scholarly journals Design and Evaluation of a Human Immunodeficiency Virus Type 1 RNA Assay Using Nucleic Acid Sequence-Based Amplification Technology Able To Quantify Both Group M and O Viruses by Using the Long Terminal Repeat as Target

1999 ◽  
Vol 37 (6) ◽  
pp. 1813-1818 ◽  
Author(s):  
Michel P. de Baar ◽  
Audrey M. van der Schoot ◽  
Jaap Goudsmit ◽  
Femke Jacobs ◽  
Ron Ehren ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Nucleic Acid ◽  
Long Terminal Repeat ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nucleic Acid Sequence ◽  
Serum Samples ◽  
Immunodeficiency Virus ◽  
Hiv 1

Currently available human immunodeficiency virus type 1 (HIV-1) RNA quantification assays can detect most viruses of the group M subtypes, but a substantial number are missed or not quantified reliably. Viruses of HIV-1 group O cannot be detected by any commercially available assay. We developed and evaluated a quantitative assay based on nucleic acid sequence-based amplification (NASBA) technology, with primers and probes located in the conserved long terminal repeat (LTR) region of the HIV-1 genome. In 68 of 72 serum samples from individuals infected with HIV-1 subtypes A to H of group M, viruses could be detected and quantified. In serum samples from two patients infected with HIV-1 group O viruses, these viruses as well could be detected and quantified. In contrast, the currently used gag-based assay underestimated the presence of subtype A viruses and could not detect subtype G and group O viruses. The discrepancy between the results of the two assays may be explained by the number of mismatches found within and among the probe and primer regions of the subtype isolates. These data indicate that LTR-based assays, including the NASBA format chosen here, are better suited to monitoring HIV-1 therapy than aregag-based assays in an era in which multiple HIV-1 subtypes and groups are spreading worldwide.

scholarly journals Human Cellular Nucleic Acid-Binding Protein Zn2+ Fingers Support Replication of Human Immunodeficiency Virus Type 1 When They Are Substituted in the Nucleocapsid Protein

2003 ◽  
Vol 77 (15) ◽  
pp. 8524-8531 ◽  
Author(s):  
Connor F. McGrath ◽  
James S. Buckman ◽  
Tracy D. Gagliardi ◽  
William J. Bosche ◽  
Lori V. Coren ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Nucleic Acid ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Structural Characteristics ◽  
Nucleic Acid Binding ◽  
Genomic Rna ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT A family of cellular nucleic acid binding proteins (CNBPs) contains seven Zn2+ fingers that have many of the structural characteristics found in retroviral nucleocapsid (NC) Zn2+ fingers. The sequence of the NH2-terminal NC Zn2+ finger of the pNL4-3 clone of human immunodeficiency virus type 1 (HIV-1) was replaced individually with sequences from each of the seven fingers from human CNBP. Six of the mutants were normal with respect to protein composition and processing, full-length genomic RNA content, and infectivity. One of the mutants, containing the fifth CNBP Zn2+ finger (CNBP-5) packaged reduced levels of genomic RNA and was defective in infectivity. There appear to be defects in reverse transcription in the CNBP-5 infections. Models of Zn2+ fingers were constructed by using computational methods based on available structural data, and atom-atom interactions were determined by the hydropathic orthogonal dynamic analysis of the protein method. Defects in the CNBP-5 mutant could possibly be explained, in part, by restrictions of a set of required atom-atom interactions in the CNBP-5 Zn2+ finger compared to mutant and wild-type Zn2+ fingers in NC that support replication. The present study shows that six of seven of the Zn2+ fingers from the CNBP protein can be used as substitutes for the Zn2+ finger in the NH2-terminal position of HIV-1 NC. This has obvious implications in antiviral therapeutics and DNA vaccines employing NC Zn2+ finger mutants.

scholarly journals The Human Factors YY1 and LSF Repress the Human Immunodeficiency Virus Type 1 Long Terminal Repeat via Recruitment of Histone Deacetylase 1

2000 ◽  
Vol 74 (15) ◽  
pp. 6790-6799 ◽  
Author(s):  
Jason J. Coull ◽  
Fabio Romerio ◽  
Jian-Min Sun ◽  
Janet L. Volker ◽  
Katherine M. Galvin ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Long Terminal Repeat ◽  
Histone Deacetylase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Zinc Fingers ◽  
Terminal Repeat ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Enigmatic mechanisms restore the resting state in activated lymphocytes following human immunodeficiency virus type 1 (HIV-1) infection, rarely allowing persistent nonproductive infection. We detail a mechanism whereby cellular factors could establish virological latency. The transcription factors YY1 and LSF cooperate in repression of transcription from the HIV-1 long terminal repeat (LTR). LSF recruits YY1 to the LTR via the zinc fingers of YY1. The first two zinc fingers were observed to be sufficient for this interaction in vitro. A mutant of LSF incapable of binding DNA blocked repression. Like other transcriptional repressors, YY1 can function via recruitment of histone deacetylase (HDAC). We find that HDAC1 copurifies with the LTR-binding YY1-LSF repressor complex, the domain of YY1 that interacts with HDAC1 is required to repress the HIV-1 promoter, expression of HDAC1 augments repression of the LTR by YY1, and the deacetylase inhibitor trichostatin A blocks repression mediated by YY1. This novel link between HDAC recruitment and inhibition of HIV-1 expression by YY1 and LSF, in the natural context of a viral promoter integrated into chromosomal DNA, is the first demonstration of a molecular mechanism of repression of HIV-1. YY1 and LSF may establish transcriptional and virological latency of HIV, a state that has recently been recognized in vivo and has significant implications for the long-term treatment of AIDS.

scholarly journals Comparison of Levels of Human Immunodeficiency Virus Type 1 RNA in Plasma as Measured by the NucliSens Nucleic Acid Sequence-Based Amplification and Quantiplex Branched-DNA Assays

1999 ◽  
Vol 37 (4) ◽  
pp. 1210-1212 ◽  
Author(s):  
C. C. Ginocchio ◽  
S. Tetali ◽  
D. Washburn ◽  
F. Zhang ◽  
M. H. Kaplan
Keyword(s):  
Human Immunodeficiency Virus ◽  
Nucleic Acid ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nucleic Acid Sequence ◽  
Branched Dna ◽  
Immunodeficiency Virus ◽  
Dna Assays ◽  
Lower Limits

This study compared levels of human immunodeficiency virus type 1 RNA in plasma as measured by the Quantiplex branched-DNA and NucliSens nucleic acid sequence-based amplification assays. RNA was detectable in 118 of 184 samples (64.13%) by the Quantiplex assay and in 171 of 184 samples (92.94%) by the NucliSens assay. Regression analysis indicated that a linear relationship existed between the two sets of values (P < 0.0001), although the Quantiplex and NucliSens values were significantly different (P < 0.001), with the NucliSens values being approximately 0.323 log higher. Spearman correlation analysis indicated that the overall changes in patient viral load patterns were highly correlative between the two assays: r = 0.912,P < 0.0001. The lower limits of sensitivity were determined to be approximately 100 copies/ml and 1,200 to 1,400 copies/ml for the NucliSens and Quantiplex assays, respectively.

scholarly journals Relationship of Incremental Specimen Volumes and Enhanced Detection of Human Immunodeficiency Virus Type 1 RNA with Nucleic Acid Amplification Technology

2000 ◽  
Vol 38 (1) ◽  
pp. 85-89
Author(s):  
Donald J. Witt ◽  
M. Kemper ◽  
Andrew Stead ◽  
Christine C. Ginocchio ◽  
Angela M. Caliendo
Keyword(s):  
Human Immunodeficiency Virus ◽  
Nucleic Acid ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nucleic Acid Amplification ◽  
Copy Numbers ◽  
Nucleic Acid Amplification Technology ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The relationship between specimen input volume and the frequency of reported human immunodeficiency virus type 1 (HIV-1) RNA copy numbers by nucleic acid amplification technology (the NASBA HIV-1 RNA QT system) was investigated. Results obtained with both clinical specimens and dilution panels indicated that both the absolute number of reported results and the reported HIV-1 RNA copy number were directly proportional to the specimen input volumes evaluated (0.1, 0.5, and 1.0 ml). Conversion of the reported HIV-1 RNA copy numbers to a constant 1.0-ml volume indicated that the numerical relationship among the specimen input volumes and the HIV-1 RNA copy numbers was multiplicative. The HIV-1 RNA copy numbers reported for the 0.5-ml input volume were approximately 5-fold increased over those reported for the 0.1-ml input volume, and those reported for the 1.0-ml input volume were 10-fold increased over those reported for the 0.1-ml input volume. For the specimen input volumes investigated, a common linear range of 264 to 5,400,000 HIV-1 RNA copies was observed. The use of increased specimen input volumes did not result in a loss of assay specificity, as the results reported for specimens from 50 seronegative blood donors were negative at all three specimen input volumes. In conclusion, an increase in the input volume of specimens analyzed by nucleic acid amplification technology can be useful for the enhanced detection of HIV-1 RNA.

Spread of distinct human immunodeficiency virus type 1 AG recombinant lineages in Africa

Microbiology ◽  
2000 ◽  
Vol 81 (2) ◽  
pp. 515-523 ◽  
Author(s):  
Marion Cornelissen ◽  
Remco van den Burg ◽  
Fokla Zorgdrager ◽  
Jaap Goudsmit
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Rt Pcr ◽  
Serum Samples ◽  
African Countries ◽  
Immunodeficiency Virus ◽  
Subtype A ◽  
Hiv 1

To identify new subtype G human immunodeficiency virus type 1 (HIV-1) strains and AG recombinant forms, we collected 28 serum samples from immigrants to the Netherlands from 12 countries throughout Africa. Based on the gag sequences 22 isolates were identified as subtype A or G. Phylogenetic analysis of discontinuous regions of the gag (726 nt), pol (1176 nt) and env (276 nt) genes revealed 13 AG recombinants with the mosaic structure A gag /G pol /A env , three with A gag /G pol /G env and one other with A gag /G pol /G env , in addition to ‘pure’ subtypes A gag /A pol /A env (n=1) and G gag /G pol /G env (n=4). To analyse the crossover points in more detail, a new RT–PCR was developed resulting in a large contiguous sequence of 2600 nt from the gag region to half the pol region. All the 13 A gag /G pol /A env recombinants appeared to belong to the circulating recombinant form (CRF) AG (IbNG). The three A gag /G pol /G env recombinants differed from the CRF AG (IbNG) subtype, suggesting the identification of a new CRF subtype. The recovery of AG recombinants from African countries a thousand miles apart indicates the active spread of new recombinants.

scholarly journals Azido-Containing Diketo Acid Derivatives Inhibit Human Immunodeficiency Virus Type 1 Integrase In Vivo and Influence the Frequency of Deletions at Two-Long-Terminal-Repeat-Circle Junctions

2004 ◽  
Vol 78 (7) ◽  
pp. 3210-3222 ◽  
Author(s):  
Evguenia S. Svarovskaia ◽  
Rebekah Barr ◽  
Xuechun Zhang ◽  
Godwin C. G. Pais ◽  
Christophe Marchand ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Long Terminal Repeat ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Mechanisms Of Action ◽  
Sequencing Analysis ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT We previously found that azido-containing β-diketo acid derivatives (DKAs) are potent inhibitors of human immunodeficiency virus type 1 (HIV-1) integrase (IN) (X. Zhang et al., Bioorg. Med. Chem. Lett., 13:1215-1219, 2003). To characterize the intracellular mechanisms of action of DKAs, we analyzed the antiviral activities of two potent azido-containing DKAs with either a monosubstitution or a disubstitution of azido groups, using single- and multiple-replication-cycle assays. Both azido-containing DKAs significantly inhibited HIV-1 infection in 293T, CEM-SS, and H9 cells (50% inhibitory concentration = 2 to 13 μM) and exhibited low cytotoxicity (50% cytotoxic concentration = 60 to 600 μM). Inhibition of HIV-1 IN in vivo was demonstrated by the observation that previously described L-708,906 resistance mutations in HIV-1 IN (T66I and T66I/S153Y) also conferred resistance to the azido-group-containing DKAs. In vitro assays and in vivo analysis indicated that the DKAs did not significantly inhibit the 3′ processing and selectively inhibited the strand transfer reaction. In addition, quantitative PCR indicated that two-long-terminal-repeat (2-LTR) circles were elevated in the presence of the azido-containing DKAs, confirming that HIV-1 IN was the intracellular target of viral inhibition. To gain insight into the mechanism by which the DKAs increased 2-LTR-circle formation of 3′-processed viral DNAs, we performed extensive DNA sequencing analysis of 2-LTR-circle junctions. The results indicated that the frequency of deletions at the circle junctions was elevated from 19% for the untreated controls to 32 to 41% in the presence of monosubstituted (but not disubstituted) DKAs. These results indicate that the structure of the DKAs can influence the extent of degradation of viral DNA ends by host nucleases and the frequency of deletions at the 2-LTR-circle junctions. Thus, sequencing analysis of 2-LTR-circle junctions can elucidate the intracellular mechanisms of action of HIV-1 IN inhibitors.

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