Hepatitis E Virus Produced from Cell Culture Has a Lipid Envelope

Area of Science:

• Virology
• Molecular Biology
• Biochemistry

Background:

• A productive cell culture system for Hepatitis E virus (HEV) was lacking, hindering detailed structural and protein composition analysis.
• Understanding HEV structure is crucial for developing effective diagnostics and therapeutics.

Purpose of the Study:

• To compare the structural characteristics and protein composition of HEV produced in cell culture versus that found in infected primate feces.
• To investigate the role of specific viral proteins and lipids in HEV infectivity and cell binding.

Main Methods:

• Purification of HEV from cell culture supernatant and infected monkey feces using ultra-centrifugation.
• Analysis of viral fractions using density gradient centrifugation and molecular weight determination.
• Infectivity assays in PLC/PRF/5 cells.
• Assessment of protein-RNA interactions and lipid association.

Main Results:

• HEV ORF2 protein, a major structural component, was found in the top fractions of both cell culture and fecal samples, with glycosylated ORF2 (88 kDa) from cell culture being non-infectious.
• HEV RNA was protected by ORF2 protein, forming RNA-ORF2 complexes with similar sedimentation coefficients to fecal HEV.
• Infectious viral RNA fractions differed in density (1.15 g/cm³ for cell culture vs. 1.21 g/cm³ for feces).
• Cell culture-derived infectious HEV contained ORF3 protein and lipid, while fecal HEV had minimal ORF3 and no detectable lipid.
• Lipid on the viral surface did not affect cell binding, but ORF3 protein interfered with it.

Conclusions:

• Most secreted HEV ORF2 protein is not associated with HEV RNA.
• HEV produced in cell culture exhibits structural differences compared to fecal HEV, notably the presence of a lipid envelope and infectious ORF3 protein in the latter.
• ORF3 protein plays a role in modulating HEV binding to host cells.