Abstract
A well conserved feature of human immunodeficiency virus, type 1 (HIV-1) and simian immunodeficiency virus (SIV) Nef is the interaction with and activation of the human p21-activated kinase 2 (PAK2). The conservation of this interaction in other species and its significance for Nef pathogenesis in vivo are poorly documented. In the present study, we measured these parameters in Nef-expressing thymocytes, macrophages, and dendritic cells of a transgenic (Tg) mouse model of AIDS (CD4C/HIV). We found that Nef binds to and activates PAK2, but not PAK1 and -3, in these three cell subsets. Nef associates with only a small fraction of PAK2. The Nef-PAK2 complex also comprises beta-PIX-COOL. The impact of the Nef-PAK2 association on disease development was also analyzed in Tg mice expressing 10 different Nef mutant alleles. CD4C/HIV Tg mice expressing Nef alleles defective in Nef-PAK2 association (P69A, P72A/P75A, R105A/R106A, Delta56-66, or G2A (myristoylation site)) failed to develop disease of the non-lymphoid organs (kidneys and lungs). Among these, only Tg mice expressing Nef(P69A) and Nef(G2A) showed some depletion of CD4(+) T cells, although a down-regulation of the CD4 surface protein was documented in all these Tg lines, except those expressing Nef(Delta56-66). Among other Tg mice expressing Nef mutants having conserved the Nef-PAK2 association (RD35AA, D174K, P147A/P150A, Delta8-17, and Delta25-65), only Tg mice expressing Nef(Delta8-17) develop kidney and lung diseases, but all showed partial CD4(+) T cell depletion despite some being defective for CD4 down-regulation (RD35AA and D174K). Therefore, Nef can activate murine PAK2 and associate with a small fraction of it, as in human cells. Such activation and binding of PAK2 is clearly not sufficient but may be required to induce a multiorgan AIDS-like disease in Tg mice.
Highlights
Nef is an accessory protein of HIV3 and simian immunodeficiency virus (SIV), which has been found to be essential for high levels of viral replication and disease progression
Nef-associated kinase (NAK) Activity Is Present in Thymocytes, Macrophages, and dendritic cells (DCs) of CD4C/HIVNef Tg Mice—We evaluated the association of Nef with NAK and its subsequent activation by an in vitro kinase assay (IVKA) performed on total lysates from thymocytes, macrophages, and DCs
Nef was immunoprecipitated with associated proteins using polyclonal anti-Nef serum followed by IVKA, and phosphorylated substrates were visualized by SDS-PAGE and autoradiography
Summary
Nef is an accessory protein of HIV3 and SIV, which has been found to be essential for high levels of viral replication and disease progression. Nef has been shown to mediate down-regulation of the cell-surface expression of CD4 molecules [19, 20], a function apparently independent of its role in enhancing viral infectivity [21]. Several studies, mostly using the SIV Nef allele, have linked this association to efficient pathogenesis and enhancement of viral infectivity [62, 63]. These observations were challenged by other studies showing that interaction of SIV Nef with PAK was not a prerequisite for SIV to achieve a high viral load and pathogenesis (59, 64 – 66) (see below)
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