CD4 downregulation by the human immunodeficiency virus type 1 Nef protein is dispensable for optimal output and functionality of viral particles in primary T cells.

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.

The HIV-1 Nef protein has a dual role in T cell receptor signaling in infected CD4+ T lymphocytes.

The phenotypic changes that are induced by immune activation in CD4(+) T lymphocytes provide an optimal environment for efficient HIV-1 replication in these cells. The pathogenic Nef protein of HIV-1 modulates the T cell receptor (TCR) signaling, but whether this has a positive or negative effect on cellular activation is a matter of debate. Here we have investigated the response to TCR stimulation of primary CD4(+) T lymphocytes infected with wt or Nef-deficient HIV-1. Results show that, in freshly isolated quiescent T cells, Nef superinduces NFAT and IL-2 production bypassing early TCR effector molecules. Conversely, the early phosphorylation of PLC-γ1, the induction of NFAT, and the expression of IL-2 are impaired by Nef in sub-optimally activated/resting T cells. Our data indicate that Nef has a dual role in the modulation of TCR signaling aimed at favoring HIV-1 replication and spread in both quiescent and metabolically active CD4(+) T lymphocytes.

Internalization and intracellular retention of CD4 are two separate functions of the human immunodeficiency virus type 1 Nef protein.

The pathogenic Nef protein of the human immunodeficiency virus type 1 (HIV-1) downregulates CD4 by inducing its endocytosis and by inhibiting the transport of the receptor to the cell membrane. By means of in vivo-selected mutations, we show that L37, P78 and E177 residues of Nef are required for its effect on CD4 internalization and recycling but dispensable for Nef-induced retention and degradation of intracellular CD4. Of note, the function of Nef on the anterograde transport of newly synthesized CD4 molecules is irrelevant in cells with a slow constitutive CD4 turnover such as T cell lines. Moreover, we show that a mutated CD4 that is unresponsive to Nef-mediated endocytosis, CD4LL(144)AA, is retained intracellularly and degraded by Nef like wild-type CD4. Thus, Nef's abilities to enhance endocytosis and induce intracellular retention of CD4 are mediated by separate protein surfaces and occur through distinct mechanisms.

The Pro78 residue regulates the capacity of the human immunodeficiency virus type 1 Nef protein to inhibit recycling of major histocompatibility complex class I molecules in an SH3-independent manner.

The Nef protein is a crucial pathogenicity factor of human immunodeficiency virus type 1 (HIV-1) that contains a proline-rich motif consisting of four conserved prolines: Pro69 (P69), P72, P75 and P78. P72 and P75 were shown to bind Src homology domains 3 (SH3) and have been implicated in many biological functions of Nef, including downmodulation of cell-surface major histocompatibility complex class I (MHC-I). P78 is involved together with P69 in positioning of the Nef-SH3 complex and it has been shown to be essential for Nef activity of MHC-I downmodulation. It is shown here that alteration of P78 affects recycling of MHC-I molecules to the cell surface, but does not interfere with SH3 binding. In addition, it is demonstrated that P72 and P75, and thus the SH3-binding capacity, are fully dispensable for Nef activity on MHC-I.