Interfacial domains in Sindbis virus 6K protein. Detection and functional characterization

Area of Science:

• Virology
• Molecular Biology
• Membrane Biology

Background:

• Alphavirus 6K protein is a short, integral membrane protein essential for viral particle assembly and release.
• The N-terminal region of 6K contains conserved interfacial domains critical for its function.
• Understanding 6K's role in membrane permeabilization and budding is key to alphavirus replication.

Purpose of the Study:

• To investigate the functional significance of the conserved interfacial sequence motif in the Alphavirus 6K protein.
• To determine how mutations in these interfacial domains affect virus budding and membrane permeability.
• To elucidate the role of 6K's interfacial region in Sindbis virus replication.

Main Methods:

• Construction and characterization of Sindbis virus 6K mutants with altered interfacial domains (9YLW11xAAA, 18FWV20xAAA, 9YLW11xAAA/18FWV20xAAA).
• Analysis of virus budding and glycoprotein processing in reconstituted mutant viruses.
• Assessment of membrane permeability changes in Escherichia coli and baby hamster kidney cells using single 6K expression and Sindbis virus replicons.

Main Results:

• Mutant viruses exhibited defects in virus budding but not glycoprotein processing.
• Single 6K expression in E. coli showed interfacial mutants had reduced membrane permeability modification and toxicity.
• Sindbis virus replicons with mutated 6K demonstrated significantly impaired membrane permeabilization activity, with the double mutant showing the most severe defect.

Conclusions:

• The interfacial sequence of Alphavirus 6K protein is vital for efficient virus budding.
• This interfacial region plays a critical role in modulating cellular membrane permeability during viral infection.
• Targeting the 6K interfacial sequence offers potential strategies for controlling alphavirus replication.