HIV-1 Nef alters the expression of beta II and epsilon isoforms of proteinkinase C and the activation of the long terminal repeat promoter in human astrocytoma cells

In the human immunodeficiency virus type 1 (HIV-1)-infected brain, the viru s does not replicate in astrocytes, but a synthesis of viral regulatory pro teins occurs in these cells, leading to accumulation of Nef. As an approach to understand the effects of Nef on astrocyte functional activity, we anal yzed whether intracellular Nef interferes with the expression and activatio n of the enzyme protein kinase C (PKC), which is an important regulator of astroglial functions and HIV-1 replication. Astrocytoma clones (U251 MG) no t expressing Nef(Neo), or expressing wild-type Nef (Bru) or nonmyristoylate d Nef(TH) were used to monitor the expression and activation of 10 PKC isof orms. The same clones were used to evaluate the effect of Nef on the viral long terminal repeat (LTR) promoter after activation of PKC with the phorbo l ester 12-myristate 13-acetate (PMA.). PKC intracellular distribution and activation were evaluated by Western blot analysis of cytosolic and membran e fractions of control and Nef-expressing clones. PMA-induced LTR activatio n was analyzed in clones transfected with a plasmid encoding for the CAT re porter gene controlled by the LTR promoter, by using an enzyme-linked immun osorbent assay to measure CAT expression. Nef selectively downregulated the expression and activation of beta II and epsilon PKC isoforms in astrocyto ma cells. Such downregulation correlated with an inhibition of LTR activati on after PMA stimulation. The myristoylation of Nef and its membrane locali zation were essential for these effects. These results suggest that Nef may alter astrocytic functions by interfering with PKC expression and activati on and contribute to the restriction of HIV-1 replication in astrocytes. (C ) 1999 Wiley-Liss, Inc.