HIV-1 Nef interferes with M-CSF receptor signaling through Hck activation and inhibits M-CSF bioactivities

Abstract

IntroductionNef is an accessory protein of HIV-1, a causative virus for AIDS. A number of reports, including studies of HIV-1-infected patients and of animal models, have demonstrated that the Nef protein is a major determinant of the pathogenicity of HIV-1. [1][2][3][4] Transgenic mice expressing the complete coding sequences of HIV-1 under the regulatory sequences of human CD4 gene developed severe AIDS-like abnormalities: loss of CD4 ϩ T cells, thymus atrophy, failure to thrive, diarrhea, wasting, premature death, interstitial pneumonitis, and tubulo-interstitial nephritis. 4 Using this mouse model and the introduction of mutation into selected HIV-1 gene(s), Hanna et al 4 clearly demonstrated that Nef harbored a major disease determinant. Therefore, much attention has been given to Nef to explain its contribution to HIV-1 pathogenesis and to investigate it as a target for antiviral drug development.CD4 ϩ T cells and monocytes/macrophages are the principal target cells for HIV-1, and the functions of Nef in CD4 ϩ T cells are generally accepted as accounting for many aspects of viral pathogenesis (reviewed in Fackler and Baur 5 and Peterlin and Trono 6 ). For example, Nef has been shown to cause the downregulation of cell surface molecules such as cell surface receptor CD4 7 and major histocompatibility complex (MHC) class I in CD4 ϩ T cells. 8 The down-regulation of MHC class I is considered to diminish the recognition of HIV-1-infected cells by cytotoxic T cells. In contrast, the contribution of monocytes/macrophages to viral pathogenesis is less well understood. Several lines of evidence support the idea that monocytes/macrophages and CD4 ϩ T cells are important for the development and progression of AIDS. Recent studies have demonstrated that Nef induces the production of CC chemokines (macrophage inflammatory proteins-1␣ and -1␤) and soluble forms of CD23 and intracellular adhesion molecule-1 by macrophages. 9,10 These Nef-induced factors from macrophages might stimulate the chemotaxis and activation of resting CD4 ϩ T cells, thereby promoting the permissiveness of CD4 ϩ T cells to HIV-1 infection. 9,10 This Nef function is likely to be mediated by the activation of NF-B transcription factor. 10 Another important feature of Nef is the binding at high affinity to myeloid lineage-specific Src family kinase Hck to activate its kinase activity. 11,12 The proline-rich (PxxP) motif in Nef binds to the Src homology 3 (SH3) domain of Hck. 11 Interestingly, Hck was found to bind preferentially and with higher affinity to Nef than other Src kinases. 11 The pathologic relevance of the molecular interaction in vivo was revealed by studies of HIV-1 transgenic mice. The mutation in the SH3-binding motif of Nef abolished the development of AIDS-like disease in the HIV-1 transgenic mice. 13 Moreover, the breeding of transgenic mice expressing the complete coding sequences of HIV-1 on a hck Ϫ/Ϫ background resulted in the delay of disease development. 13 These studies suggest that the modulation of macrophage functions by ...

IL-34 and M-CSF share the receptor Fms but are not identical in biological activity and signal activation

“…These results raised a possibility that IL-34 and M-CSF were different in receptor recognition. To test this possibility, we used the flow cytometry-based ligand binding analysis, 24 in which target cells were sequentially incubated with Flag-tagged human IL-34 or human M-CSF, biotin-labeled anti-Flag antibody and PE-labeled streptavidin. In this analysis, the fluorescence Taken all together, our present study showed that IL-34 and M-CSF indeed resembled but were not necessarily identical in their biological activity and signal activation kinetics/strength, which might be caused by the difference in their structure, Fms domains that they bound and the rate of Fms downregulation.…”

“…Human M-CSF-Flag was prepared as described previously. 24 Using the full-length IL-34 cDNA obtained by PCR as a template, we prepared IL-34-Flag cDNA also by PCR.…”

“…The insert cDNAs were subcloned into pcDNA3.1 expression vector (Invitrogen), and the expression plasmids were transfected into 293T cells using Lipofectamine 2000 as described previously. 16,24 The supernatants collected after 2 days were analyzed for the expression of Flagtagged proteins by western blotting with anti-Flag antibody (clone M2; Sigma) and the activity to stimulate the growth of TF-1-fms cells, or subjected to the ligand binding analysis. The sequence alignment was performed using the ClustalW algorithm.…”

“…The binding of IL-34-Flag or M-CSF-Flag proteins to hematopoietic cell lines was assessed by the flow cytometry-based analysis. 24 Before assay, the supernatants of transfected 293T cells were analyzed for the amount of IL-34-Flag or M-CSF-Flag proteins by western blotting with anti-Flag M2 antibody, and appropriately diluted with DMEM medium-10% FCS. TF-1 cells maintained with rhGM-CSF, and TF-1-fms and M-NFS-60 cells maintained with rhM-CSF were factor-depleted in RPMI 1640-10% FCS for 6 h at 371C.…”

HIV-1 Nef Triggers Macrophage Fusion in a p61Hck- and Protease-Dependent Manner

“…The binding affinity between Nef and Hck is the highest among the SH3-mediated interactions (22,23); however, very little is known about the functional consequences of Hck activation by Nef in macrophages. It has been shown to interfere with M-CSF signaling (24,25) and to activate Stat3 (26). Moreover, in a long-term HIV-positive nonprogressor, Trible et al (27) identified an unusual Nef variant that failed to activate Hck, indicating that Hck/ Nef interaction is important for AIDS progression.…”