RESUMEN
The HIV-1 accessory protein Nef is considered to play an important role in the development of a podocyte phenotype in HIV-1 associated nephropathy. We hypothesized that Nef may be altering the podocyte phenotype both structurally and functionally. To elucidate the involved mechanisms, podocyte proteins interacting with Nef were identified using GST pull down assay and yeast two hybrid assay. The GST pull down assay on protein extracts made from stable colonies of conditionally immortalized human podocytes expressing Nef (Nef/CIHP) displayed a band at 45 kD, which was identified as actin by mass spectrometry. Yeast two hybrid assay identified the following Nef-interacting proteins: syntrophin, filamin B, syntaxin, translational elongation factor 1, and zyxin. The Nef-actin and Nef-zyxin interactions were confirmed by co-localization studies on Nef/CIHP stable cell lines. The co-localization studies also showed that Nef/CIHP stable cell lines had a decreased number of actin filaments (stress fibers), displayed formation of lamellipodia, and increased number of podocyte projections (filopodia). Nef/CIHP displayed an enhanced cortical F-actin score index (P<0.001) and thus indicated a reorganization of F-actin in the cortical regions. Microarray analysis showed that Nef enhanced the expression of Rac1, syndecan-4, Rif, and CDC42 and attenuated the expression of syndecan-3 and syntenin. In addition, Nef/CIHPs displayed a diminished sphingomyelinase (ASMase) activity. Functionally, Nef/CIHPs displayed diminished attachment and enhanced detachment to their substrate. These findings indicate that Nef interaction with actin compromises the podocyte cytoskeleton integrity.