TSG101 and ALIX both function in HIV budding and in vesicle formation at the multivesicular body (MVB), where they interact with other Endosomal Sorting Complex Required for Transport (ESCRT) pathway factors required for release of viruses and vesicles. Proteomic analyses revealed that ALIX and TSG101/ESCRT-I also bind a series of proteins involved in cytokinesis, including CEP55, CD2AP, ROCK1, and IQGAP1. ALIX and TSG101 concentrate at centrosomes and are then recruited to the midbodies of dividing cells through direct interactions between the central CEP55 'hinge' region and GPP-based motifs within TSG101 and ALIX. ESCRT-III and VPS4 proteins are also recruited, indicating that much of the ESCRT pathway localizes to the midbody. Depletion of ALIX and TSG101/ESCRT-I inhibits the abscission step of HeLa cell cytokinesis, as does VPS4 overexpression, confirming a requirement for these proteins in cell division. Furthermore, ALIX point mutants that block CEP55 and CHMP4/ESCRT-III binding also inhibit abscission, indicating that both interactions are essential. These experiments suggest that the ESCRT pathway may be recruited to facilitate analogous membrane fission events during HIV budding, MVB vesicle formation, and the abscission stage of cytokinesis.

Type 1 interferon (IFN) inhibits the release of HIV-1 virus particles via poorly defined mechanisms. Here, we show that IFNalpha induces retention of viral particles on the surface of fibroblasts, T cells, or primary lymphocytes infected with HIV-1 lacking the Vpu protein. Retained particles are tethered to cell surfaces, can be endocytosed, appear fully assembled, exhibit mature morphology, and can be detached by protease. Strikingly, expression of the HIV-1 Vpu protein attenuates the ability of human cells to adhere to, and thereby retain, nascent HIV-1 particles upon IFNalpha treatment. Vpu also counteracts the IFNalpha-induced retention of virus-like particles assembled from the Ebola virus matrix protein. Furthermore, levels of IFNalpha that suppress replication of Vpu-defective HIV-1 have little effect on wild-type HIV-1. Thus, we propose that HIV-1 expresses Vpu to counteract an IFNalpha-induced, general host defense that inhibits dissemination of enveloped virions from the surface of infected cells.

We wish to report that we have discovered an error in the AD-ROCK1 yeast two hybrid construct used in the experiment displayed in Figure 1D. This error introduced a stop codon after ROCK1 amino acid 123, so that the two hybrid interactions that we reported as being between TSG101 and full-length ROCK1 were actually between TSG101 and a truncated construct that expressed only the first 123 residues of ROCK1. This error does not affect the corresponding ROCK1-TSG101 co-immunoprecipitation experiment displayed in Figure 1B of the original paper. Based on the co-immunoprecipitation experiment, the conclusion that ROCK1 can interact with TSG101 stands.The authors apologize for any inconvenience caused.

IntroductionVectors derived from retroviruses offer particularly flexible properties in gene transfer applications given the numerous possible associations of various viral surface glycoproteins (GPs; determining cell tropism) with different types of viral cores (determining genome replication and integration). 1 For example, association of the vesicular stomatitis virus G (VSV-G) GP with viral cores derived from lentiviruses results in vector pseudotypes that have broad tropism and can integrate into nonproliferating target cells. 2 They have proved useful for the transduction of several cell types ex vivo and in vivo. [3][4][5][6][7] Yet there is considerable interest in exploring the properties of lentiviral vectors pseudotyped with alternative viral GPs. [8][9][10][11][12][13][14] This parameter is likely to modulate the physicochemical properties of the vectors, their interaction with the host immune system, and their host range. Several studies have indeed shown that the transduction efficiency of target cells is dependent on the type of GP used to coat retroviral vectors. [15][16][17][18][19][20] Additionally, some in vivo gene transfer applications will require vectors that are targeted for specific cell entry or gene expression (or both) after systemic administration. 21 Due to the wide distribution of its receptor, a lipid component of the plasma membrane, 22 VSV-G pseudotypes may bind to the surface of all cells encountered after inoculation before reaching the target cells. Moreover, VSV-Gpseudotyped vectors are rapidly inactivated by human serum 23 and this might impose a limitation on the use of VSV-G as a GP to pseudotype vectors for systemic gene delivery.Lentiviral vectors derived from simian immunodeficiency virus (SIV) have been generated in several laboratories, 1 including our own. 24 Characterization of these vectors has indicated that they are similar to those derived from human immunodeficiency virus 1 (HIV-1) with respect to the insertion of transgenes in nonproliferating cells, although SIV vectors perform better than HIV-1 vectors in simian cells. 24 Here, we report the properties of SIVmac-derived vectors pseudotyped with a panel of GPs derived from different membrane-enveloped viruses. In particular, we examined stability in human or macaque sera and gene transfer in primary hematopoietic cells including peripheral blood lymphocytes (PBLs) and CD34 ϩ cells. Materials and methods CellsThe 293T human embryo kidney cell line (American Type Culture Collection, Rockville, MD, CRL-1573) and the TE671 human rhabdomyosarcoma cell line (ATCC CRL-8805) were grown in Dulbecco modified Eagle medium (DMEM; Life Technologies, Cergy-Pontoise, France) supplemented with 10% fetal calf serum (FCS).Human and cynomolgus macaque (Macaca fascicularis) CD34 ϩ cells were obtained according to the institutional guidelines of the ethic commission from mobilized blood and bone marrow samples, respectively, as described previously. 25-27 CD34 ϩ cells were recovered after Ficoll-Paque (Amersham-Pharmacia Biot...