Viral bimolecular fluorescence complementation: a novel tool to study intracellular vesicular trafficking pathways.
The Human Immunodeficiency Virus type 1 (HIV-1) accessory protein Nef interacts with a multitude of cellular proteins, manipulating the host membrane trafficking machinery to evade immune surveillance. Nef interactions have been analyzed using various in vitro assays, co-immunoprecipitation studies, and more recently mass spectrometry. However, these methods do not evaluate Nef interactions in the context of viral infection nor do they define the sub-cellular location of these interactions. In this report, we describe a novel bimolecular fluorescence complementation (BiFC) lentiviral expression tool, termed viral BiFC, to study Nef interactions with host cellular proteins in the context of viral infection. Using the F2A cleavage site from the foot and mouth disease virus we generated a viral BiFC expression vector capable of concurrent expression of Nef and host cellular proteins; PACS-1, MHC-I and SNX18. Our studies confirmed the interaction between Nef and PACS-1, a host membrane trafficking protein involved in Nef-mediated immune evasion, and demonstrated co-localization of this complex with LAMP-1 positive endolysosomal vesicles. Furthermore, we utilized viral BiFC to localize the Nef/MHC-I interaction to an AP-1 positive endosomal compartment. Finally, viral BiFC was observed between Nef and the membrane trafficking regulator SNX18. This novel demonstration of an association between Nef and SNX18 was localized to AP-1 positive vesicles. In summary, viral BiFC is a unique tool designed to analyze the interaction between Nef and host cellular proteins by mapping the sub-cellular locations of their interactions during viral infection.
Department of Microbiology and Immunology, The University of Western Ontario, Schulich School of Medicine and Dentistry, London, Ontario, Canada.
Brennan S
BS
Dirk
Department of Microbiology and Immunology, The University of Western Ontario, Schulich School of Medicine and Dentistry, London, Ontario, Canada.
Rajesh Abraham
RA
Jacob
Department of Microbiology and Immunology, The University of Western Ontario, Schulich School of Medicine and Dentistry, London, Ontario, Canada.
Aaron L
AL
Johnson
Department of Microbiology and Immunology, The University of Western Ontario, Schulich School of Medicine and Dentistry, London, Ontario, Canada.
Emily N
EN
Pawlak
Department of Microbiology and Immunology, The University of Western Ontario, Schulich School of Medicine and Dentistry, London, Ontario, Canada.
P Craig
PC
Cavanagh
Department of Microbiology and Immunology, The University of Western Ontario, Schulich School of Medicine and Dentistry, London, Ontario, Canada.
Logan
L
Van Nynatten
Department of Microbiology and Immunology, The University of Western Ontario, Schulich School of Medicine and Dentistry, London, Ontario, Canada.
S M Mansour
SM
Haeryfar
Department of Microbiology and Immunology, The University of Western Ontario, Schulich School of Medicine and Dentistry, London, Ontario, Canada.
Jimmy D
JD
Dikeakos
D015153:Blotting, Western; D005434:Flow Cytometry; D005453:Fluorescence; D005805:Genes, MHC Class I / Q000502:physiology; D057809:HEK293 Cells; D015497:HIV-1 / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D016086:Lentivirus; D021381:Protein Transport / Q000502:physiology; D058305:Sorting Nexins / Q000502:physiology; D018808:Transcription Factor AP-1 / Q000502:physiology; D022161:Transport Vesicles / Q000502:physiology* / Q000821:virology; D033921:Vesicular Transport Proteins / Q000502:physiology; D016662:Virus Integration / Q000502:physiology*; D014779:Virus Replication / Q000502:physiology; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000502:physiology*
C491914:PACS1 protein, human; C530791:SNX18 protein, human; D058305:Sorting Nexins; D018808:Transcription Factor AP-1; D033921:Vesicular Transport Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus; C514837:nef protein, Human immunodeficiency virus 1