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Hepatitis E Virus Produced from Cell Culture Has a Lipid Envelope.

Qi Y, Zhang F, Zhang L, Harrison TJ, Huang W, Zhao C, Kong W, Jiang C, Wang Y - PLoS ONE (2015)

Bottom Line: The common feature of the two samples after ultracentrifugation was that the ORF2 protein mainly remained in the top fractions.The ORF2 protein in this fraction can bind to and protect HEV RNA from digestion by RNase A.Both were infectious in PLC/PRF/5 cells.

View Article: PubMed Central - PubMed

Affiliation: National Engineering Laboratory for AIDS Vaccine, Jilin University, Changchun, 130012, China; Division of HIV/AIDS and Sexually-Transmitted Virus Vaccines, National Institutes for Food and Drug Control, Beijing, 100050, China.

ABSTRACT
The absence of a productive cell culture system hampered detailed analysis of the structure and protein composition of the hepatitis E virion. In this study, hepatitis E virus from a robust HEV cell culture system and from the feces of infected monkeys at the peak of virus excretion was purified by ultra-centrifugation. The common feature of the two samples after ultracentrifugation was that the ORF2 protein mainly remained in the top fractions. The ORF2 protein from cell culture system was glycosylated, with an apparent molecular weight of 88 kDa, and was not infectious in PLC/PRF/5 cells. The ORF2 protein in this fraction can bind to and protect HEV RNA from digestion by RNase A. The RNA-ORF2 product has a similar sedimentation coefficient to the virus from feces. The viral RNA in the cell culture supernatant was mainly in the fraction of 1.15 g/cm3 but that from the feces was mainly in the fraction of 1.21 g/cm3. Both were infectious in PLC/PRF/5 cells. And the fraction in the middle of the gradient (1.06 g/cm3) from the cell culture supernatant,but not that from the feces, also has ORF2 protein and HEV RNA but was not infectious in PLC/PRF/5.The infectious RNA-rich fraction from the cell culture contained ORF3 protein and lipid but the corresponding fraction from feces had no lipid and little ORF3 protein. The lipid on the surface of the virus has no effect on its binding to cells but the ORF3 protein interferes with binding. The result suggests that most of the secreted ORF2 protein is not associated with HEV RNA and that hepatitis E virus produced in cell culture differs in structure from the virus found in feces in that it has a lipid envelope.

No MeSH data available.


Related in: MedlinePlus

Specific Competitive Inhibition EMSA of HEV RNA and captured ORF2 protein.The biotin-RNA without ORF2 protein was as negative control. The assay was carried out as described under “Materials and Methods”. The non-biotin labeled RNA in lane 2 and 3 were 4 fold and 8 fold to biotin labeled RNA. The lane 4 was shown the nonspecific competitive inhibition of 0.2μg total PLC/PRF/5 cell RNA.
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pone.0132503.g005: Specific Competitive Inhibition EMSA of HEV RNA and captured ORF2 protein.The biotin-RNA without ORF2 protein was as negative control. The assay was carried out as described under “Materials and Methods”. The non-biotin labeled RNA in lane 2 and 3 were 4 fold and 8 fold to biotin labeled RNA. The lane 4 was shown the nonspecific competitive inhibition of 0.2μg total PLC/PRF/5 cell RNA.

Mentions: We carried out a competitive inhibition electrophoretic mobility shift assay to elucidate the binding specificity of captured ORF2 and HEV RNA. Fig 5 shows the HEV RNA binding activity of captured ORF2 protein as a function of increasing amount of non-biotin-labeled HEV RNA as a competitor. The 80% competition was observed in the presence of 4-fold non-biotin-labeled HEV RNA, and the binding activity of the biotin-RNA with the captured ORF2 protein was replaced completely with non- biotin-RNA in the presence of 8-fold non-biotin-labeled HEV RNA. The biotin-RNAs without binding proteins were partially degraded with the enzymes produced by lysed cells. The total RNA of PLC/PRF/5 does not affect the binding action because of the lack of specific binding sites.


Hepatitis E Virus Produced from Cell Culture Has a Lipid Envelope.

Qi Y, Zhang F, Zhang L, Harrison TJ, Huang W, Zhao C, Kong W, Jiang C, Wang Y - PLoS ONE (2015)

Specific Competitive Inhibition EMSA of HEV RNA and captured ORF2 protein.The biotin-RNA without ORF2 protein was as negative control. The assay was carried out as described under “Materials and Methods”. The non-biotin labeled RNA in lane 2 and 3 were 4 fold and 8 fold to biotin labeled RNA. The lane 4 was shown the nonspecific competitive inhibition of 0.2μg total PLC/PRF/5 cell RNA.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4498737&req=5

pone.0132503.g005: Specific Competitive Inhibition EMSA of HEV RNA and captured ORF2 protein.The biotin-RNA without ORF2 protein was as negative control. The assay was carried out as described under “Materials and Methods”. The non-biotin labeled RNA in lane 2 and 3 were 4 fold and 8 fold to biotin labeled RNA. The lane 4 was shown the nonspecific competitive inhibition of 0.2μg total PLC/PRF/5 cell RNA.
Mentions: We carried out a competitive inhibition electrophoretic mobility shift assay to elucidate the binding specificity of captured ORF2 and HEV RNA. Fig 5 shows the HEV RNA binding activity of captured ORF2 protein as a function of increasing amount of non-biotin-labeled HEV RNA as a competitor. The 80% competition was observed in the presence of 4-fold non-biotin-labeled HEV RNA, and the binding activity of the biotin-RNA with the captured ORF2 protein was replaced completely with non- biotin-RNA in the presence of 8-fold non-biotin-labeled HEV RNA. The biotin-RNAs without binding proteins were partially degraded with the enzymes produced by lysed cells. The total RNA of PLC/PRF/5 does not affect the binding action because of the lack of specific binding sites.

Bottom Line: The common feature of the two samples after ultracentrifugation was that the ORF2 protein mainly remained in the top fractions.The ORF2 protein in this fraction can bind to and protect HEV RNA from digestion by RNase A.Both were infectious in PLC/PRF/5 cells.

View Article: PubMed Central - PubMed

Affiliation: National Engineering Laboratory for AIDS Vaccine, Jilin University, Changchun, 130012, China; Division of HIV/AIDS and Sexually-Transmitted Virus Vaccines, National Institutes for Food and Drug Control, Beijing, 100050, China.

ABSTRACT
The absence of a productive cell culture system hampered detailed analysis of the structure and protein composition of the hepatitis E virion. In this study, hepatitis E virus from a robust HEV cell culture system and from the feces of infected monkeys at the peak of virus excretion was purified by ultra-centrifugation. The common feature of the two samples after ultracentrifugation was that the ORF2 protein mainly remained in the top fractions. The ORF2 protein from cell culture system was glycosylated, with an apparent molecular weight of 88 kDa, and was not infectious in PLC/PRF/5 cells. The ORF2 protein in this fraction can bind to and protect HEV RNA from digestion by RNase A. The RNA-ORF2 product has a similar sedimentation coefficient to the virus from feces. The viral RNA in the cell culture supernatant was mainly in the fraction of 1.15 g/cm3 but that from the feces was mainly in the fraction of 1.21 g/cm3. Both were infectious in PLC/PRF/5 cells. And the fraction in the middle of the gradient (1.06 g/cm3) from the cell culture supernatant,but not that from the feces, also has ORF2 protein and HEV RNA but was not infectious in PLC/PRF/5.The infectious RNA-rich fraction from the cell culture contained ORF3 protein and lipid but the corresponding fraction from feces had no lipid and little ORF3 protein. The lipid on the surface of the virus has no effect on its binding to cells but the ORF3 protein interferes with binding. The result suggests that most of the secreted ORF2 protein is not associated with HEV RNA and that hepatitis E virus produced in cell culture differs in structure from the virus found in feces in that it has a lipid envelope.

No MeSH data available.


Related in: MedlinePlus