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

We have investigated the optimal reaction conditions and the limiting sensitivity for detection of HIV-1 DNA by PCR. The amplification systems studied were gag (SK38/SK39); pol (P3/P4); and two other systems described here for the first time, LTR (LTR1/LTR2) and nef (Nef1/Nef2), which amplify fragments of 115 bp, 308 bp, 632 bp and 643 bp, respectively. Two PCR profiles were assayed, and the requirements for deoxynucleoside triphosphate and MgCl2 concentrations for each amplification reaction were determined. Optimal reaction conditions were oriented toward selecting maximal amplification of the expected size fragment. Limiting sensitivity was estimated by testing the decreasing copy number of a plasmid containing HIV-1 genome and obtaining a positive amplification signal with at least 5, 5, 10 and 5 copies for LTR, gag, pol and nef, respectively. We conclude that the establishment of the detection sensitivity on a PCR is an important parameter to be considered for the interpretation of results on HIV-1 infection.
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PMID:Influence of PCR parameters on amplifications of HIV-1 DNA: establishment of limiting sensitivity. 193 Oct 39

The Tat protein of the human immunodeficiency virus type 1 (HIV-1) is required for efficient viral gene expression. By means of mutational analyses, several domains of the Tat protein that are required for complete activation of HIV-1 gene expression have been defined. These include an amino-terminal activating domain, a cysteine-rich dimerization domain, and a basic domain important in the binding of Tat to the trans-activation response element (TAR) and in Tat nuclear localization. Recently, we described a mutation, known as delta tat, which resulted in a protein with a truncated basic domain. This protein had a "trans-dominant" phenotype in that it inhibited wild-type Tat activation of the HIV-1 LTR. To further characterize the requirements for generating a Tat trans-dominant phenotype, we constructed a variety of Tat proteins with truncations or substitutions in the basic domain. A number of these proteins showed a trans-dominant phenotype. These Tat mutants also inhibited activation of the HIV-1 LTR by a protein composed of Tat fused to the prokaryotic R17 (phage MS2) RNA-binding protein in which the R17 recognition element was inserted in the HIV-1 LTR in place of TAR. Thus, an intact TAR element was not required for this inhibition. We also studied the cellular localization of Tat and a trans-dominant Tat mutant by means of immunofluorescence staining with the use of antibodies reactive to different domains of the Tat protein. The results indicated that Tat becomes localized predominantly in the nucleus both in the presence and absence of the trans-dominant Tat construct, suggesting that the trans-dominant mutant does not inhibit Tat nuclear localization. These studies further define the requirements for the creation of trans-dominant Tat mutants, and suggest that the mechanism of trans-dominant Tat inhibition may be either the formation of an inactive complex between wild-type and mutant Tat or sequestration of cellular factors involved in regulating HIV-1 gene expression.
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PMID:Trans-dominant Tat mutants with alterations in the basic domain inhibit HIV-1 gene expression. 193 22

The TAR element extending from -17 to +80 in the human immunodeficiency virus long terminal repeat (HIV LTR) is required for activation of gene expression by the tat trans-activator protein. TAR RNA forms a stable stem-loop structure, and mutagenesis studies indicate that the stem structure, the primary sequence of the loop, and the bulge element are the major determinants for tat activation. RNA gel retardation analysis demonstrates that both tat and cellular proteins bind to TAR RNA, but the mechanism by which these proteins increase HIV gene expression is unknown. We have fractionated HeLa cell nuclear extracts in an attempt to identify cellular proteins that bind to TAR RNA and are involved in regulating HIV gene expression. RNA gel retardation and UV cross-linking reveal that a cellular protein of 185 kD, which we designate TAR RNA-binding protein 185 (TRP-185), binds with both high affinity and marked specificity to TAR RNA. RNA gel retardation and competition analyses indicate that TRP-185 binding is strongly dependent on the TAR RNA loop sequences. The binding of TRP-185 is modulated by both a set of cellular cofactors and the tat protein. Highly purified preparations of TRP-185 are capable of activating in vitro transcription of wild-type, but not mutated, HIV LTR chloramphenicol acetyltransferase (CAT) constructs. These results characterize a positively acting cellular RNA-binding factor, TRP-185, which is involved in the regulation of HIV gene expression.
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PMID:tat regulates binding of the human immunodeficiency virus trans-activating region RNA loop-binding protein TRP-185. 193 97

Amplification of DNA by polymerase chain reaction (PCR) is influenced by the homology of oligonucleotide primers with the DNA template. We have developed a procedure, termed anchored PCR, whereby nucleotide sequence alterations in the template can be directly related to the quantity of amplified product. Genetic variation in the human immunodeficiency virus HIV-1 has been studied using anchored PCR. In four field isolates of the virus, the 3'LTR was compared both by PCR analysis of DNA from virus cultures and DNA sequencing. DNA templates that matched the primers varied less than threefold in PCR product yield, whereas significant 3' end primer-template mispairing decreased PCR product 10- to 100-fold. Using these guidelines for genetic variability manifested through PCR, 40 PCR primers encompassing the GAG, ENV, and 3' LTR segments of the genome were used to compare sequential HIV-1 isolates form six patients. Some primers were apparently located in genomic regions without significant interisolate variability, as they yielded equivalent amounts of amplified DNA from all the isolates. The quantity of amplified DNA obtained with other primers varied 10- to 100-fold among patients, but was consistent for sequential isolates from an individual patient. Two African HIV-1 isolates were readily distinguished from a panel of North American isolates by the same method. Systematic classification of HIV-1 genetic variants may be possible by anchored PCR.
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PMID:Genetic comparison of human immunodeficiency virus (HIV-1) isolates by polymerase chain reaction. 194 29

To study the effect of methylation of O6-guanine on the binding of cellular factors to different DNA sequences, modified oligonucleotides were constructed, in which O6-Methylguanine (O6-MeG) replaced some guanines. The DNA sequences utilized were: the region of the c-fos promoter containing the binding site for serum response factor (SRF); the region of the HIV LTR containing two binding sites for the transcription factor NF kappa B; the region of the HIV LTR containing three binding sites for the cellular factor sp1. After incubation of labeled oligonucleotides, either unmodified or containing O6-MeG, with nuclear extracts obtained from different cell lines, gel retardation assays indicated that the presence of O6-MeG resulted in inhibition of binding of cellular factors to DNA sequences located in the promoter regions of genes. This inhibition was not the same for all modified oligonucleotides but dependent on the position in which O6-MeG was located. The results obtained indicate that alkylation of O6-guanine affects the binding of transcription factors and thereby possibly the regulation of genes expression.
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PMID:O6-methylguanine inhibits the binding of transcription factors to DNA. 194 51

In order to establish whether the Polymerase Chain Reaction (PCR) may constitute a new marker of evolution towards AIDS in symptomless HIV infected subjects, we used PCR with three primer pairs (in the gag, pol and LTR regions) in 223 seropositive individuals at different stages of HIV infection. Among 176 symptomless seropositive individuals, 174 (98.8%) were positive with at least one primer pair. The subjects negative with at least one primer pair had a CD4 lymphocyte count significantly higher (p less than 0.01), and serum immunoglobulin G, immunoglobulin A, neopterin and beta-2-microglobulin concentrations significantly lower (p less than 0.01) than the individuals positive with the three primer pairs. Among 73 seropositive individuals followed over a two year period, 59 presented the same PCR pattern over this time period, while PCR showed different results in 14. Forty-seven AIDS patients were positive with the three primer pairs. The number of PCR negative with at least one primer pair was significantly fewer (p less than 0.001) in symptomatic individuals than in symptomless individuals. We conclude that the percentage of positive PCR results in HIV infected individuals is linked to the clinical stage of infection and to the disease progression.
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PMID:Polymerase chain reaction (PCR) in various stages of HIV infection. Relationship to disease progression. 195 61

The extent of latent HIV-1 infection in blood T cells and monocytes of 23 seropositive individuals was examined using DNA amplification (PCR) of HIV-1 sequences. Amplified DNA was found in at least one cell type in all seropositives tested, including 13 asymptomatic, 5 ARC, and 5 AIDS patients. Amplification with two or more primer sets from the gag, env, LTR occurred in 21 (91%) patients' T cells and 17 (74%) patients' monocytes. However, amplification with the LTR primers in monocytes was uncommon. Among four patients tested, amplified DNA continued to be detected after a greater than one thousand-fold dilution (less than 500 cells) of both T cell and monocyte lysates. Repeat analysis after 7-9 mo in five seropositives yielded similar findings in T cells and monocytes, but some variation in the efficacy of amplification with individual primers occurred. There was no difference in those 10 patients who were taking AZT, compared to those who were untreated. Our results indicate that a fraction (less than 1%) of both T cells and monocytes in blood carry a latent infection in all stages of HIV-1 disease and can serve as reservoirs throughout AZT therapy.
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PMID:Latent HIV-1 infection in enriched populations of blood monocytes and T cells from seropositive patients. 198 1

Three molecular clones of HIV-1, derived from a single isolate (AL1), exhibited distinct replicative and cytopathic properties during propagation in a human T cell line. The phenotypic differences observed were attributable, in large part, to changes affecting the viral LTR. Nucleotide sequence and PCR analyses demonstrated the presence of novel duplications or deletions involving the NF-kappa B motif. These changes in the enhancer element were identified in the original AL1 virus stock. Subcloning of the variant NF-kappa B segments into LTR-driven CAT expression vectors confirmed a correlation between promoter activity and replicative/cytopathic capacity.
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PMID:A novel HIV-1 isolate containing alterations affecting the NF-kappa B element. 199 72

The evidence presented here indicates that the domain containing the COOH-terminal part of the Saccharomyces cerevisiae SCD25 gene product (C-domain), which is homologous to the COOH-terminal part of CDC25 protein, can elicit activation of mammalian ras proteins in CHO cells. Transfection of expression vectors carrying the C-domain of SCD25, but not of CDC25, promotes the GTP-bound form of ras proteins as determined by analysis of the guanine nucleotides bound to ras proteins immunoprecipitated by Y13-259 mAb, and enhances transcription of a HIV-LTR-CAT construct. This is the first demonstration of the activation of ras proteins by transfection of a single heterologous gene.
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PMID:The COOH-domain of the product of the Saccharomyces cerevisiae SCD25 gene elicits activation of p21-ras proteins in mammalian cells. 200 Feb 28

The finding of dual HTLV-I and HIV-1 infection in populations at risk for AIDS raises the possibility that interaction between the two viruses might have clinical significance. It was shown that HTLV-I enhances HIV-1 expression, but whether HIV-1 activates HTLV-I remains to be demonstrated. To study HTLV-I behaviour following HIV-1 infection, we superinfected cells from two HTLV-I transformed cell lines with HIV-1 (strain IIIB). Viral RNA analysis indicated that HTLV-I expression in the doubly infected cells was moderately enhanced. Moreover, CAT assays in HTLV-I transformed cells transiently transfected with HTLV-I LTR-CAT disclosed higher activity in the HIV-1 superinfected cultures. This enhancement was observed only after infection with active HIV-1 virus, but not following exposure to inactivated viral particles or transfection with HIV-1 tat gene.
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PMID:Reciprocal activation of human T-lymphotropic viruses in HTLV-I-transformed cells superinfected with HIV-1. 200 72


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