Screening And Mechanisms Investigation Of Inhibitors Against Nef Protein Of HIV-1

Object Acquired Immunodeficiency Syndrome(AIDS)is a chronic infectious disease caused by human immunodeficiency virus type 1(HIV-1)infection.The main routes of transmission include blood contact,vertical transmission,and sexual transmission.AIDS is one of the severe diseases threatening human health because of its lethality and lack of effective cure drugs.The existence of viral reservoir is the majorobstacle for not achieving a cure.Current combined antiretroviral therapy(c ART)mainly targets 3 of 15 HIV-1 proteins leaving many potential viral vulnerabilities unexploited.To purge the HIV-1 latent reservoir,various strategies including “shock and kill” have been developed.A key question remains: how to restore impaired immune surveillance? HIV-1 protein Nef has long been known to mediate the downregulation of cell-surface MHC-1 and assist HIV-1 to evade the immune system.It is of great theoretical and practical value to seek and develop a novel specific Nef antagonist to down-regulate MHC-1 antiviral drugs.The focus of this study is to search for antiviral drug(s)that can specifically antagonize Nef protein from the FDA-approved compound library,restore the expression of MHC-1 on the cell surface,increase the response of the immune system to HIV-1,and provide a new strategy for enhancing the immune surveillance of HIV-1 in clinical practice.Research Method Initially,we used molecular cloning techniques toconstruct HIV-1-Nef-GFP expression vector.Through the cell transfectionand flow cytometry,the effect of Nef-GFP expression upon the expression of MHC-I in HEK293 T cellswas evaluated.By high throughput screening methods,lovastatinhas been identified to inhibitthe Nef-mediated MHC-1 down-regulation.Through virus packaging and amplification in vitro,overexpression of Nef and SERINC5,as well as ELISA,we also examined whether lovastatin could inhibit CD4 and SERINC5,which were also down-regulated by Nef,and inhibit the intrinsic infectivity of virions by Nef.By conducting infection invitroand co-culture,we detectedwhether lovastatin may promote CTLs to eliminate infected cells.Further,we test whether lovastatin inhibits the viral rebound of CD4+ T lymphocytes isolated from HIV-1-infected individuals.Finally,the mechanism of lovastatin specific inhibit Nef mediated MHC-I downregulation to restore MHC-I expression was investigated by molecular docking and site-directed mutagenesis,and followed by co-immunoprecipitation experiments.Results Through high throughout screening of Food and Drug Administration(FDA)-approved drugs,we identified that lovastatin,a statin drug,is able to signicantly antagonize Nef to downregulate MHC-I,CD4,and SERINC5,and inhibit the intrinsic infectivity of virions.In addition,lovastatin boosted autologous CTLs to eradicate the infected cells and effectively inhibit the subsequent viral rebound in CD4+ T-lymphocytes isolated from HIV-1-infected individuals receiving suppressive c ART.Furthermore,we found that lovastatin inhibits Nef-induced MHC-I downregulation by directly binding with Nef and disrupting the Nef-MHC-1-AP-1 complex.After in vitro autologous HIV-1 infection,CD4+ T cells were pre-treated with lovastatin.The cells were then co-cultured with autologous CD8+ T cells pre-stimulated with a mixture of Gag-antigen epitope peptides that have been described as unmutated epitopes in chronically HIV-1–infected patients,to assess HIV-1-specific CTL response and cytolytic activity.We found that after treatment with lovastatin,viral replication in infected cells was effective and sustained.The inhibition of lovastatin can effectively inhibit the endogenous infectivity of viralparticles.According to the results of cell killing experiments,we found that after treatment with lovastatin,the infected CD4 + T cells was eliminated by CTLs significantly,whichindicated that lovastatin can also promote the anti-viral effect of autologous CTLs and inhibit the viral rebound of CD4 + T lymphocytesisolated from HIV-1-infected patientseffectively.In addition,we calculated the possible sites of lovastatin and Nef-MHC-I complex through molecular docking experiments,and found that lovastatin interferes with the Nef-MHC-1-AP-1 complex by directly interacting with Nef protein.Through molecular mutation experiments,we further confirmed the directinteraction between lovastatin and Nef,which affects the formation of Nef-MHC-1-AP-1 complex and subsequently inhibits the down-regulation of MHC-I mediated by Nef protein.Conclusions In this study,we have identified a promising anti-HIV-1 druglovastatin for counteracting Nef-mediated downregulation of MHC-I,CD4,and SERINC5.Lovastatin could potentially be used in the clinic to enhance the anti-HIV-1 immune surveillance.Respiratory syncytial virus(RSV)is the most common respiratory virus,and it is associated with pediatric pneumonia,causing bronchiolitis and significant mortality in infants and young children.Micro RNAs(mi RNAs)are endogenous noncoding small RNAs that function in gene regulation and are associated with host immune responseand disease progression.In the present study,to identify the potential biomarkers andinvestigate the molecular mechanisms of severe RSV-associated pediatric pneumonia,we profiled the global transcriptome and mi RNAome of whole blood samples fromchildren with mild or severe RSV-associated pneumonia.Expression profiles of wholeblood micro RNAs and m RNAs were altered and distinctly different in children withsevere RSV-associated pneumonia.The four most significantly upregulated mi RNAswere identified including hsa-mi R-1271-5p,hsa-mi R-10a-3p,hsa-mi R-125b-5p,andhsa-mi R-30b-3p in children with severe RSV-associated pneumonia.The severe RSV-associated pneumonia specific differentially expressed mi RNAs-targetsinteraction network was contrasted.These target genes were further analyzed withgene ontology(GO)enrichment analysis.Interestingly,we found that most of thetarget genes were involved in inflammatory and immune responses,including the NF-k B signaling pathway,the MAPK signaling pathway and T cell receptor signaling.Our findings will contribute to the identification of biomarkers and new drug designstrategies to treat severe RSV-associated pediatric pneumonia.