The HIV Nef Protein Alters Ca²⁺ Signaling in Myelomonocytic Cells through SH3-mediated Protein-Protein Interactions*

Abstract

Human immunodeficiency virus Nef plays an important role in AIDS pathogenesis. In addition to the well known down-regulation of cell surface receptors (CD4, MHCI), Nef is able to alter cellular signaling. Of particular interest for this study is the ability of Nef to bind with a very high affinity to SH3 domains of myelomonocyte-specific protein-tyrosine kinases of the Src family (Src-like PTK). We have therefore investigated Ca²⁺signaling in HL60 cells retrovirally transduced with wild type Nef or with a Nef mutant deficient in the SH3-interacting proline-rich motif (Nef_{(P XX P)4⁻}). In differentiated HL60 cells, Nef markedly altered cellular Ca²⁺ signaling; the amount of intracellularly stored Ca²⁺ was increased, and as a consequence, store-operated Ca²⁺-influx was decreased. This effect was not observed in undifferentiated HL60 cells or in CEM T-lymphocytes and correlated with the differentiation-induced up-regulation of Src-like PTK. The Nef effect on Ca²⁺ signaling depended entirely on the integrity of its PXXP motif. The Src-like PTK p56/59^hckco-immunoprecipitated with both Nef and with the inositol 1,4,5-trisphosphate receptor, providing a possible mechanistic link between the viral protein and intracellular Ca²⁺ stores of the host cell. Collectively, our results demonstrate that the human immunodeficiency virus 1 Nef protein manipulates intracellular Ca²⁺ stores through SH3-mediated interactions in myelomonocytic cells.