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Elucidating virus entry using a tetracysteine-tagged virus.

Bjorn-Patrick Mohl¹, Polly Roy¹

¹Department of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK.

Methods (San Diego, Calif.)

|August 14, 2017

Summary

Researchers developed a new method to fluorescently tag Bluetongue virus (BTV) for in vivo imaging. This technique reveals how viral proteins VP2 and VP5 separate during cell entry, aiding virus-host interaction studies.

Keywords:

Biarsenical tetracysteine Bluetongue virus Double-stranded RNA Fluorescent protein Reverse genetics

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Area of Science:

Virology
Cell Biology
Molecular Imaging

Background:

Fluorescent tags are crucial for studying virus-host interactions.
Challenges include tag interference with protein function, toxicity, and imaging limitations.
Bluetongue virus (BTV) entry mechanisms were not fully understood due to lack of tagged viruses.

Purpose of the Study:

To develop and validate a method for fluorescently tagging Bluetongue virus (BTV).
To investigate the roles of outer capsid proteins VP2 and VP5 during BTV cell entry and trafficking.
To establish a transferable technology for labeling other complex non-enveloped viruses.

Main Methods:

Genetic modification of the segmented BTV genome to incorporate fluorescent tags.
In vivo imaging of tagged BTV within host cells.
Analysis of viral protein dissociation and localization during endosomal trafficking.

Main Results:

Successful generation and validation of fluorescently tagged BTV.
Demonstrated sequential dissociation of VP2 and VP5 proteins.
Observed protein retention in late endosomes correlated with decreasing pH and viral progression.

Conclusions:

The developed tagging technology enables detailed visualization of BTV entry and trafficking.
The study elucidates the pH-dependent dissociation of BTV outer capsid proteins.
This methodology offers a promising approach for studying other complex non-enveloped viruses.