Association of Nef with p21-Activated Kinase 2 Is Dispensable for Efficient Human Immunodeficiency Virus Type 1 Replication and Cytopathicity in Ex Vivo-Infected Human Lymphoid Tissue

Abstract

Interaction of the human immunodeficiency virus type 1 (HIV-1) Nef protein with p21-activated kinase 2 (PAK2) has been proposed to play a role in T-cell activation, viral replication, apoptosis, and progression to AIDS. However, these hypotheses were based on results obtained using Nef mutants impaired in multiple functions. Recently, it was reported that Nef residue F191 is specifically involved in PAK2 binding. However, only a limited number of Nef activities were investigated in these studies. To further evaluate the role of F191 in Nef function and to elucidate the biological relevance of Nef-PAK2 interaction, we performed a comprehensive analysis of HIV-1 Nef mutants carrying F191H and F191R mutations. We found that the F191H mutation reduces and the F191R mutation disrupts the association of Nef with PAK2. Both mutants upregulated the major histocompatibility complex II (MHC-II)-associated invariant chain and downregulated CD4, MHC-I, and CD28, although with reduced efficiency for the latter. Furthermore, the F191H/R changes neither affected the levels of interleukin-2 receptor expression and apoptosis of HIV-1-infected primary T cells nor reduced Nef-mediated induction of NFAT. Unexpectedly, the F191H change markedly reduced and the F191R mutation disrupted the ability of Nef to enhance virion infectivity in P4-CCR5 indicator cells but not in TZM-bl cells or peripheral blood mononuclear cells. Most importantly, all HIV-1 Nef mutants replicated efficiently and caused CD4؉ T-cell depletion in ex vivo-infected human lymphoid tissue. Altogether, our data show that the interaction of Nef with PAK2 does not play a major role in T-cell activation, viral replication, and apoptosis.

Optimized ReplicatingRenillaLuciferase Reporter HIV-1 Utilizing Novel Internal Ribosome Entry Site Elements for Native Nef Expression and Function

“…MHC-I down-regulation was less apparent when compared to virusunexposed (mock infected) cultures of PBMCs as well as two CD4 + T cell lines. Interestingly, similar findings were reported by Brown et al in infected PBMCs (7 days postinfection) and primary monocyte-derived macrophages (10-13 days postinfection), 29 and to a lesser degree by Kirchhoff and colleagues in PBMCs 77 but not Jurkat cells. 75 Our flow cytometric analysis of endogenous MHC-I following HIV-1 infection (like analyses done by others 75 ) utilized a pan-reactive antibody that did not distinguish between HLA-A, HLA-B, and HLA-C.…”

Role of Nef in primate lentiviral immunopathogenesis

“…In agreement with a relevant role in vivo Nef-mediated infectivity enhancement is conserved between different groups of primate lentiviruses [12,18,122] and apparently contributes to efficient spread of SIVmac in infected rhesus macaques [53]. However, usage of HeLaderived cell indicator lines is a caveat of most studies on Nef-mediated infectivity enhancement and recent data suggest that the effects may be less pronounced in primary CD4 + T cells [123]. To avoid possible artifacts it will be important to further define the effects of Nef on virion infectivity in producer and target cells relevant for viral spread in vivo in the infected host.…”

Inhibition of the Nef regulatory protein of HIV-1 by a single-domain antibody

“…20,22,23 Our experiments show that coexpression of sdAb19 with WT Nef was phenotypically comparable to the expression of the NefF195A alone. Because this Nef mutant specifically lacks association with PAK2 activity without impairment of any other Nef function, 52 these results suggest a direct interference of sdAb19 upstream or at the level of PAK2 recruitment by Nef. Conceivably, this inhibition may result from blocking of protein interactions of the F195 residue that is in close proximity with the di-Leu motif required for CD4 internalization.…”