Heterologous Src homology 4 domains support membrane anchoring and biological activity of HIV-1 Nef.
The HIV-1 pathogenicity factor Nef enhances viral replication by modulation of multiple host cell transport and signaling pathways. Nef associates with membranes via an N-terminal Src homology 4 (SH4) domain, and membrane association is believed to be essential for its biological functions. At which subcellular site(s) Nef exerts its different functions and how kinetics of membrane interactions contribute to its biological activity are unknown. To address how specific characteristics of Nef membrane association affect its biological properties, the SH4 domain of Nef was replaced by heterologous membrane targeting domains. The use of a panel of heterologous SH4 domains resulted in chimeric Nef proteins with distinct steady state subcellular localization, membrane association efficiency, and anterograde transport routes. Irrespective of these modifications, cardinal Nef functions affecting host cell vesicular transport and actin dynamics were fully preserved. In contrast, stable targeting of Nef to the surface of mitochondria, peroxisomes, or the Golgi apparatus, and thus prevention of plasma membrane delivery, caused potent and broad loss of Nef activity. These results support the concept that Nef adopts its active conformation in the membrane-associated state but exclude that membrane-associated Nef simply acts by recruiting soluble factors independently of its local microenvironment. Rather than its steady state subcellular localization or membrane affinity, the ability to undergo dynamic anterograde and internalization cycles appear to determine Nef function. These results reveal that functional membrane interactions of Nef underlie critical spatiotemporal regulation and suggest that delivery to distinct subcellular sites via such transport cycles provides the basis for the multifunctionality of Nef.
From the Department of Infectious Diseases, Integrative Virology and.
Miriam M
MM
Geist
From the Department of Infectious Diseases, Integrative Virology and.
Xiaoyu
X
Pan
Molecular Virology, University Hospital Heidelberg,69120 Heidelberg, Germany and.
Silke
S
Bender
Molecular Virology, University Hospital Heidelberg,69120 Heidelberg, Germany and.
Ralf
R
Bartenschlager
the Biochemistry Center, Heidelberg University, 69120 Heidelberg, Germany.
Walter
W
Nickel
From the Department of Infectious Diseases, Integrative Virology and oliver.fackler@med.uni-heidelberg.de.
Oliver T
OT
Fackler
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D015497:HIV-1; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D019169:Jurkat Cells; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus
Host-Pathogen Interactions; Human Immunodeficiency Virus; Membrane Trafficking; Nef; Plasma Membrane; SH4 Domain; Viral Protein