Molecular Mechanism For Novel Host Restriction Factor SERINC5 Antagonism By HIV-1Nef

AIDS prevalence is increasing worldwide,seriously threatening public health and social development.Because human immunodeficiency virus type 1?HIV-1?is characterized by rapid mutation,strong resistance and difficulty in elimination,there is still no effective prevention and antiviral therapy.Therefore,it's imminent to develop antiretroviral therapy based on novel mechanisms.Recently,SERINC5?Ser5?was reported as a novel host restriction factor to inhibit viral infectivity,which can be antagonized by the HIV-1 auxiliary protein Nef.However,we don't know the specific molecular mechanism of Ser5 antagonism by Nef.This study aimed to elucidate it and offer the reference for the development of novel antiviral therapy and drugs.Firstly,to explore the effect of Ser5 antagonism by HIV-1Nef,we investigated the effect of Ser5 on HIV-1 infectivity.Our results showed that Ser5 overexpression selectively reduced Nef-deficient HIV-1infectivity by 20 fold and Ser5 increased in the.For 293T and HIV-1 target cell Jurkat-Tag,Ser5 from progeny virions,surface of producing cells and the whole cells were significantly reduced by Nef.We found that Nef directly degraded Ser5 after bafilomycin A1 treatment.The interaction between Nef and Ser5 was first detected in living cells using a bimolecular fluorescence complementation?Bi FC?assay,by which some motifs required for interaction were comfirmed.These results demonstrate that Nef excludes Ser5 from budding progeny virions with the downregulation of Ser5 on the membrane,indicating that Nef counteracts Ser5 antiviral activity.In addition,to identify the pathway of Ser5 degraded by Nef,we found that Nef first initiated receptor-dependent endocytosis during Ser5 downregulation from cell surface,which is mediated by the AP-2 adaptor complex,of which the AP-2?2 subunit played an important role.Then,we detected that the internalized Nef-Ser5 complex was targeted to Rab5+early,Rab7+late,and Rab11+recycling endosomes.Manipulation of AP-2,Rab5,Rab7 and Rab11 expression levels affected the Nef-dependent Ser5 degradation.We found that Ser5 itself can be ubiquitinated by BiFC and immunoprecipitation.Ubiquitination can target membrane protein from late endosome to lysosome.Although Nef did not promote the ubiquitination of Ser5,it could be involved in the downregulation of ubiquitinated Ser5from the membrane.Both both K48-and K63-specific ubiquitin linkages were required for the degradation of Ser5.Finally,we found that Nef could trigger Ser5 to lysosomes for final degradation,which were rescued by NH₄Cl or bafilomycin A1 treatment.Ser5 was redistributed into cytoplasm and showed a strong colocalization with LAMP1.Collectively,these results show that HIV-1 Nef antagonizes Ser5 restriction by downregulation of Ser5 via the endosome-lysosome system.Taken together,we demonstrate that Nef downregulates cell surface Ser5 and alters its intracellular transport pathways,resulting in disruption of its antiviral activity.Subsequently,Nef internalizes Ser5from the plasma membrane via AP-2-mediated endocytosis,and targets Ser5 to endosomal-lysosomal pathway for degradation,suggesting that HIV-1 auxiliary proteins can evade the host innate immunity by actively degrading restriction factor.Revealing this evasion mechanism will provide new insights into developing novel specific therapies.