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Methods to modulate hepatitis E virus assembly and capsid protein orf2 stability

A virus and protein technology, applied in the fields of molecular biology and virology, can solve problems such as unknown host regulation

Active Publication Date: 2022-07-08
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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  • Methods to modulate hepatitis E virus assembly and capsid protein orf2 stability
  • Methods to modulate hepatitis E virus assembly and capsid protein orf2 stability
  • Methods to modulate hepatitis E virus assembly and capsid protein orf2 stability

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0223] Example 1: HEV capsid protein ORF2 is acetylated at the conserved amino acid residue K411.

[0224] 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

[0226] Example 2: Formation of HEV inclusion bodies is dependent on K411 acetylation of ORF2

[0227] 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

[0230] Example 3: Dynamic assessment of the effect of mutation K411R on inclusion body formation

[0231] 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, ORF2 inclusion body formation was analyzed by live-cell imaging of wild-type ORF2 and acetylation-depleted ORF2 in HeLa cells. 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 of the de...

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Abstract

The present invention relates to methods for improving hepatitis E virus production, HEV virus replication, viral ORF2 protein stability, and HEV virus-like particle formation by HDAC6 inhibitors. The present invention also relates to cell culture systems, and cell cultures for the production of hepatitis E virus, virus-like particles and ORF2 protein. In addition, the present invention also relates to methods of treating HEV virus infection by increasing the expression and / or activity of HDAC6 in cells.

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): C12N7/00C12N5/10C07K14/18A61K39/29A61K45/00A61P31/14
CPCA61K39/12A61K45/00A61P31/14C12N7/00C07K14/005A61K2039/5258C12N2770/24252C12N2770/24222C12N2770/24223Y02A50/30
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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