title: Two sorting motifs, a ubiquitination motif and a tyrosine motif, are involved in HIV-1 and simian immunodeficiency virus Nef-mediated receptor endocytosis.
abstract: HIV-1 and SIV Nef proteins downregulate cell surface CD4 and MHC class I (MHC-I) molecules of infected cells, which are necessary for efficient viral replication and pathogenicity. We previously reported that K144 in HIV-1 Nef is di-ubiquitinated, and K144R substitution impairs Nef-mediated CD4 downregulation. In this report, we extend the role of ubiquitination at this lysine residue from Nef-mediated CD4 downregulation to Nef-mediated MHC-I downregulation and from HIV Nef to SIV Nef. All HIV-1 Nef mutants that contain K144R substitution are inactive in MHC-I downregulation. Tested MHC-I alleles include HLA-ABC endogenously expressed and HLA-A2 exogenously expressed in Jurkat T cells. CD4 downregulation by SIV Nef involves K176 that aligns with K144 in HIV-1 Nef, as well as an N-terminal tyrosine motif Y28Y39 not present in HIV-1 Nef. Dual mutation at K176 and Y28Y39 completely impaired SIV Nef-mediated CD4 and MHC-I downregulation, whereas a single mutation at K176 or Y28Y39 did not. The involvement of tyrosine motif in SIV Nef-mediated CD4 and MHC-I downregulation prompted us to investigate a putative tyrosine motif (Y202Y/F203) in HIV-1 Nef that is conserved among HIV-1 species. Single mutation at the tyrosine motif Y202F203 in HIV-1 Nef (NA7) greatly impaired Nef-mediated CD4 downregulation, which is similar to what we observed previously with the single mutation at lysine K144. Thus, our study demonstrated that Nef-mediated receptor endocytosis involves the ubiquitination motif and tyrosine motif.
authors.affiliation: Department of Oncological Sciences, Mount Sinai School of Medicine, New York, NY 10029, USA.
authors.forename: Catherine Yi
authors.initials: CY
authors.lastname: Cai
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Zhang
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Sinko
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Burakoff
authors.forename: Yong-Jiu
authors.initials: YJ
authors.lastname: Jin
mesh_terms: D020816:Amino Acid Motifs; D015704:CD4 Antigens / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D004705:Endocytosis; D015693:Gene Products, nef / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005805:Genes, MHC Class I; D015497:HIV-1 / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015789:HLA-A2 Antigen; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D019169:Jurkat Cells; D015302:Simian Immunodeficiency Virus / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D054875:Ubiquitination; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D015704:CD4 Antigens; D015693:Gene Products, nef; D015789:HLA-A2 Antigen; D054311:nef Gene Products, Human Immunodeficiency Virus