Newly Established Genetic System for Functional Analysis of MetSV

Area of Science:

• Virology
• Microbiology
• Molecular Biology

Background:

• Methanosarcina spherical virus (MetSV) possesses a linear chromosome with 22 open reading frames (ORFs), many with unknown functions.
• Functional annotation of MetSV ORFs using standard tools identified only a few genes, such as DNA polymerase and capsid proteins.
• Biochemical or genetic methods are crucial for verifying the functions of the remaining MetSV ORFs.

Purpose of the Study:

• To establish a functional genetic system for MetSV, the first for a methanoarchaeal virus.
• To enable detailed investigation of MetSV protein functions and virus-host interactions.

Main Methods:

• Cloning the MetSV genome into an *E. coli* plasmid (pCR-XL-2) to create a genetic system.
• Utilizing Nanopore sequencing to compare plasmids with the MetSV reference and identify mutations.
• Generating linear MetSV inserts via BamHI restriction and transforming *Methanosarcina mazei* using optimized liposome-mediated transformation.
• Analyzing MetSV virions using Transmission Electron Microscopy (TEM) and infection experiments.
• Generating and testing a deletion mutant (ΔMetSVORF09) for infectivity.

Main Results:

• A functional genetic system for MetSV was successfully established.
• Plasmid-derived MetSV virions showed comparable morphology and lytic behavior to native MetSV.
• A ΔMetSVORF09 mutant demonstrated retained infectivity, validating the system's functionality.
• Nanopore sequencing revealed mutations in plasmid constructs impacting protein-coding sequences.

Conclusions:

• The developed genetic system is the first functional system for a virus infecting methanoarchaea.
• This system provides a powerful tool for future research into MetSV protein functions.
• It will facilitate deeper understanding of virus-host interactions in methanoarchaeal systems.