Tetherin is an IFN-inducible restriction factor that inhibits HIV-1 particle release in the absence of the HIV-1 countermeasure, viral protein U (Vpu). Although ubiquitous in HIV-1 and simian immunodeficiency viruses from chimpanzees, greater spot nosed monkeys, mustached monkeys, and Mona monkeys, other primate lentiviruses do not encode a Vpu protein. Here we demonstrate that SIV from Tantalus monkeys (SIVtan) encodes an envelope glycoprotein (SIVtan Env) able to counteract tetherin from Tantalus monkeys, rhesus monkeys, sooty mangabeys, and humans, but not from pigs. We show that sensitivity to Vpu but not SIVtan Env can be transferred with the human tetherin transmembrane region. We also identify a mutation in the tetherin extracellular domain, which almost completely abolishes sensitivity of human tetherin to SIVtan Env without compromising antiviral activity or sensitivity to Vpu. SIVtan Env expression results in a reduction of surface tetherin, as well as reduction in tetherin co-localization with mature surface-associated virus. Immuno-electron microscopy reveals co-localization of SIVtan Env with tetherin in intracellular tubulo-vesicular structures, suggesting that tetherin is sequestered away from budding virions at the cell surface. Along with HIV-1 Vpu and SIV Nef, envelope glycoprotein is the third and most broadly active lentiviral-encoded tetherin countermeasure to be described. Our observations emphasize the importance of tetherin in protecting mammals against viral infection and suggest that HIV-1 Vpu inhibitors may select active envelope mutants.HIV ͉ restriction ͉ innate immunity

The chemokine receptor CXCR3 is expressed on the surface of both resting and activated T- lymphocytes. We describe here a study of the endocytosis of CXCR3 using T-lymphocytes and CXCR3 transfectants. Chemokine-induced CXCR3 downregulation occurred in a rapid, dose-dependent manner, with CXCL11 the most potent and efficacious ligand. Endocytosis was mediated in part by arrestins, but appeared to occur independently of clathrin and caveolae. In contrast to other chemokine receptors, which are largely recycled to the cell surface within an hour, cell surface replenishment of CXCR3 occurred over several hours and was dependent upon mRNA transcription, de novo protein synthesis and transport through the ER and Golgi. Confocal microscopy and Western blotting confirmed the fate of endocytosed CXCR3 to be degradation, mediated in part by lysosomes and proteosomes. Site-directed mutagenesis of the CXCR3 C-terminus revealed that internalization and degradation were independent of phophorylation, ubiquitination or a conserved LL motif. CXCR3 was found to be efficiently internalized in the absence of ligand, a process involving a YXXL motif at the extreme of the C-terminus. Although freshly isolated T-lymphocytes expressed moderate cell surface levels of CXCR3, they were only responsive to CXCL11 with CXCL9 and CXCL10 only having significant activity on activated T-lymphocytes. Thus, the activities of CXCR3 are tightly controlled following mRNA translation. Since CXCR3+ cells are themselves a source of IFN-γ, which potently induces the expression of CXCR3 ligands, such tight regulation of CXCR3 may serve as a control to avoid the unnecessary amplification of activated T-lymphocyte recruitment.

SummaryInnate immune factors may restrict hematopoietic stem cell (HSC) genetic engineering and contribute to broad individual variability in gene therapy outcomes. Here, we show that HSCs harbor an early, constitutively active innate immune block to lentiviral transduction that can be efficiently overcome by cyclosporine H (CsH). CsH potently enhances gene transfer and editing in human long-term repopulating HSCs by inhibiting interferon-induced transmembrane protein 3 (IFITM3), which potently restricts VSV glycoprotein-mediated vector entry. Importantly, individual variability in endogenous IFITM3 levels correlated with permissiveness of HSCs to lentiviral transduction, suggesting that CsH treatment will be useful for improving ex vivo gene therapy and standardizing HSC transduction across patients. Overall, our work unravels the involvement of innate pathogen recognition molecules in immune blocks to gene correction in primary human HSCs and highlights how these roadblocks can be overcome to develop innovative cell and gene therapies.

Innate resistance to retroviral infection and replication is induced by interferons (IFNs). IFN-inducible factors restricting viral replication include the cytidine deaminase APOBEC3G (40, 60) and the E3 ubiquitin ligase TRIM5 (1), both of which target replication primarily during the process of viral entry. A third IFN-inducible activity, tetherin (BST-2/ CD317/HM1.24), acts to restrict viral release (13,35,36,41,62). The importance of these factors in controlling viral replication is underlined by the requirement for lentiviral genomes to encode trans-acting countermeasures; lentiviral Vif proteins (33,54,55) and spumaviral Bet proteins (28, 42, 51) counteract APOBECs whereas HIV-1 Vpu, HIV-2 Nef, and HIV-2 and simian immunodeficiency virus (SIV) Envs may counteract tetherins (15,18,26,35,36,62,65).Tetherin is a type II single-pass transmembrane protein. It is anchored to the cell membrane by both N-terminal transmembrane domain and C-terminal glycophosphatidylinositol (GPI) anchors that are linked by an extracellular coiled-coil domain that promotes dimerization of adjacent tetherin molecules. Accordingly, tetherin in both the cell membrane and the envelope of the budding virus can prevent virion release either by direct cross-linking or by the formation of dimers between adjacent coiled-coil domains (41). The primary role for tetherin remains unclear; however, it is likely that, by trapping enveloped viruses at the cell surface, tetherin prevents the further dissemination of nascent virions. However, given the constitutive high-level expression of tetherin on plasmacytoid dendritic cells (pDC [type I IFN-producing cells]) (5), tetherin may play a more fundamental role in the initiation and perpetuation of a virus-specific immune response (58).The domestic cat lineage has faced multiple invasions by viruses from the family Retroviridae. In addition to an exogenous gammaretrovirus (feline leukemia virus [FeLV]), a lentivirus (feline immunodeficiency virus [FIV]), and a spumavirus (feline foamy virus [FFV]), cats also harbor the endogenous RD114 gamma retrovirus (47, 48) and full-length endogenous FeLVs (50). While lentiviruses have spread throughout the Felidae, from lions in Africa to pumas in North America and Pallas cats in Mongolia (61), the gamma retroviruses are restricted solely to domestic cats (3,4,47,48), although occasional cross-species transmission events have been recorded in Florida panthers (37) and Iberian lynxes (30). The limited distribution of the gamma retroviruses among felids suggests that they entered the domestic cat population after the divergence of the Felis lineage from the other felids circa 6.2 million years ago (19). The presence of three exogenous members and one endogenous member of the Retroviridae in domestic cats offers an intriguing insight into the retrovirus-host interaction. As cats express a truncated TRIM5 lacking a capsid-binding B30.2/SPRY domain (29), their ability to suppress retroviral replication may be impaired. If tetherin is to have a major role in the contro...