| HIV Nef is an important determinant of progression to AIDS. Nef functions include downregulation of CD4 and MHC-1, enhancement of viral infectivity, enhancement of replication in resting T cells, and modulation of cellular signaling pathways. Activation of p21-activated kinase 2 (Pak2), a cellular serine/threonine kinase, by Nef may be important for T cell activation and AIDS progression, but the mechanism by which Nef activates Pak2 is unknown. Furthermore, little is known about compartmentalization of nef sequences in HIV-1-infected patients in vivo, nor about functional characteristics of Nefs in specific tissue sites such as brain. This thesis addresses two poorly understood aspects of HIV-1 Nef. (1) the mechanism by which Nef associates with and activates Pak2; and (2) the compartmentalization and relative conservation of 3 Nef functions, CD4 and MHC-1 downregulation and Pak2 activation, in brain versus lymphoid tissues of AIDS patients. We demonstrated that Nef amino acids at positions 85, 89, 187, 188 and 191 are critical for Pak2 association. Mutation of these residues reduced association with Pak2 without affecting other Nef functions. Compensation and covariation occurred at residues 89 and 191, suggesting that covariation at these positions confers a selective advantage in vivo. Thus, Nef residues 85, 89, 187, 188, and 191 may form part of a unique binding surface specifically important for association with activated Pak2. This work provides insights into the mechanism of Pak2 activation and identifies valuable mutants with which to study the importance of Nef-Pak2 association for T cell activation and HIV-1 pathogenesis. To better understand functional characteristics of Nefs in HIV-1-infected patients, we cloned and characterized brain- and lymphoid-derived Nef alleles from two patients with late stage AIDS. Genetic compartmentalization of brain versus lymphoid nef sequences was demonstrated. CD4 and MHC-I downregulation activity were conserved in primary Nef clones, but MHC-I downregulation by brain-derived Nefs was weaker than downregulation by lymphoid-derived Nefs. Motifs KEEE- or EKEE- at the PACS-1 binding site involved in MHC-I downregulation represented brain-specific signature patterns and contributed to the reduced MHC-I downregulation by brain-derived Nefs. Pak2 association was less well conserved and did not correlate with brain or lymphoid origin. These studies provide insights into the relative conservation and variability of Nef functions in vivo, and establish groundwork for further investigation of the relative contributions of various Nef functions to HIV-1 pathogenesis. |
