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Acetylated HEV (Hepatitis E Virus) capsid protein ORF2 (Open Reading Frame 2) and application thereof

A technology for acetylating type E and hepatitis viruses, which is applied in the fields of molecular biology and virology, and can solve problems such as unknown host regulation.

Active Publication Date: 2018-11-06
NAT INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although ORF2 is important in the HEV life cycle, how it is regulated in the host remains unclear

Method used

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  • Acetylated HEV (Hepatitis E Virus) capsid protein ORF2 (Open Reading Frame 2) and application thereof
  • Acetylated HEV (Hepatitis E Virus) capsid protein ORF2 (Open Reading Frame 2) and application thereof
  • Acetylated HEV (Hepatitis E Virus) capsid protein ORF2 (Open Reading Frame 2) and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0224] Example 1: HEV capsid protein ORF2 is acetylated at the conserved amino acid residue K411.

[0225] The N-terminus of ORF2 is not essential for its antigenicity and virion assembly (39). ORF2(112-660) shares the same biological properties as wild-type virus (40). Therefore, using this truncated ORF2, the interaction of HEV ORF2 with host proteins was studied and several molecules interacting with ORF2 were successfully identified (12). To test whether ORF2 undergoes post-translational modifications, including acetylation, GFP-tagged genotype 1 and type 4 ORF2 truncated fragments (GFP-1-ORF2(112-660) and GFP-4-ORF2(112-660)) Introduced into 293T cells. Then, ORF2 was immunoprecipitated using GFP antibody and analyzed by acetylation mass spectrometry ( figure 1 A, 1C, 1D). Mass spectrometry results showed that GFP-1-ORF2 and GFP-4-ORF2 were in the same peptide EPTVK 411 The K411 position of LYTSVEN is acetylated, and this peptide is a very conserved sequence in the n...

Embodiment 2

[0227] Example 2: HEV inclusion body formation depends on K411 acetylation of ORF2

[0228] Due to the important role of lysine acetylation in the viral life cycle, it was decided to evaluate the function of K411 acetylation of HEV ORF2. Wild-type 1-ORF2(112-660), 4-ORF2(112-660), mutant 1-ORF2 fused with GFP marker K411R (112-660), or 4-ORF2 K411R (112-660), transfected HeLa cells. Cells were fixed, immunofluorescence stained with anti-GFP antibody and anti-tubulin antibody, and DNA was stained by DAPI. Fluorescence microscopy to determine the localization of ORF2 in cells. It was surprisingly found that GFP-1-ORF2 K411R (112-660) and GFP-4-ORF2 K411R (112-660) was mainly dispersed homogeneously in the cytoplasm, while wild-type GFP-ORF2(112-660) of genotype 1 and genotype 4 both showed strong inclusion body formation ( figure 2 A, 2B). To test whether the enhanced inclusion body formation was due to N-terminal truncation, the full-length ORF2 (ORF2(1-660)) was tested...

Embodiment 3

[0231] Example 3: Dynamic evaluation of the effect of mutation K411R on inclusion body formation

[0232] It appears that acetylation of HEV ORF2 is involved in regulating inclusion body formation ( figure 2 ). However, the inclusion body formation process of HEV ORF2 remains unknown. To determine the impact of K411 acetylation on this process, wild-type ORF2 and acetylation-deficient ORF2 were subjected to live-cell imaging in HeLa cells, and ORF2 inclusion body formation was analyzed. Live cell imaging started 12 hours after GFP-1-ORF2(112-660) transfection and continued for up to 8 hours. Cells had few detectable inclusions at the start of imaging and few detectable inclusions formed after approximately 30-90 minutes ( image 3 A). Afterwards, as time went on, the assembly produced stronger and stronger large-volume inclusion bodies ( image 3 A). These results indicate that acetylation of K411 greatly affects the self-assembly of ORF2 inclusion bodies. To test whet...

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Abstract

The invention relates to acetylated HEV (Hepatitis E Virus) capsid protein ORF2 (Open Reading Frame 2), a method for increasing the production of the acetylated HEV capsid protein ORF2 and a cell culture. The invention also relates to application of the acetylated HEV capsid protein ORF2 to promotion of ORF2 polymerizaiton and / or virus-like particle assembling.

Description

technical field [0001] The present invention relates to the fields of molecular biology and virology. Specifically, the present invention relates to acetylation of hepatitis E virus (Hepatitis E virus, HEV) capsid protein ORF2, and a method for regulating the acetylation level of HEV ORF2 polypeptide. The present invention also relates to the use of inhibitors of HDAC6 in improving ORF2 polypeptide stability, HEV virus formation, HEV virus replication and HEV virus-like particle formation. The present invention also relates to methods of treating HEV virus infection by altering the acetylation level of HEV ORF2 in cells. Background technique [0002] Hepatitis E virus (HEV) infects humans and animals worldwide, causing enterically transmitted viral hepatitis that is fatal in 25% of pregnant women. Hepatitis E virus sequences from patients with enterically transmitted non-A non-B hepatitis were reported in 1989 and were similar to sequences isolated from pigs, rabbits, rats...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/08C12N15/86C12N5/10A61K38/16A61K39/29A61P1/16A61P31/14
CPCA61K39/12A61P1/16A61P31/14C12N15/86C07K14/005A61K38/00C12N2770/28134C12N2770/28122C12N2770/28123C12N2770/28143C12N2770/28151
Inventor 王佑春许楠黄维金赵晨燕张黎
Owner NAT INST FOR FOOD & DRUG CONTROL
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