Abstract
Tunneling nanotubes (TNTs) are intercellular structures that allow for the passage of vesicles, organelles, genomic material, pathogenic proteins and pathogens. The unconventional actin molecular motor protein Myosin-X (Myo10) is a known inducer of TNTs in neuronal cells, yet its role in other cell types has not been examined. The Nef HIV-1 accessory protein is critical for HIV-1 pathogenesis and can self-disseminate in culture via TNTs. Understanding its intercellular spreading mechanism could reveal ways to control its damaging effects during HIV-1 infection. Our goal in this study was to characterize the intercellular transport mechanism of Nef from macrophages to T cells. We demonstrate that Nef increases TNTs in a Myo10-dependent manner in macrophages and observed the transfer of Nef via TNTs from macrophages to T cells. To quantify this transfer mechanism, we established an indirect flow cytometry assay. Since Nef expression in T cells down-regulates the surface receptor CD4, we correlated the decrease in CD4 to the transfer of Nef between these cells. Thus, we co-cultured macrophages expressing varying levels of Nef with a T cell line expressing high levels of CD4 and quantified the changes in CD4 surface expression resulting from Nef transfer. We demonstrate that Nef transfer occurs via a cell-to-cell dependent mechanism that directly correlates with the presence of Myo10-dependent TNTs. Thus, we show that Nef can regulate Myo10 expression, thereby inducing TNT formation, resulting in its own transfer from macrophages to T cells. In addition, we demonstrate that up-regulation of Myo10 induced by Nef also occurs in human monocyte derived macrophages during HIV-1 infection.