RESUMO
The interaction between the human immunodeficiency virus (HIV) integrase (IN) and its cellular cofactor lens epithelium-derived growth factor (LEDGF/p75) is crucial for HIV replication. While recently discovered LEDGINs inhibit HIV-1 replication by occupying the LEDGF/p75 pocket in IN, it remained to be demonstrated whether LEDGF/p75 by itself can be targeted. By phage display we identified cyclic peptides (CPs) as the first LEDGF/p75 ligands that inhibit the LEDGF/p75-IN interaction. The CPs inhibit HIV replication in different cell lines without overt toxicity. In accord with the role of LEDGF/p75 in HIV integration and its inhibition by LEDGINs, CP64, and CP65 block HIV replication primarily by inhibiting the integration step. The CPs retained activity against HIV strains resistant to raltegravir or LEDGINs. Saturation transfer difference (STD) NMR showed residues in CP64 that strongly interact with LEDGF/p75 but not with HIV IN. Mutational analysis identified tryptophan as an important residue responsible for the activity of the peptides. Serial passaging of virus in the presence of CPs did not yield resistant strains. Our work provides proof-of-concept for direct targeting of LEDGF/p75 as novel therapeutic strategy and the CPs thereby serve as scaffold for future development of new HIV therapeutics.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Fármacos Anti-HIV/farmacologia , Técnicas de Visualização da Superfície Celular , HIV-1/fisiologia , Peptídeos Cíclicos/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Replicação Viral , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Fármacos Anti-HIV/química , Sítios de Ligação , Sequência Conservada , Avaliação Pré-Clínica de Medicamentos , Farmacorresistência Viral , Integrase de HIV/química , HIV-1/efeitos dos fármacos , HIV-2/efeitos dos fármacos , HIV-2/fisiologia , Células HeLa , Humanos , Biblioteca de Peptídeos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/genética , Ligação Proteica , Fatores de Transcrição/química , Internalização do VírusRESUMO
A deoxyribonucleic acid (DNA) fragment encoding the cholera toxin B subunit (CTB) was linked 5' to the simian immunodeficiency virus (SIVmac) Gag p27 capsid gene (CTB-Gag). The fusion gene was transferred into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation methods and transformed plants regenerated. The CTB-Gag gene fusion was detected in transformed potato leaf genomic DNA by polymerase chain reaction-mediated DNA amplification. The results of immunoblot analysis with anti-CTB and anti-Gag antibodies verified the synthesis of biologically active CTB-Gag fusion protein in transformed leaf and tuber tissues. Synthesis and assembly of the CTB-Gag fusion protein into oligomeric structures of pentamer size was confirmed by GM1-ganglioside-enzyme-linked immunosorbent assay (GM1-ELISA) of transformed potato tuber tissue extracts. The binding of CTB-Gag fusion protein oligomers to intestinal epithelial cell membrane receptors quantified by GM1-ELISA showed that CTB-Gag fusion protein made up approx 0.016-0.022% of the total soluble tuber protein. The synthesis of CTB-Gag monomers and their assembly into biologically active CTB-Gag fusion protein oligomers in potato tuber tissues provides the opportunity for employment of the carrier and adjuvant properties of CTB for the development of edible plant-based subunit mucosal vaccines for enhanced mucosal immunity against SIV in macaques.
Assuntos
Toxina da Cólera/genética , Produtos do Gene gag/genética , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Solanum tuberosum/genética , Agrobacterium tumefaciens/genética , Proteínas do Capsídeo/genética , Toxina da Cólera/biossíntese , DNA/análise , Produtos do Gene gag/biossíntese , Técnicas de Transferência de Genes , Folhas de Planta/química , Folhas de Planta/imunologia , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/genética , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Solanum tuberosum/metabolismo , Solanum tuberosum/microbiologiaRESUMO
A cDNA encoding the Simian immunodeficiency virus type (SIV(mac)) Gag capsid protein was introduced into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation methods. The gag gene was detected in the genomic DNA of transformed leaf tissues by PCR DNA amplification. Immunoblot analysis of transformed potato plant extracts with anti-Gag monoclonal antibody showed that biologically active Gag protein was synthesized in transformed tuber tissues. Based on ELISA results, recombinant Gag protein made up 0.006-0.014% of total soluble tuber protein. The synthesis of SIV Gag in transformed potato tubers opens the way for development of Gag-based edible plant vaccines for protection against SIV and potentially HIV-1 infection.
Assuntos
Vacinas contra a AIDS/isolamento & purificação , Proteínas do Capsídeo/isolamento & purificação , Produtos do Gene gag/isolamento & purificação , Infecções por HIV/prevenção & controle , Vacinas contra a SAIDS/isolamento & purificação , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vírus da Imunodeficiência Símia/genética , Vacinas contra a AIDS/genética , Agrobacterium tumefaciens/genética , Proteínas do Capsídeo/genética , Expressão Gênica , Produtos do Gene gag/genética , Humanos , Vacinas contra a SAIDS/genética , Solanum tuberosum/genéticaRESUMO
A cDNA encoding the simian-human immunodeficiency virus (SHIV 89.6p) Tat regulatory element protein was fused to the c-terminus of the cholera toxin B subunit gene (ctxB-tat) and introduced into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation methods. The fusion gene was detected in the genomic DNA of transformed potato leaf cells by PCR DNA amplification. Synthesis and assembly of the CTB-Tat fusion protein into oligomeric structures of pentamer size was detected in transformed tuber extracts by immunoblot analysis. The binding of CTB-Tat fusion protein pentamers to intestinal epithelial cell membrane glycolipid receptors was quantified by G(M1)-ganglioside enzyme-linked immunosorbent assay (G(M1)-ELISA). Based on the ELISA results, CTB-Tat fusion protein made up about 0.005-0.007% of total soluble tuber protein or approximately 4.6mg per 100g potato tuber tissue. The synthesis and assembly of CTB-Tat monomers into biologically active oligomers in transformed potato tuber tissues demonstrates the feasibility of using viral pathogen antigens synthesized in edible plants for mucosal immunization against HIV-1 infection.