Heterologous Src Homology 4 Domains Support Membrane Anchoring and Biological Activity of HIV-1 Nef [Membrane Biology]

April 4th, 2014 by Geist, M. M., Pan, X., Bender, S., Bartenschlager, R., Nickel, W., Fackler, O. T.

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 is unknown. To address how specific characteristics of Nefs membrane association affect its biological properties, Nefs SH4 domain 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 spatio-temporal regulation and suggest that delivery to distinct subcellular sites via such transport cycles provides the basis for the multi-functionality of Nef.