Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus

Yunrong Gao; Claire Ogilvie; Anirudh Raghavan; Chloe Von Hoffmann; Bo Liang

doi:10.3791/62010

JoVE Journal
Biochemistry

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JoVE Journal Biochemistry

Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus

호흡기 싱크량 바이러스의 바이러스 특이적 뉴클레오캡시드의 생성 및 조립

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09:08 min

July 27, 2021

DOI: 10.3791/62010-v

Yunrong Gao¹, Claire Ogilvie¹, Anirudh Raghavan¹, Chloe Von Hoffmann¹, Bo Liang¹

¹Department of Biochemistry,Emory University School of Medicine

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Overview

This article presents a method for the in vitro assembly of respiratory syncytial virus (RSV) nucleocapsids using the phosphoprotein (P) as a chaperone for the RNA-free nucleoprotein (N). The approach addresses challenges in recombinant viral protein expression and can be applied to other non-segmented negative sense RNA viruses.

Key Study Components

Area of Science

Virology
Molecular Biology
Viral Protein Expression

Background

Respiratory syncytial virus (RSV) is a significant respiratory pathogen.
Understanding RSV RNA synthesis is crucial for developing therapeutic strategies.
Chaperone proteins can facilitate the proper folding and assembly of viral proteins.
Challenges exist in expressing recombinant viral proteins effectively.

Purpose of Study

To develop a protocol for RSV nucleocapsid assembly.
To utilize the phosphoprotein (P) as a chaperone for RNA-free nucleoprotein (N).
To provide a method applicable to other non-segmented negative sense RNA viruses.

Methods Used

Coexpression of phosphoprotein (P) and RNA-free nucleoprotein (N).
In vitro assembly of virus-specific nucleocapsids.
Ligation independent cloning for acquiring co-expression constructs.
Electron microscopy for visualizing assembled structures.

Main Results

Successful assembly of RNA-free RSV nucleoproteins into nucleocapsids.
Demonstrated the effectiveness of using P as a chaperone.
Method showed potential for application with other viruses.
Electron microscopy confirmed the assembly of large structures.

Conclusions

The study provides a valuable protocol for RSV research.
Utilizing chaperone proteins can enhance viral protein expression.
This method may facilitate further studies on non-segmented negative sense RNA viruses.

Frequently Asked Questions

What is the significance of RSV?

RSV is a major cause of respiratory illness in infants and young children.

How does the chaperone protein P function?

P assists in the proper folding and assembly of the nucleoprotein N.

Can this method be applied to other viruses?

Yes, it can be adapted for other non-segmented negative sense RNA viruses.

What techniques were used to visualize the nucleocapsids?

Electron microscopy was employed to check the assembled structures.

What challenges does this method address?

It tackles issues related to recombinant viral protein expression.

What is the role of RNA-free nucleoprotein N?

N is essential for the assembly of the viral nucleocapsid.

호흡기 싱크량 바이러스(RSV) RNA 합성의 심층 기계분석의 경우, 바이러스 특이적 뉴클레오캡시드(NC)의 후속 체외 조립을 위해 RNA-프리 뉴클레오프로틴(N0)의 공동발현을 위해 샤페론 인포단(P)을 활용하는 프로토콜을 보고한다.

이 방법은 RNA가 없는 N 단백질을 얻기 위해 또 다른 바이러스 단백질 N'을 위한 샤페론으로서 하나의 바이러스 성 단백질 P'를 사용하여 재조합 바이러스 단백질 발현 문제를 해결하는 다양한 방법을 활용합니다. RNA-free RSV N 단백질은 바이러스 특이적 RNA를 체외에서 뉴클레오캡시드로 조립하기 전에 수득된다. 이 방법은 또한 다른 비분할 음성 감각 RNA 바이러스와 함께 사용될 수 있다.

리칭 독립적 복제 방법은 공동 식 구문 수집을 위한 빠른 기술입니다. 왜 또는 선택 얼룩 전자 현미경은 원시 세포에서 큰 어셈블리를 확인하는 빠른 방법입니다. 우리는 당신에게 두 가지 팁을 제공하고 싶습니다.

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