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 serine kinase (E.T. Sawal et al., Proc. Natl. Acad. Sci. USA 91, 1539-1543, 1994). In the present study, we further define the Nef sequence requirements for this kinase association and investigate the effect of this kinase association on functions of HIV-1 Nef. We observe that, in addition to the membrane targeting signal and the conserved arg-arg residues within the core region, mutations in the proline-rich domain of Nef also affect its ability to associate with the serine kinase activity. 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 previously been observed for the membrane targeting site at the N-terminus of Nef. In contrast, the proline-rich region of Nef is found to be involved in mediating efficient proviral DNA synthesis and the enhanced virion-infectivity function, but is not necessary for CD4 down-modulation by Nef. Thus, it appears that serine kinase association of Nef is necessary for efficient proviral DNA synthesis and for promotion of virion infectivity of Nef viruses, but is dispensable for down-regulation of the CD4 receptor by Nef. These findings define three functional domains of Nef that are required for its interaction with the serine kinase activity and suggest that the cellular interaction events via the myristoylation and arg-arg regions of Nef lie upstream of the interaction event via the proline-rich domain.