title: Identification of SERINC5-001 as the Predominant Spliced Isoform for HIV-1 Restriction.
abstract: Among the five serine incorporator (SERINC) family members, SERINC5 (Ser5) was reported to strongly inhibit HIV-1 replication, which is counteracted by Nef.Ser5produces 5 alternatively spliced isoforms: Ser5-001 has 10 putative transmembrane domains, whereas Ser5-004, -005, -008a, and -008b do not have the last one. Here, we confirmed the strong Ser5 anti-HIV-1 activity and investigated its isoforms' expression and antiviral activities. It was found thatSer5-001transcripts were detected at least 10-fold more than the other isoforms by real-time quantitative PCR. When Ser5-001 and its two isoforms Ser5-005 and Ser5-008a were expressed from the same mammalian expression vector, only Ser5-001 was stably expressed, whereas the others were poorly expressed due to rapid degradation. In addition, unlike the other isoforms, which are located mainly in the cytoplasm, Ser5-001 is localized primarily to the plasma membrane. To map the critical determinant, Ser5 mutants bearing C-terminal deletions were created. It was found that the 10th transmembrane domain is required for Ser5 stable expression and plasma membrane localization. As expected, only Ser5-001 strongly inhibits HIV-1 infectivity, whereas the other Ser5 isoforms and mutants that do not have the 10th transmembrane domain show very poor activity. It was also observed that the Nef counteractive activity could be easily saturated by Ser5 overexpression. Thus, we conclude that Ser5-001 is the predominant antiviral isoform that restricts HIV-1, and the 10th transmembrane domain plays a critical role in this process by regulating its protein stability and plasma membrane targeting.IMPORTANCEHuman immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) express a small protein, Nef, to enhance viral pathogenesisin vivoNef has an importantin vitrofunction, which is to make virus particles more infectious, but the mechanism has been unclear. Recently, Nef was reported to counteract a novel anti-HIV host protein, SERINC5 (Ser5). Ser5 has five alternatively spliced isoforms, Ser5-001, -004, -005, -008a, and -008b, and only Ser5-001 has an extra C-terminal transmembrane domain. We now show that the Ser5-001 transcripts are produced at least 10-fold more than the others, and only Ser5-001 produces stable proteins that are targeted to the plasma membrane. Importantly, only Ser5-001 shows strong anti-HIV-1 activity. We further demonstrate that the extra transmembrane domain is required for Ser5 stable expression and plasma membrane localization. These results suggest that plasma membrane localization is required for Ser5 antiviral activity, and Ser5-001 is the predominant isoform that contributes to the activity.
authors.affiliation: Harbin Veterinary Research Institute, CAAS-Michigan State University Joint Laboratory of Innate Immunity, State Key Laboratory of Veterinary Biotechnology, Chinese Academy of Agricultural Sciences, Harbin, China.
authors.forename: Xianfeng
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA.
authors.forename: Tao
authors.initials: T
authors.lastname: Zhou
authors.affiliation: Harbin Veterinary Research Institute, CAAS-Michigan State University Joint Laboratory of Innate Immunity, State Key Laboratory of Veterinary Biotechnology, Chinese Academy of Agricultural Sciences, Harbin, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Yang
authors.affiliation: Harbin Veterinary Research Institute, CAAS-Michigan State University Joint Laboratory of Innate Immunity, State Key Laboratory of Veterinary Biotechnology, Chinese Academy of Agricultural Sciences, Harbin, China.
authors.forename: Yumei
authors.initials: Y
authors.lastname: Lin
authors.affiliation: Harbin Veterinary Research Institute, CAAS-Michigan State University Joint Laboratory of Innate Immunity, State Key Laboratory of Veterinary Biotechnology, Chinese Academy of Agricultural Sciences, Harbin, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Shi
authors.affiliation: Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA.
authors.forename: Xihe
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA.
authors.forename: Dylan A
authors.initials: DA
authors.lastname: Frabutt
authors.affiliation: College of Wildlife Resources, Northeast Forestry University, Harbin, China.|Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA.
authors.forename: Xiangwei
authors.initials: X
authors.lastname: Zeng
authors.affiliation: Harbin Veterinary Research Institute, CAAS-Michigan State University Joint Laboratory of Innate Immunity, State Key Laboratory of Veterinary Biotechnology, Chinese Academy of Agricultural Sciences, Harbin, China.
authors.forename: Sunan
authors.initials: S
authors.lastname: Li
authors.affiliation: Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Venta
authors.affiliation: Harbin Veterinary Research Institute, CAAS-Michigan State University Joint Laboratory of Innate Immunity, State Key Laboratory of Veterinary Biotechnology, Chinese Academy of Agricultural Sciences, Harbin, China zhengyo@msu.edu.|Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA.
authors.forename: Yong-Hui
authors.identifier: http://orcid.org/0000-0002-1098-7385
authors.initials: YH
authors.lastname: Zheng
mesh_terms: D000818:Animals; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D054884:Host-Pathogen Interactions; D006801:Humans; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms; D012326:RNA Splicing; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000378:metabolism
chemical_list: D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D020033:Protein Isoforms; D011956:Receptors, Cell Surface; C103949:SERINC3 protein, human; C000602387:SERINC5 protein, human; D054311:nef Gene Products, Human Immunodeficiency Virus
keywords: HIV-1; Nef; SERINC3; SERINC5; infectivity; restriction factor