title: The Vpu-regulated endocytosis of HIV-1 Gag is clathrin-independent.
abstract: Recent results by us and others have shown that the accessory protein Vpu determines plasma membrane versus endosomal accumulation of the HIV-1 core protein Gag and progeny virions in the HeLa model of HIV-1 infection, since Vpu suppresses endocytosis of cell surface-associated Gag. In this report, we used pulse-chase studies and subcellular fractionations to investigate endocytosis of newly synthesized Gag in HeLa H1 cells. The uptake of Gag in Delta Vpu-virus background was not blocked by inhibitors of clathrin-mediated endocytosis and macropinocytosis. The cholesterol-sequestering drug filipin inhibited the uptake, but only if the drug was applied before extensive multimerization of Gag had taken place. Thus, the uptake mechanism most likely is only indirectly dependent on cholesterol. Our results also indicated that targeting phenotype of Gag was different in confluent versus subconfluent cell cultures, which could perhaps explain some of the controversies in intracellular targeting of Gag.
authors.affiliation: Department of Virology, Haartman Institute, PO Box 21, FIN-000 14 University of Helsinki, Finland.
authors.forename: Kirsi
authors.initials: K
authors.lastname: Harila
authors.forename: Antti
authors.initials: A
authors.lastname: Salminen
authors.forename: Ian
authors.initials: I
authors.lastname: Prior
authors.forename: Jorma
authors.initials: J
authors.lastname: Hinkula
authors.forename: Maarit
authors.initials: M
authors.lastname: Suomalainen
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D002784:Cholesterol / Q000502:physiology; D002966:Clathrin / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D033922:Clathrin Heavy Chains / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D005372:Filipin / Q000494:pharmacology; D015658:HIV Infections / Q000503:physiopathology / Q000821:virology; D015497:HIV-1 / Q000472:pathogenicity / Q000502:physiology*; D006367:HeLa Cells; D054298:Human Immunodeficiency Virus Proteins / Q000502:physiology*; D006801:Humans; D010873:Pinocytosis / Q000187:drug effects / Q000502:physiology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D054334:Viral Regulatory and Accessory Proteins / Q000502:physiology*; D019065:Virus Assembly; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000502:physiology*; D000086082:Viroporin Proteins