindromic repeats screen><dihydroceramide desaturase><exosome><experiment><experimental research><experimental study><experiments><extracellular vesicles><fat metabolism><genome scale><genome-wide><genomewide><glucocerebroside synthetase><glucosylceramide synthase><glycosylceramide synthetase><hepatitis virus infection><human pathogen><late endosome><life course><lipid metabolism><lipidomics><member><membrane structure><neglect><new approaches><novel approaches><novel strategies><novel strategy><pathogenic virus><permissiveness><social role><thermolability><thermostability><trafficking><viral RNA><viral infection><viral multiplication><viral pathogen><viral replication><viral transmission><virus RNA><virus infection><virus multiplication><virus pathogen><virus transmission><virus-induced disease>