title: HIV-1 Vif interaction with APOBEC3 deaminases and its characterization by a new sensitive assay.
abstract: The human APOBEC3 (A3) cytidine deaminases, such as APOBEC3G (A3G) and APOBEC3F (A3F), are potent inhibitors of Vif-deficient human immunodeficiency virus type 1 (HIV-1). HIV-1 Vif (viral infectivity factor) binds A3 proteins and targets these proteins for ubiquitination and proteasomal degradation. As such, the therapeutic blockage of Vif-A3 interaction is predicted to stimulate natural antiviral activity by rescuing APOBEC expression and virion packaging. In this study, we describe a successful application of the Protein Fragment Complementation Assay (PCA) based on the enzyme TEM-1 β-lactamase to study Vif-A3 interactions. PCA is based on the interaction between two protein binding partners (e.g., Vif and A3G), which are fused to the two halves of a dissected marker protein (β-lactamase). Binding of the two partners reassembles β-lactamase and hence reconstitutes its activity. To validate our assay, we studied the effect of well-described Vif (DRMR, YRHHY) and A3G (D128K) mutations on the interaction between the two proteins. Additionally, we studied the interaction of human Vif with other members of the A3 family: A3F and APOBEC3C (A3C). Our results demonstrate the applicability of PCA as a simple and reliable technique for the assessment of Vif-A3 interactions. Furthermore, when compared with co-immunoprecipitation assays, PCA appeared to be a more sensitive technique for the quantitative assessment of Vif-A3 interactions. Thus, with our results, we conclude that PCA could be used to quantitatively study specific domains that may be involved in the interaction between Vif and APOBEC proteins.
authors.affiliation: URIA-IMM, Faculdade de Farmácia da Universidade Lisboa, Av. Das Forças Armadas, 1649-059 Lisbon, Portugal.
authors.forename: Iris
authors.initials: I
authors.lastname: Cadima-Couto
authors.forename: Nuno
authors.initials: N
authors.lastname: Saraiva
authors.forename: Ana Catarina C
authors.initials: AC
authors.lastname: Santos
authors.forename: Joao
authors.initials: J
authors.lastname: Goncalves
mesh_terms: D000071478:APOBEC Deaminases; D003564:Cytidine Deaminase; D043525:Cytosine Deaminase / Q000378:metabolism*; D057809:HEK293 Cells; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D040081:Protein Array Analysis / Q000379:methods / Q000592:standards*; D011485:Protein Binding / Q000502:physiology; D054320:vif Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*
chemical_list: D054320:vif Gene Products, Human Immunodeficiency Virus; D043525:Cytosine Deaminase; D000071478:APOBEC Deaminases; C502889:APOBEC3 proteins, human; D003564:Cytidine Deaminase