Nef is a multifunctional protein of the human immunodeficiency virus type 1 (HIV-1) required for high viral replication and disease progression. Several findings indicate that the capacity of Nef to downregulate surface CD4 is essential for the protein's pathogenic activity, although the mechanisms that link the two functions are yet unclear. It is believed that, by reducing surface CD4 levels, Nef counteracts the receptor's negative effects on virion infectivity and release. Here, we show that, in 293T cells co-expressing CD4 and HIV-1, the capacity of Nef to enhance the virion incorporation of Env products and release of viral particles was mediated by retention-degradation of neo-synthesized CD4 rather than by accelerated receptor endocytosis. Different results were observed in primary CD4+ T lymphocytes in which Nef-mediated CD4 downregulation occurs primarily by accelerated internalization. In HIV-infected T cell cultures, Nef was crucial for the removal of surface CD4 at the beginning of the infection, while later on maximal CD4 downregulation was achieved in a Nef-independent manner. Moreover, by means of in vivo selected Nef mutants, we observed that CD4 downregulation is not essential for Nef ability to enhance Env incorporation into virions and increase viral infectivity or replication in CD4+ T lymphocytes. Notably, Nef expression itself was dispensable for efficient release of HIV-1 particles by T cells. In conclusion, we propose that the CD4 downregulation activity of Nef plays a role before the late productive phases of HIV-1 replication.
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