ABSTRACT
The Tat protein from HIV-1, when fused with heterologous proteins or peptides, can traverse biological membranes in a process called "protein transduction," delivering its cargo into cells. A Tat-eGFP fusion protein was purified from bacteria to study the transduction kinetics of Tat fusion proteins into cultured myoblasts and in the muscle tissue. Correctly folded Tat-eGFP reaches a maximum intracellular level in nearly 30 min, while its endogenous fluorescence is first detected only after 14 h. The nuclear localization signal from the basic domain of Tat was not sufficient to confer nuclear localization to Tat-eGFP, suggesting that the nuclear import pathway used by the exogenously added Tat-eGFP might be sensitive to the folding state of eGFP. In mice, the direct delivery to the muscle tissue using subcutaneous injections or the intra-arterial pathway led to few positive fibers in the muscle periphery or surrounding the blood vessels. Muscles injected with Tat-eGFP showed intense labeling of the extracellular matrix (ECM), suggesting that, although Tat fusion proteins can transduce muscle fibers, their binding by components of the ECM surrounding myofibers could interfere with the intracellular transduction process.
Subject(s)
Gene Products, tat/metabolism , HIV-1 , Muscle, Skeletal/metabolism , Animals , Antimicrobial Cationic Peptides , Biological Transport , Cells, Cultured , Gene Products, tat/genetics , Gene Transfer Techniques , Green Fluorescent Proteins , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Mice , Mice, Inbred BALB C , Recombinant Fusion Proteins/administration & dosage , Transcriptional Activation , Transduction, Genetic , Transfection , tat Gene Products, Human Immunodeficiency VirusABSTRACT
A quantitative reverse transcriptase PCR assay with automated detection by nonradioactive hybridization was developed for the determination of human immunodeficiency virus (HIV) type 1 RNA levels. This assay is based on the use of an external standard curve with an internal standard. The accuracy of quantification was verified by comparison with reference commercial tests, the Chiron branched-DNA and Roche AMPLICOR HIV MONITOR assays. This assay was able to quantify viremia in patients with CD4 cell numbers below and above 500/mm3 and to quantify some HIV strains which could not be titrated by the MONITOR assay.
Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , HIV Infections/blood , HIV-1/isolation & purification , Polymerase Chain Reaction/methods , DNA Primers , HIV Infections/virology , HIV-1/immunology , Humans , Sensitivity and SpecificityABSTRACT
Zidovudine treatment of human immunodeficiency virus (HIV) infection induces drug-resistant viral strains harbouring specific amino acid substitutions in the reverse transcriptase (RT). To investigate whether this phenomenon could be observed in the case of human T lymphotropic virus type I (HTLV-I) infection, we analysed the HTLV-I RT proviral gene sequence in five HTLV-I/HIV-1 co-infected patients treated with zidovudine for HIV-1 infection and in one untreated co-infected subject. In the 816 bp of HTLV-I pol gene sequence determined, no particular nucleotide mutation associated with zidovudine therapy could be identified in the treated subjects. Moreover, the dominant HTLV-1 deduced amino acid sequences determined in treated subjects were identical to that from the untreated subject. Our data show that in the co-infected patients already presenting well-defined mutations associated with zidovudine resistance in HIV-1, no mutations were observed in a part of the pol gene coding for the RT activity of HTLV-I.
