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Query: UMLS:C0019693 (HIV)
 170,526 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two new T-cell-based reporter cell lines were established to measure human immunodeficiency virus type 1 (HIV-1) infectivity. One cell line naturally expresses CD4 and CXCR4, making it susceptible to X4-tropic viruses, and the other cell line, in which a CCR5 expression vector was introduced, is susceptible to both X4- and R5-tropic viruses. Reporter cells were constructed by transfecting the human T-cell line HPB-Ma, which demonstrates high susceptibility to HIV-1, with genomes expressing two different luciferase reporters, HIV-1 long terminal repeat-driven firefly luciferase and cytomegalovirus promoter-driven renilla luciferase. Upon HIV infection, the cells expressed firefly luciferase at levels that were highly correlated (r2=0.91 to 0.98) with the production of the capsid antigen p24. The cells also constitutively expressed renilla luciferase, which was used to monitor cell numbers and viability. The reliability of the cell lines for two in vitro applications, drug resistance phenotyping and drug screening, was confirmed. As HIV-1 efficiently replicated in these cells, they could be used for multiple-round replication assays as an alternative method to a single-cycle replication protocol. Coefficients of variation for drug susceptibility evaluated with the cell lines ranged from 17 to 41%. The new cell lines were beneficial for evaluating antiretroviral drug resistance. Firefly luciferase gave a wider dynamic range for evaluating virus infectivity, and the introduction of renilla luciferase improved assay reproducibility. The cell lines were also beneficial for screening new antiretroviral agents, as false inhibition caused by the cytotoxicity of test compounds was easily detected by monitoring renilla luciferase activity.
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PMID:Use of new T-cell-based cell lines expressing two luciferase reporters for accurately evaluating susceptibility to anti-human immunodeficiency virus type 1 drugs. 1718 60

Replication of human immunodeficiency virus type 1 (HIV-1) is controlled by a variety of viral and host proteins. The viral protein Tat acts in concert with host cellular factors to stimulate transcriptional elongation from the viral long terminal repeat (LTR) through a specific interaction with a 59-residue stem-loop RNA known as the trans-activation responsive element (TAR). Inhibitors of Tat-TAR recognition are expected to block transcription and suppress HIV-1 replication. In previous studies, we showed that 2'-O-methyl (OMe) oligonucleotide mixmers containing locked nucleic acid (LNA) residues are powerful steric block inhibitors of Tat-dependent trans-activation in a HeLa cell reporter system. Here we compare OMe/LNA mixmer oligonucleotides with oligonucleotides containing tricyclo-DNAs and their mixmers with OMe residues in four different assays: (1) binding to the target TAR RNA, (2) Tat-dependent in vitro transcription from an HIV-1 DNA template directed by HeLa cell nuclear extract, (3) trans-activation inhibition in HeLa cells containing a stably integrated firefly luciferase reporter gene under HIV-1 LTR control, and (4) an anti-HIV beta-galactosidase reporter assay of viral infection. Although tricyclo-DNA oligonucleotides bound TAR RNA more weakly, they were as good as OMe/LNA oligonucleotides in suppressing in vitro transcription and trans-activation in HeLa cells when delivered by cationic lipid. No inhibition of in vitro transcription and trans-activation in HeLa cells was observed for tricyclo-DNA/OMe mixmers, even though their affinities to TAR RNA were strong and their cell distributions did not differ from oligonucleotides containing all or predominantly tricyclo-DNA residues. Tricyclo-DNA 16-mer showed sequence-specific inhibition of beta-galactosidase expression in an anti-HIV HeLa cell reporter assay.
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PMID:Tricyclo-DNA containing oligonucleotides as steric block inhibitors of human immunodeficiency virus type 1 tat-dependent trans-activation and HIV-1 infectivity. 1746 63

HIV-1 uses a programmed -1 ribosomal frameshift to synthesize the precursor of its enzymes, Gag-Pol. The frameshift efficiency that is critical for the virus replication, is controlled by an interaction between the ribosome and a specific structure on the viral mRNA, the frameshift stimulatory signal. The rate of cap-dependent translation initiation is known to be altered by the TAR RNA structure, present at the 5' and 3' end of all HIV-1 mRNAs. Depending upon its concentration, TAR activates or inhibits the double-stranded RNA-dependent protein kinase (PKR). We investigated here whether changes in translation initiation caused by TAR affect HIV-1 frameshift efficiency. CD4+ T cells and 293T cells were transfected with a dual-luciferase construct where the firefly luciferase expression depends upon the HIV-1 frameshift. Translation initiation was altered by adding TAR in cis or trans of the reporter mRNA. We show that HIV-1 frameshift efficiency correlates negatively with changes in the rate of translation initiation caused by TAR and mediated by PKR. A model is presented where changes in the rate of initiation affect the probability of frameshifting by altering the distance between elongating ribosomes on the mRNA, which influences the frequency of encounter between these ribosomes and the frameshift stimulatory signal.
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PMID:The presence of the TAR RNA structure alters the programmed -1 ribosomal frameshift efficiency of the human immunodeficiency virus type 1 (HIV-1) by modifying the rate of translation initiation. 1798 74

In vitro translation systems are used to investigate translational mechanisms and to synthesize proteins for characterization. Most available mammalian cell-free systems have reduced efficiency due to decreased translation initiation caused by phosphorylation of the initiation factor eIF2alpha on Ser51. We describe here a novel cell-free protein synthesis system using extracts from cultured mouse embryonic fibroblasts that are homozygous for the Ser51 to- Ala mutation in eIF2alpha (A/A cells). The translation efficiency of a capped and polyadenylated firefly luciferase mRNA in A/A cell extracts was 30-fold higher than in wild-type extracts. Protein synthesis in extracts from A/A cells was active for at least 2 h and generated up to 20 microg/mL of luciferase protein. Additionally, the A/A cell-free system faithfully recapitulated the selectivity of in vivo translation for mRNA features; translation was stimulated by a 5'-end cap (m7GpppN) and a 3'-end poly(A) tail in a synergistic manner. The system also showed similar efficiencies of cap-dependent and IRES-mediated translation (EMCV IRES). Significantly, the A/A cell-free system supported the post-translational modification of proteins, as shown by glycosylation of the HIV type-1 gp120 and cleavage of the signal peptide from beta-lactamase. We propose that cell-free systems from A/A cells can be a useful tool for investigating mechanisms of mammalian mRNA translation and for the production of recombinant proteins for molecular studies. In addition, cell-free systems from differentiated cells with the Ser51Ala mutation should provide a means for investigating cell type-specific features of protein synthesis.
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PMID:An efficient in vitro translation system from mammalian cells lacking the translational inhibition caused by eIF2 phosphorylation. 1823 Jul 59

The luciferase reporter phages (LRP) show great promise for diagnostic mycobacteriology. Though conventional constructs developed from lytic phages such as D29 and TM4 are highly specific, they lack sensitivity. We have isolated and characterized Che12, the first true temperate phage infecting M. tuberculosis. Since the tuberculosis (TB) cases among HIV infected population result from the reactivation of latent bacilli, it would be useful to develop LRP that can detect dormant bacteria. During dormancy, pathogenic mycobacteria switch their metabolism involving divergent genes than during normal, active growth phase. Since the promoters of these genes can potentially function during dormancy, they were exploited for the construction of novel mycobacterial luciferase reporter phages. The promoters of hsp60, isocitrate lyase (icl), and alpha crystallin (acr) genes from M. tuberculosis were used for expressing firefly luciferase gene (FFlux) in both Che12 and TM4 phages and their efficiency was evaluated in detecting dormant bacteria from clinical isolates of M. tuberculosis. These LRP constructs exhibited detectable luciferase activity in dormant as well as in actively growing M. tuberculosis. The TM4 ts mutant based constructs showed about one log increase in light output in three of the five tested clinical isolates and in M. tuberculosis H37Rv compared to conventional lytic reporter phage, phAE129. By refining the LRP assay format further, an ideal rapid assay can be designed not only to diagnose active and dormant TB but also to differentiate the species and to find their drug susceptibility pattern.
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PMID:Construction and evaluation of luciferase reporter phages for the detection of active and non-replicating tubercle bacilli. 1827 45

Integrating lentiviral vectors based on the human immunodeficiency virus type-1 (HIV-1) can transduce quiescent cells, which in lung account for almost 95% of the epithelial cell population. Pseudotyping lentiviral vectors with the envelope glycoprotein from the Ebola Zaire virus, the lymphocytic choriomeningitis virus (LCMV), the Mokola virus, and the vesicular stomatitis virus (VSV-G) resulted in transduction of mouse alveolar epithelium, but gene expression in the lung of C57BL/6 and BALB/c mice waned within 90 days of vector injection. Intratracheal delivery of the four pseudotyped lentiviral vectors resulted in transgene-specific T-cell activation in both mouse strains, albeit lower than that achieved by intramuscular injection of the vectors. We performed an adoptive transfer of luciferase-specific T cells, isolated from spleen or lung of donor mice injected with VSV-G-pseudotyped lentivirus vector expressing luciferase into the muscle or lung, respectively, into recipient recombination-activating gene (RAG)-deficient mice transduced in lung with adenovirus expressing firefly luciferase (ffluc2). Gene expression declined within 7 days of adoptive transfer approaching background levels by day 36. Taken together, our results suggest that the loss of transduced cells in lung is due to VSV-G.HIV vector-mediated activation of transgene-specific T cells rather than as result of normal turnover of airway cells.
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PMID:Activation of transgene-specific T cells following lentivirus-mediated gene delivery to mouse lung. 1972 65

To evaluate the interaction between HIV-1 envelope glycoprotein (Env) and target cell receptors, various cell-cell-fusion assays have been developed. In the present study, we established a novel fusion system. In this system, the expression of the sensitive reporter gene, firefly luciferase (FL) gene, in the target cells was used to evaluate cell fusion event. Simultaneously, constitutively expressed Renilla luciferase (RL) gene was used to monitor effector cell number and viability. FL gave a wider dynamic range than other known reporters and the introduction of RL made the assay accurate and reproducible. This system is especially beneficial for investigation of potential entry-influencing agents, for its power of ruling out the false inhibition or enhancement caused by the artificial cell-number variation. As a case study, we applied this fusion system to observe the effect of a serine protease, thrombin, on HIV Env-mediated cell-cell fusion and have found the fusion enhancement activity of thrombin over two R5-tropic HIV strains.
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PMID:A sensitive HIV-1 envelope induced fusion assay identifies fusion enhancement of thrombin. 2004 72

Defensins are antimicrobial peptides expressed by plants and animals. In mammals there are three subfamilies of defensins, distinguished by structural features: alpha, beta and theta. Alpha and beta-defensins are linear peptides with broad anti-microbial activity that are expressed by many mammals including humans. In contrast, theta-defensins are cyclic anti-microbial peptides made by several non-human primates but not humans. All three defensin types have anti-HIV-1 activity, but their mechanisms of action differ. We studied the anti-HIV-1 activity of one defensin from each group, HNP-1 (alpha), HBD-2 (beta) and RTD-1 (theta). We examined how each defensin affected HIV-1 infection and demonstrated that the cyclic defensin RTD-1 inhibited HIV-1 entry, while acyclic HNP-1 and HBD-2 inhibited HIV-1 replication even when added 12 hours post-infection and blocked viral replication after HIV-1 cDNA formation. We further found that all three defensins downmodulated CXCR4. Moreover, RTD-1 inactivated X4 HIV-1, while HNP-1 and HBD-2 inactivated both X4 and R5 HIV-1. The data presented here show that acyclic and cyclic defensins block HIV-1 replication by shared and diverse mechanisms. Moreover, we found that HNP-1 and RTD-1 directly inhibited firefly luciferase enzymatic activity, which may affect the interpretation of previously published data.
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PMID:Cyclic and acyclic defensins inhibit human immunodeficiency virus type-1 replication by different mechanisms. 2030 15

Human immunodeficiency virus type 1 (HIV-1) Tat plays an important role in HIV-associated neuropathogenesis; the underlying mechanisms are still evolving. We have recently shown that HIV-1 Tat induces expression of glial fibrillary acidic protein (GFAP), a characteristic of HIV-1 infection of the central nervous system. We have also shown that the Tat-induced GFAP expression in astrocytes is regulated by p300 and that deletion of the early growth response 1 (Egr-1) cis-transacting element within the p300 promoter abolishes Tat-induced GFAP expression. In this study, we further examined the relationship between Tat and Egr-1 in astrocytes. We found increased Egr-1 protein expression in Tat-expressing human astrocytoma cells and mouse primary astrocytes. Using the Egr-1 promoter-driven firefly luciferase reporter gene assay and the site-directed mutagenesis, we demonstrated that Tat increased Egr-1 expression by transactivating the Egr-1 promoter and involving specific serum response elements within the promoter. Consistent with these data, we showed that Tat transactivation of the Egr-1 promoter was abrogated when astrocytes were cultured in serum-reduced media. Taken together, these results reveal that Tat directly transactivates Egr-1 expression and suggest that Tat interaction with Egr-1 is probably one of the very upstream molecular events that initiate Tat-induced astrocyte dysfunction and subsequent Tat neurotoxicity.
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PMID:Activation of Egr-1 expression in astrocytes by HIV-1 Tat: new insights into astrocyte-mediated Tat neurotoxicity. 2041 33

The direct detection of native proteins in heterogeneous solutions remains a challenging problem. Standard methodologies rely on a separation step to circumvent nonspecific signal generation. We hypothesized that a simple and general method for the detection of native proteins in solution could be achieved through ternary complexation, where the conditional signal generation afforded by split-protein reporters could be married to the specificity afforded by either native receptors or specific antibodies. Toward this goal, we describe a solution phase split-luciferase assay for native protein detection, where we fused fragmented halves of firefly luciferase to separate receptor fragments or single-chain antibodies, allowing for conditional luciferase complementation in the presence of several biologically significant protein targets. To demonstrate the utility of this strategy, we have developed and validated assay platforms for the vascular endothelial growth factor, the gp120 coat protein from HIV-1, and the human epidermal growth factor receptor 2 (HER2), a marker for breast cancer. The specificities of the recognition elements, CD4 and the 17b single-chain antibody, employed in the gp120 sensor allowed us to parse gp120s from different clades. Our rationally designed HER2 sensing platform was capable of discriminating between HER2 expression levels in several tumor cell lines. In addition, luminescence from reassembled luciferase was linear across a panel of cell lines with increasing HER2 expression. We envision that the proof of principle studies presented herein may allow for the potential detection of a broad range of biological analytes utilizing ternary split-protein systems.
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PMID:A general approach for receptor and antibody-targeted detection of native proteins utilizing split-luciferase reassembly. 2068 84


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