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Acetylated hepatitis E virus capsid protein orf2 and use 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: 2022-07-15
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 hepatitis E virus capsid protein orf2 and use thereof
  • Acetylated hepatitis E virus capsid protein orf2 and use thereof
  • Acetylated hepatitis E virus capsid protein orf2 and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0227] Example 1: HEV capsid protein ORF2 is acetylated at the conserved amino acid residue K411.

[0228] The N-terminus of ORF2 is not required for its antigenicity and viral particle assembly (39). ORF2 (112-660) shares the same biological properties as the wild-type virus (40). Therefore, using this truncated ORF2, the interaction of HEV ORF2 with host proteins was studied and multiple ORF2-interacting molecules were successfully identified (12). To test whether ORF2 undergoes post-translational modifications, including acetylation, GFP-tagged genotype 1 and 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 subjected to acetylation mass spectrometry ( figure 1 A, 1C, 1D). The mass spectrometry results showed that GFP-1-ORF2 and GFP-4-ORF2 in the same peptide EPTVK 411 Acetylated at position K411 of LYTSVEN, this peptide is a highly conserved sequence among 9 exa...

Embodiment 2

[0230] Example 2: Formation of HEV inclusion bodies is dependent on K411 acetylation of ORF2

[0231] Since lysine acetylation plays an important role in the life cycle of the virus, it was decided to evaluate the function of K411 acetylation of HEV ORF2. With wild-type 1-ORF2(112-660), 4-ORF2(112-660), mutant 1-ORF2 fused with GFP tag K411R (112-660), or 4-ORF2 K411R (112-660), HeLa cells were transfected. Cells were fixed, immunofluorescently stained with anti-GFP and anti-tubulin antibodies, and DNA was stained by DAPI. Fluorescence microscopy determined the localization of ORF2 in cells. Surprisingly, GFP-1-ORF2 K411R (112-660) and GFP-4-ORF2 K411R (112-660) were mainly uniformly dispersed in the cytoplasm, whereas both genotype 1 and genotype 4 wild-type GFP-ORF2 (112-660) showed strong inclusion body formation ( figure 2 A, 2B). To test whether the enhanced inclusion body formation is due to N-terminal truncation, full-length ORF2 (ORF2(1-660)) was tested, yieldi...

Embodiment 3

[0234] Example 3: Dynamic assessment of the effect of mutation K411R on inclusion body formation

[0235] It appears that acetylation of HEV ORF2 is involved in regulating inclusion body formation ( figure 2 ). However, the process of inclusion body formation of HEV ORF2 remains unknown. To determine the effect of K411 acetylation on this process, wild-type ORF2 and acetylation-depleted ORF2 were subjected to live-cell imaging in HeLa cells to analyze ORF2 inclusion body formation. 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 a few detectable inclusions formed after approximately 30-90 min ( image 3 A). After that, over time, the assembly yielded stronger and stronger bulky inclusion bodies ( image 3 A). These results suggest that acetylation of K411 greatly affects the self-assembly of ORF2 inclusion bodies. To test whether acetylation is one...

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Abstract

The present invention relates to acetylated hepatitis E virus ORF2 protein, and methods and cell cultures for increasing the production of the acetylated ORF2 protein. The present invention also relates to the use of the acetylated ORF2 protein for promoting ORF2 multimerization and / or virus-like particle assembly.

Description

technical field [0001] The present invention relates to the fields of molecular biology and virology. Specifically, the present invention relates to the acetylation of the Hepatitis E virus (HEV) capsid protein ORF2, and a method for modulating the acetylation level of the Hepatitis E virus ORF2 polypeptide. The present invention also relates to the use of inhibitors of HDAC6 for 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 viral infection by altering the acetylation level of HEV ORF2 in cells. Background technique [0002] Hepatitis E virus (HEV) infects humans and animals worldwide, causing enteric-transmitted viral hepatitis with a fatality rate of 25% in pregnant women. Hepatitis E virus sequences from patients with intestinally transmitted non-A non-B hepatitis were reported in 1989 and were similar to sequences isolated from pigs, r...

Claims

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Application Information

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Patent Type & Authority Patents(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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