ABSTRACT
Strict regulation of HIV-1 PR function is critical for efficient production of mature viral particles. During viral protein expression and viral assembly, HIV-1 PR located within Gag-Pol precursor must be inactive to prevent premature cytoplasmic processing of the viral Gag and Gag-Pol precursors. Premature activation of HIV-1 precursors leads to major defects in viral assembly and production of viral particles. A cell-level premature activation of HIV-1 precursors assay using bioluminescence resonance energy transfer (BRET) was established. Three thousand compounds were screened to evaluate this assay. The results showed that the assay is sensitive, specific and stable (Z' factor is 0.905).
Subject(s)
Humans , Anti-HIV Agents , Pharmacology , Benzoxazines , Pharmacology , Bioluminescence Resonance Energy Transfer Techniques , Methods , Fusion Proteins, gag-pol , Genetics , Metabolism , HEK293 Cells , HIV Protease , Metabolism , Physiology , HIV-1 , High-Throughput Screening Assays , Methods , Plasmids , Genetics , Protein Precursors , Metabolism , Physiology , Pyridazines , Pharmacology , Transfection , Virion , Virus Assembly , gag Gene Products, Human Immunodeficiency Virus , Genetics , MetabolismABSTRACT
We report a 7-year HIV-1 clade A/E-infected child untreated with antiretroviral therapy who had positive HIV antibody testing but undetectable plasma HIV-1 RNA by Roche Amplicor version 1.5 and bDNA version 3.0. DNA PCR was positive by methods using gag/pol primers but not env/pol primers. The patient had strong HIV-1-specific cytotoxic T lymphocyte responses, which likely contributed to her low viral burden and undetectable plasma HIV-1 RNA.
Subject(s)
Anti-Retroviral Agents , Blotting, Western , Child , DNA Primers , DNA, Viral/blood , Enzyme-Linked Immunosorbent Assay , Female , Fusion Proteins, gag-pol/blood , HIV Infections/blood , HIV-1/genetics , Humans , Interferon-gamma/blood , Polymerase Chain Reaction , RNA, Viral/blood , Viral LoadABSTRACT
<p><b>BACKGROUND</b>Construction of replication-deficient recombinant adenovirus expressing gag-pol and env genes of human immunodeficiency virus (HIV) in mice.</p><p><b>METHODS</b>gag-polDelta and gp140TM genes were cloned into shuttle vector pAdTrack-CMV respectively, and then the plasmids containing gag-polDelta or gp140TM gene were cotransformed with the backbone of adenovirus into E.coli BJ5183. Transfections of the recombinants were performed to obtain recombinant adenoviruses. Its immunogenicity was evaluated by testing antibody levels of mice primed with DNA vaccines and boosted with recombinant adenoviruses.</p><p><b>RESULTS</b>The replication-deficient recombinant adenovirus could express Gp140TM, Gag P55 and P24 proteins correctly. The mice primed with DNA vaccines and boosted with recombinant adenoviruses elicited high titer of HIV-1-specific antibody compared with that inoculated with DNA vaccines only.</p><p><b>CONCLUSION</b>Replication-deficient recombinant adenovirus expressing gag-polDelta and gp140TM can elicit high titer HIV-1-specific antibodies.</p>