Structural and Functional Analysis of Nef Protein of Human Immunodeficiency Virus (HIV)

• Project/Area Number: 09670308
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Virology
• Research Institution: Tokyo Medical and Dental University
• Principal Investigator: KIMURA Tohru Tokyo Medical and Dental University School of Medicine Reserch Associate, 医学部, 助手 (50280962)
• Project Period (FY): 1997 – 1998
• Project Status: Completed (Fiscal Year 1998)
• Budget Amount: ¥2,900,000 (Direct Cost: ¥2,900,000); Fiscal Year 1998: ¥1,300,000 (Direct Cost: ¥1,300,000); Fiscal Year 1997: ¥1,600,000 (Direct Cost: ¥1,600,000)
• Keywords: HIV / AIDS / CD4 downregulation / Nafl (Nef-associated factor 1) / coiled-coil / protein trafficking / Naf1(Nef-associated factor1) / SH3ドメイン / Nef結合因子 / 活性化T細胞

Research Abstract

Human immunodeficiency virus (MV) is an etiological agent of acquired immunodoficiency syndrome (AIDS). The nef gene of HIV has been shown to be essential for AIDS pathogenesis in various animal models. Moreover, it has been reported that, in in vitro system, nef supports the efficient viral growth and suppresses cell surface CD4 expression. However. the molecular mechanism by which nef exerts such effects remains to be elucidated. At first, we generated various Nef mutants and identified critical sites for viral growth and pathogenesis. Secondly, we cloned a novel Nef binding protein Nafl (Nef-associated factor 1). Naf1 protein has four putative coiled-coil domains and its mRNA is highly expressed in spleen and peripheral blood lymphocytes. When its cDNA was transfected to human cells, Naf1 protein was localaized in particular membrane compartments, which seems to be enlarged by Naf1 overexpression. Furthermore, when Naf1 was overexpressed in human cells, it enhanced the cell surface CD4 expression. This Naf1-mediated CD4 upregulation was counteracted by Nef expression. Recently, many coiled-coil-containing proteins have been reported which are involved in the membrane trafficking and sorting events. These proteins are localized in specific membrane compartments such as lysosomes and, when overexpressed, alter the morphology of these compartments. Given the Naf1's structure, its subcellular localization and its ability to upregulate cell surface CD4 molecule, we envisage that Naf1 upreguaces cell surface CD4 expression presumably during trafficking and sorting, and that Nef inhibits function of Naf I through its physical association. Thus far, Nef has been shown to downregulate CD4 expression by accelerating clathrin-mediated endocytosis pathway. Our results clarified the alternative and novel mode of Nef action : Nef suppresses cell surface CD4 expression via inhibition of cellualr pathway which upregukates CD4 expression.

Research Products

• [Publications] Kawano,Y.,et al.: "Mutational analysis of human immunodeficiency virus type 1 (HIV-1) accessory genes: requirement of a site in the nef gene for HIV-1 replication in Activated CD4^+ T Cells in vitro and in vivo." J.Virol.71. 8456-8466 (1997)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Fukushi,M.et al.: "Identification and cloning of a novel cellular protein Naf1,Nef-associated factor 1,that increases cell surface CD4 expression." FEBS Letters. 442. 83-88 (1999)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Y.Kawano, Y., Tanaka, N.Misawa, R.Tanaka, J.-I.Kira, T.Kimura, M.Fukushi, K.Sano.T.Goto, M.Nakai, T.Kobayashi, N.Yamamoto, Y.Koyanagi.: "Mutational analysis of human immunodeficiency virus type 1 (HIV-1) accessory genes : requirement of a site in the nef gene for HIV-1 replication in activated CD4^+T Cells in vitro and in vivo." J.Virol.volume. 71. 8456-8566 (1997)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] M.Fukushi, J.Dixon, T.Kimura, N.Tsurutani, MJ.Dixon, N.Yamamoto: "Identification and cloning of a novel cellular protein Naf1, Nef-associated factor 1, that increases cell surface CD4 expression." FEBS Letters, volume. 442. 83-88 (1999)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Fukushi,M.et al.: "Identification and cloning of a novel cellular protein Naf1,Nef-associated factor 1,that increases cell surface CD4 expression." FEBS Letters. 442. 83-88 (1999)
• [Publications] J.Penninger et al.: "The interferon regulatory transcription factor IRF-1 controls positive and negative selection of CD8^+ thymocytes." Immunity. 7. 243-254 (1997)
• [Publications] Y.Kawano et al.: "Mutational analysis of human immunodeficiency virus type 1(HIV-1)accessory genes:requirement of a site in the nef gene for HIV-1 replication in activated CD4^+ T Cells in vitro and in vivo." J.Virol.71. 8456-8566 (1997)