Live-cell observation of cytosolic HIV-1 assembly onset reveals RNA-interacting Gag oligomers.
Assembly of the Gag polyprotein into new viral particles in infected cells is a crucial step in the retroviral replication cycle. Currently, little is known about the onset of assembly in the cytosol. In this paper, we analyzed the cytosolic HIV-1 Gag fraction in real time in live cells using advanced fluctuation imaging methods and thereby provide detailed insights into the complex relationship between cytosolic Gag mobility, stoichiometry, and interactions. We show that Gag diffuses as a monomer on the subsecond timescale with severely reduced mobility. Reduction of mobility is associated with basic residues in its nucleocapsid (NC) domain, whereas capsid (CA) and matrix (MA) domains do not contribute significantly. Strikingly, another diffusive Gag species was observed on the seconds timescale that oligomerized in a concentration-dependent manner. Both NC- and CA-mediated interactions strongly assist this process. Our results reveal potential nucleation steps of cytosolic Gag fractions before membrane-assisted Gag assembly.
Physical Chemistry, Department of Chemistry, Ludwig Maximilian University of Munich, D-81377 Munich, Germany NanoSystems Initiative Munich (NIM), Ludwig Maximilian University of Munich, D-81377 Munich, Germany Munich Center for Integrated Protein Science (CiPSM), Ludwig Maximilian University of Munich, D-81377 Munich, Germany Center for Nanoscience (CeNS), Ludwig Maximilian University of Munich, D-81377 Munich, Germany.
Jelle
J
Hendrix
Physical Chemistry, Department of Chemistry, Ludwig Maximilian University of Munich, D-81377 Munich, Germany NanoSystems Initiative Munich (NIM), Ludwig Maximilian University of Munich, D-81377 Munich, Germany Munich Center for Integrated Protein Science (CiPSM), Ludwig Maximilian University of Munich, D-81377 Munich, Germany Center for Nanoscience (CeNS), Ludwig Maximilian University of Munich, D-81377 Munich, Germany.
Viola
V
Baumgärtel
Physical Chemistry, Department of Chemistry, Ludwig Maximilian University of Munich, D-81377 Munich, Germany NanoSystems Initiative Munich (NIM), Ludwig Maximilian University of Munich, D-81377 Munich, Germany Munich Center for Integrated Protein Science (CiPSM), Ludwig Maximilian University of Munich, D-81377 Munich, Germany Center for Nanoscience (CeNS), Ludwig Maximilian University of Munich, D-81377 Munich, Germany.
Waldemar
W
Schrimpf
Physical Chemistry, Department of Chemistry, Ludwig Maximilian University of Munich, D-81377 Munich, Germany NanoSystems Initiative Munich (NIM), Ludwig Maximilian University of Munich, D-81377 Munich, Germany Munich Center for Integrated Protein Science (CiPSM), Ludwig Maximilian University of Munich, D-81377 Munich, Germany Center for Nanoscience (CeNS), Ludwig Maximilian University of Munich, D-81377 Munich, Germany.
Sergey
S
Ivanchenko
Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, Irvine, CA 92697 Development Biology Center Optical Biology Core Facility, University of California, Irvine, Irvine, CA 92697.
Michelle A
MA
Digman
Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, Irvine, CA 92697 Development Biology Center Optical Biology Core Facility, University of California, Irvine, Irvine, CA 92697.
Enrico
E
Gratton
Department of Infectious Diseases, Virology, University Hospital Heidelberg, D-69120 Heidelberg, Germany.
Hans-Georg
HG
Kräusslich
Department of Infectious Diseases, Virology, University Hospital Heidelberg, D-69120 Heidelberg, Germany.
Barbara
B
Müller
Physical Chemistry, Department of Chemistry, Ludwig Maximilian University of Munich, D-81377 Munich, Germany NanoSystems Initiative Munich (NIM), Ludwig Maximilian University of Munich, D-81377 Munich, Germany Munich Center for Integrated Protein Science (CiPSM), Ludwig Maximilian University of Munich, D-81377 Munich, Germany Center for Nanoscience (CeNS), Ludwig Maximilian University of Munich, D-81377 Munich, Germany d.lamb@lmu.de.
Don C
DC
Lamb
D000595:Amino Acid Sequence; D003593:Cytoplasm / Q000378:metabolism; D015497:HIV-1 / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D021381:Protein Transport; D059010:Single-Cell Analysis; D019065:Virus Assembly; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*
D054301:gag Gene Products, Human Immunodeficiency Virus