Random Insertional Mutagenesis of a Serotype 2 Dengue Virus Clone

Area of Science:

• Virology
• Molecular Biology
• Genetics

Background:

• Protein tagging is crucial for understanding protein function.
• Modifying positive-strand RNA virus proteins during infection is challenging due to compact genomes and multifunctional proteins.
• Existing targeted tagging methods are time-consuming and inefficient.

Purpose of the Study:

• To describe an efficient protocol for transposon mutagenesis of dengue virus serotype 2.
• To enable functional genetic studies of dengue virus proteins.
• To facilitate the generation and analysis of mutant dengue virus libraries.

Main Methods:

• Whole-genome transposon insertional mutagenesis was applied to the dengue virus 16681 strain.
• Mutant viral libraries with random small insertions were generated.
• Libraries were passaged in mammalian cells, and insertion sites were mapped using Next-Generation Sequencing (NGS).

Main Results:

• A detailed four-stage protocol for transposon mutagenesis of dengue virus was established.
• This protocol allows for the generation of mutant dengue virus libraries.
• Next-Generation Sequencing (NGS) enables mapping of insertion sites in viable viral progeny.

Conclusions:

• Transposon mutagenesis provides an efficient strategy for generating functional genetic diversity in dengue virus.
• This protocol facilitates the investigation of dengue virus protein function in the context of viral infection.
• The described method is valuable for studying RNA virus genetics and developing antiviral strategies.