ABSTRACT
HIV-1 associated neurocognitive disorders (HAND) are a major complication of HIV-1 infection. The mechanism(s) underlying HAND are not completely understood but, based on in vitro studies, the HIV-1 Tat protein may play an important role. In this study, the effect of prolonged exposure to endogenously produced Tat in the brain was investigated using a tat-transgenic (TT) mouse model constitutively expressing the HIV-1 tat gene. We found that stimulus-evoked glutamate exocytosis in the hippocampus and cortex was significantly increased in TT as compared with wild-type control (CC) mice, while GABA exocytosis was unchanged in the hippocampus and decreased in the cortex. This suggests that Tat generates a latent hyper-excitability state, which favors the detrimental effects of neurotoxic and/or excitotoxic agents. To challenge this idea, TT mice were tested for susceptibility to kainate-induced seizures and neurodegeneration, and found to exhibit significantly greater responses to the convulsant agent than CC mice. These results support the concept that constitutive expression of tat in the brain generates a latent excitatory state, which may increase the negative effects of damaging insults. These events may play a key role in the development of HAND.
Subject(s)
Brain/pathology , Nervous System Diseases/pathology , Nervous System Diseases/virology , tat Gene Products, Human Immunodeficiency Virus/metabolism , Analysis of Variance , Animals , Brain/drug effects , Brain/metabolism , Brain/virology , Disease Models, Animal , Dose-Response Relationship, Drug , Gene Products, tat/pharmacology , Kainic Acid/toxicity , Male , Mice , Mice, Transgenic , Neurotransmitter Agents/metabolism , Seizures/chemically induced , Seizures/physiopathology , Statistics, Nonparametric , Vesicular Glutamate Transport Proteins/metabolism , tat Gene Products, Human Immunodeficiency Virus/geneticsABSTRACT
Tat, the trans activation protein of HIV, is produced early upon infection to promote and expand HIV replication and transmission. However, Tat appears to also have effects on target cells, which may affect Ag recognition both during infection and after vaccination. In particular, Tat targets dendritic cells and induces their maturation and Ag-presenting functions, increasing Th1 T cell responses. We show in this work that Tat modifies the catalytic subunit composition of immunoproteasomes in B and T cells either expressing Tat or treated with exogenous biological active Tat protein. In particular, Tat up-regulates latent membrane protein 7 and multicatalytic endopeptidase complex like-1 subunits and down-modulates the latent membrane protein 2 subunit. These changes correlate with the increase of all three major proteolytic activities of the proteasome and result in a more efficient generation and presentation of subdominant MHC-I-binding CTL epitopes of heterologous Ags. Thus, Tat modifies the Ag processing and modulates the generation of CTL epitopes. This may have an impact on both the control of virally infected cells during HIV-1 infection and the use of Tat for vaccination strategies.
Subject(s)
Cysteine Endopeptidases/metabolism , Epitopes, T-Lymphocyte/biosynthesis , Gene Products, tat/physiology , HIV-1/immunology , Lymphocyte Activation/immunology , Multienzyme Complexes/metabolism , T-Lymphocytes, Cytotoxic/enzymology , T-Lymphocytes, Cytotoxic/virology , Antigen Presentation/immunology , Catalytic Domain , Cell Line, Transformed , Cysteine Endopeptidases/isolation & purification , Cytotoxicity Tests, Immunologic , Enzyme Activation/immunology , Epitopes, T-Lymphocyte/immunology , Epitopes, T-Lymphocyte/metabolism , Epstein-Barr Virus Nuclear Antigens/biosynthesis , Epstein-Barr Virus Nuclear Antigens/immunology , Epstein-Barr Virus Nuclear Antigens/metabolism , Gene Products, tat/biosynthesis , Gene Products, tat/genetics , Genetic Vectors , Humans , Hydrolysis , Immunodominant Epitopes/biosynthesis , Immunodominant Epitopes/immunology , Immunodominant Epitopes/metabolism , Jurkat Cells , Multienzyme Complexes/isolation & purification , Peptide Fragments/biosynthesis , Peptide Fragments/immunology , Peptide Fragments/metabolism , Proteasome Endopeptidase Complex , Protein Subunits/isolation & purification , Protein Subunits/metabolism , T-Lymphocytes, Cytotoxic/immunology , tat Gene Products, Human Immunodeficiency VirusABSTRACT
A novel class of cationic block copolymers constituted by a neutral hydrophilic poly(ethylene glycol) (PEG) block and a positively charged poly(dimethylamino)ethyl methacrylate block was prepared for delivery of DNA. These block copolymers spontaneously assemble with DNA to give in aqueous medium micellar-like structures. Five of these novel block copolymers (K1-5), differing in the length of both the PEG chain and the linear charge density of the poly(dimethylamino)ethyl methacrylate block, were prepared and analyzed for gene delivery, gene expression and safety. All five block copolymers protected DNA from DNAse I digestion and delivered the DNA into the cell. However, only three of them (K1, K2 and K5) released the DNA at level allowing efficient gene expression into cells. No toxic effects of both the copolymers alone or their DNA complexes were observed in vitro or in mice. In addition, copolymers were scarcely immunogenic. These results indicate that this novel class of cationic block copolymers is safe and possesses the biological characteristics required for DNA delivery, thus, representing promising vehicles for DNA vaccination.
