Study Of The Role Of HIV-1 Nef In TPN-dependent MHC Class ? Antigen Presentation

Human immunodeficiency virus 1(HIV-1)is the etiologic agent of acquired immunodeficiency syndrome(AIDS),and there is currently no cure and effective vaccine for HIV infection.HIV-1 Nef protein is necessary for high viral loads and for timely progression to AIDS,and has been reported to play important roles in virus transmission,pathogenesis and immune evasion.Tapasin(TPN)is a key component of MHC class I peptide loading complex(PLC),and the main function of TPN is promoting the binding of MHC class I molecules in ER with high-affinity peptides(i.e.peptide optimization),ensuring the MHC class I-peptide cargo can be translocated to cell surface for recognition by cytotxic T lymphocytes.Here,we find that Nef interacts with TPN,and interferes with TPN-dependent peptide optimization and MHC class I antigen presentation moderately.Firstly,we confirmed the interaction between TPN and Nef utilizing yeast two-hybrid assay,Co-immunoprecipitation and pull down assay,which was initially identified by yeast two-hybrid screen.Then,we investigated the effect of Nef on the interactions of PLC components and observed that Nef attenuates the interaction between TPN and TAP1(transporter associated with antigen processing 1).The biologic effect of the identified interaction was explored subsequently.The MHC gene in human(human leukocyte antigen,HLA)is highly polymorphic,different MHC class I allele displays a distinctive dependence on TPN for surface expression and antigen presentation,and we chose HLA-B4402(B44)and HLA-B2705(B27)for further study as representatives for TPN-dependent and-independent class I molecules,respectively.Utilizing the well-known correlation between the thermostability of MHC class I complexes and the affinity of their peptide cargo,we determined the effect of Nef on thermostability of MHC I complexes and found that Nef reduces the thermostability of B44 to some extent,but not the thermostability of B27,which suggests that Nef interferes with the peptide optimization of TPN-dependent MHC class I molecules.It is reported that impaired peptide optimization can lead to the retention of MHC class I molecules in ER.Therefore,we performed endo-H assay and observed that Nef impairs the intracellular maturation of TPN-dependent MHC class I molecules from ER to cell surface.We further determined the effect of Nef on the expression of MHC class I molecules on cell surface,and found that Nef reduces the expression of both B27 and B44 on cell surface,but the downregulation of B44 is stronger than the downregulation of B27,which indicates that Nef may reduce the expression of TPN-dependent MHC class I molecules on cell surface through it's interaction with TPN,and involve other mechanisms to reduce the expression of both TPN-dependent and –independent MHC class I molecules on cell surface.