Viral bimolecular fluorescence complementation: a novel tool to study intracellular vesicular trafficking pathways

Area of Science:

• Virology
• Cell Biology
• Molecular Biology

Background:

• The Human Immunodeficiency Virus type 1 (HIV-1) Nef protein manipulates host cell processes to evade immune detection.
• Existing methods for studying Nef interactions lack context of viral infection and sub-cellular localization.

Purpose of the Study:

• To develop and validate a novel lentiviral bimolecular fluorescence complementation (BiFC) tool, termed viral BiFC, for studying Nef interactions in infected cells.
• To map the sub-cellular locations of Nef interactions with host proteins PACS-1, MHC-I, and SNX18 during viral infection.

Main Methods:

• Development of a lentiviral expression vector utilizing the F2A cleavage site for co-expression of Nef and host proteins.
• Application of viral BiFC to visualize and localize interactions between Nef and PACS-1, MHC-I, and SNX18.
• Co-localization studies with endolysosomal markers LAMP-1 and AP-1.

Main Results:

• Viral BiFC confirmed the interaction between Nef and PACS-1, localizing the complex to LAMP-1 positive endolysosomal vesicles.
• The Nef/MHC-I interaction was localized to AP-1 positive endosomal compartments using viral BiFC.
• A novel interaction between Nef and the membrane trafficking regulator SNX18 was identified and localized to AP-1 positive vesicles.

Conclusions:

• Viral BiFC is an effective tool for studying host-pathogen interactions in the context of viral infection.
• This method provides crucial insights into the sub-cellular localization of Nef-host protein interactions, aiding the understanding of HIV-1 immune evasion strategies.
• The identified Nef-SNX18 interaction offers new avenues for research into HIV-1 pathogenesis and host manipulation.