HIV-1 NEF ASSOCIATION WITH CELLULAR SERINE KINASE CORRELATES WITH ENHANCED VIRION INFECTIVITY AND EFFICIENT PROVIRAL DNA-SYNTHESIS

We previously reported on the association of Nef with a cellular serin e kinase (E. T. Sawai at al., Proc. Natl. Acad. Sei. USA 91, 1539-1543 , 1994). In the present study, we further define the Nef sequence requ irements for this kinase association and investigate the effect of thi s kinase association on functions of HIV-I Nef. We observe that, in ad dition to the membrane targeting signal and the conserved arg-arg resi dues within the core region, mutations in the proline-rich domain of N ef also affect its ability to associate with the serine kinase activit y. The region encompassing the arg-arg residues of Nef is shown to be important for Nef-mediated cell-surface CD4 down-modulation as well as enhancement of viral growth properties. This is similar to what has p reviously been observed for the membrane targeting site at the N-termi nus of Nef. In contrast, the proline-rich region of Nef is found to be involved in mediating efficient proviral DNA synthesis and the enhanc ed virion-infectivity function, but is not necessary for CD4 down-modu lation by Nef. Thus, it appears that serine kinase association of Nef is necessary for efficient proviral DMA synthesis and for promotion of virion infectivity of Nef(+) viruses, but is dispensable for down-reg ulation of the CD4 receptor by Nef. These findings define three functi onal domains of Nef that are required for its interaction with the ser ine kinase activity and suggest that the cellular interaction events v ia the myristoylation and arg-arg regions of Nef lie upstream of the i nteraction event via the proline-rich domain. (C) 1996 Academic Press, Inc.