Bone marrow stromal antigen 2 (BST-2, also known as tetherin) is a recently identified interferon-inducible host restriction factor that can block the production of enveloped viruses by trapping virus particles at the cell surface. This antiviral effect is counteracted by the human immunodeficiency virus type 1 (HIV-1) accessory protein viral protein U (Vpu). Here we show that HIV-1 Vpu physically interacts with BST-2 through their mutual transmembrane domains and leads to the degradation of this host factor via a lysosomal, not proteasomal, pathway. The degradation is partially controlled by a cellular protein, ␤-transducin repeat-containing protein (␤TrCP), which is known to be required for the Vpu-induced degradation of CD4. Importantly, targeting of BST-2 by Vpu occurs at the plasma membrane followed by the active internalization of this host protein by Vpu independently of constitutive endocytosis. Thus, the primary site of action of Vpu is the plasma membrane, where Vpu targets and internalizes cell-surface BST-2 through transmembrane interactions, leading to lysosomal degradation, partially in a ␤TrCP-dependent manner. Also, we propose the following configuration of BST-2 in tethering virions to the cell surface; each of the dimerized BST-2 molecules acts as a bridge between viral and cell membranes. Viral protein U (Vpu)2 is an 81-amino acid type I integral membrane phosphoprotein expressed by human immunodeficiency virus type 1 (HIV-1) (1, 2) and several simian immunodeficiency viruses (3-6). Vpu is not incorporated into virus particles (7), indicating that it acts exclusively in virus-producer cells. Indeed, Vpu is known to play two distinct roles during the later stages of infection. First, Vpu interacts with newly synthesized CD4 molecules complexed with the gp160 envelope glycoprotein precursor in the endoplasmic reticulum (8, 9) and recruits the ␤-transducin repeat-containing protein 1 (␤TrCP-1) subunit of the Skp1-Cullin1-F-box ubiquitin ligase complex (10) as well as ␤TrCP-2 (11) through its phosphoserine residues at positions 52 and 56 in the cytoplasmic (CT) domain (12,13). This event results in proteasome-mediated degradation of CD4 (10, 14, 15) allowing gp160 to resume transport toward the cell surface for virion incorporation. Second, Vpu mediates the enhancement of virion release (16 -18) in a cell type-dependent manner (e.g. HeLa cells require Vpu, whereas COS7 cells do not (19,20)), and its absence leads to the accumulation of viral particles at the cell surface (21).In contrast to the effect of Vpu on CD4 degradation, little had been known about the mechanism by which Vpu enhances the release of virions. The finding that HeLa-COS7 heterokaryons exhibited HeLa-type properties suggested that Vpu-responsive HeLa cells might harbor endogenous a restriction factor(s) that could be counteracted by this viral protein (22), as seen in Vifresponsive cells harboring the antiretroviral factor APOBEC3G counteracted by Vif (23). Neil et al. (24) showed that Vpu-deficient viral particles accumulated ...

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Membrane-associated RING-CH 8 (MARCH8) is one of 11 members of the recently discovered MARCH family of RING (really interesting new gene)-finger E3 ubiquitin ligases. MARCH8 downregulates several host transmembrane proteins, including major histocompatibility complex (MHC)-II, CD86, interleukin (IL)-1 receptor accessory protein, TNF-related apoptosis-inducing ligand (TRAIL) receptor 1 and the transferrin receptor. However, its physiological roles remain largely unknown. Here we identify MARCH8 as a novel antiviral factor. The ectopic expression of MARCH8 in virus-producing cells does not affect levels of lentivirus production, but it does markedly reduce viral infectivity. MARCH8 blocks the incorporation of HIV-1 envelope glycoprotein into virus particles by downregulating it from the cell surface, probably through their interaction, resulting in a substantial reduction in the efficiency of viral entry. The inhibitory effect of MARCH8 on vesicular stomatitis virus G-glycoprotein is even more remarkable, suggesting a broad-spectrum inhibition of enveloped viruses by MARCH8. Notably, the endogenous expression of MARCH8 is high in monocyte-derived macrophages and dendritic cells, and MARCH8 knockdown or knockout in macrophages significantly increases the infectivity of virions produced by these cells. Our findings thus indicate that MARCH8 is highly expressed in terminally differentiated myeloid cells, and that it is a potent antiviral protein that targets viral envelope glycoproteins and reduces their incorporation into virions.

HM1.24/Bst2/CD317 is a protein highly expressed in multiple myeloma cells and has unique topology with two membrane anchor domains, an NH 2 -terminal transmembrane domain and a glycosylphosphatidylinositol attached to the COOH terminus. We show here that human HM1.24 is localized not only on the cell surface but also in the trans-Golgi network and/or recycling endosomes, where it resides in detergent-resistant microdomains, lipid rafts. In contrast to other glycosylphosphatidylinositol-anchored proteins, HM1.24 was internalized from lipid rafts on the cell surface by clathrin-mediated endocytosis. Interestingly, a non-canonical tyrosine-based motif, which contains two tyrosine residues, Tyr-6 and Tyr-8, present in the NH 2 -terminal cytoplasmic tail, was essential for endocytosis through interaction with an ␣-adaptin, but not 2-subunit, of the AP-2 complex. Indeed, an appendage domain of ␣-adaptin was identified as a protein interacting with the cytoplasmic tail of HM1.24. Furthermore, overexpression of the appendage domain of ␣-adaptin in cells depleted of ␣-adaptin could rescue the clathrin-mediated endocytosis of HM1.24 but not of the transferrin receptor. Taken together, our findings suggest that clathrin-dependent endocytosis of human HM1.24 from the cell surface lipid rafts is mediated by direct interaction with ␣-adaptin.

SKD1 is a member of the family of ATPases associated with cellular activities whose yeast homologue Vps4p has been implicated in endosomal/vacuolar membrane transports. When a mutant of SKD1 that lacks ATPase activity [SKD1(E235Q)] was overexpressed in mammalian cells, it induced a dominant negative phenotype characterized by aberrant endosomal structures (denoted as E235Q compartments). Expression of SKD1(E235Q) caused an accumulation of basolateral recycling receptors, such as asialoglycoprotein receptor and low-density lipoprotein in polarized hepatocytes and Madin-Darby canine kidney cells, respectively, in E235Q compartments. In addition, SKD1(E235Q) also abrogated, via endosomes, transport to the trans-Golgi network, as indicated by an accumulation of TGN38 in E235Q compartments. Three lines of evidence further demonstrated that SKD1 participates in the membrane transport from early endosomes to late endosomes/lysosomes: (1) a redistribution of a late endosomal and lysosomal membrane protein endolyn in E235Q compartments; (2) an inhibition of epidermal growth factor receptor degradation, due to an accumulation of the receptors in E235Q compartments; and (3) a mis-sorting of and defect in the proteolytic processing of newly synthesized cathepsin D. An intriguing finding was that the expression of SKD1(E235Q) caused the number of lysosomes to decrease (to one-sixth of control numbers) but their size to increase (2.4-fold larger in diameter than control lysosomes). Indeed, an ultrastructural analysis revealed that the expression of SKD1(E235Q) causes an accumulation of hybrid organelles formed by direct fusion between late endosomes and lysosomes. We conclude that SKD1 regulates multiple steps of membrane transport out of early endosomes and the reformation of lysosomes from a hybrid organelle.