Human immunodeficiency virus type-1 (HIV-1) DNA and messenger RNA sequences in both cell lines and blood obtained directly from HIV-1-infected patients were amplified by polymerase chain reaction and hybridized to fluorescein-labeled probes in situ, and the individually labeled cells were analyzed by flow cytometry. After flow cytometric analysis, heterogeneous cell populations were reproducibly resolved into HIV-1-positive and -negative distributions. Fluorescence microscopy showed that the cellular morphology was preserved and intracellular localization of amplified product DNA was maintained. Retention of nonspecific probe was not observed. Analysis of proviral DNA and viral messenger RNA in cells in the blood of HIV-1-infected patients showed that the HIV-1 genome persists in a large reservoir of latently infected cells. With the use of this technique it is now possible to detect single-copy DNA or low-abundance messenger RNA rapidly and reproducibly in a minor subpopulation of cells in suspension at single-cell resolution and to sort those cells for further characterization.
Three nonradioisotopic polymerase chain reaction (PCR)-based detection techniques were evaluated for sensitivity and specificity in detecting human immunodeficiency virus type 1 (HIV-1) proviral DNA in peripheral blood mononuclear cells. The Roche prototype HIV-1 PCR assay, the Du Pont enzyme-linked oligonucleotide sandwich assay (ELOSA), and the Gen-Probe hybridization protection assay (HPA) were compared with a standard radioisotopic oligonucleotide solution hybridization (OSH) technique. A panel of 111 well-characterized clinical samples that included peripheral blood mononuclear cells from 48 healthy, low-risk, HIV-1 antibody-negative subjects, 24 antibody-positive subjects with stable CD4 counts of less than 200/mm3, and 39 antibody-positive subjects with stable CD4 counts of greater than 800/mm3 were studied. Each method demonstrated good specificity, ranging between 96 and 100%; those of the OSH and ELOSA (Du Pont) were 1O00%, those of the HPA (Gen-Probe) were 1O00 with one probe and 96% with the other probe, and that of the HIV-1 PCR assay (Roche) was 96%. Sensitivities ranged from 96 to 100% for the low-CD4-count group, with the OSH, the HIV-1 PCR assay (Roche), and the HPA (Gen-Probe) all attaining a sensitivity of 100%o. For the high-CD4-count group, sensitivities ranged from 69 to 97%, with the OSH attaining a sensitivity of 97% and the HPA attaining sensitivities of 97% with one probe and 95% with the other probe. These data indicate that the nonradioisotopic techniques are sensitive and specific for the detection of HIV-1 proviral DNA in clinical samples.
This study reports the rates of phenotypic switching in strains of Candida albicans isolated from superficial and invasive infections. Of 19 invasive strains, 68% showed switching activity, often at very high rates, compared with only 28% of 40 strains isolated from superficial sites (P = 0.004).
The third variable domain (V3 domain) of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 is a major determinant of phenotypic variability. The V3 domain of HIV-1 has many basic amino acid residues. Lymphocytotropic HIV-1 tends to have a V3 domain with a higher density of positive charge than does monocytotropic HIV-1. The importance of basic residues in the V3 domain for the HIV-1 infectivity, however, has not been well investigated. Here we show that mutation of basic amino acid residues at positions 303, 306, 309, 313, and 325 in the V3 domain of the lymphocytotropic isolate NL4-3 results in a dramatic elimination of both virus infectivity and syncytium-inducing ability. Three basic amino acid substitutions (at position 306, 309, and 313) induced a decrease in the binding ability of two kinds of neutralizing antibodies (NEA9284 and 0.5 beta) that recognize a different site in the V3 domain. This suggests that the basic residues play an important role in maintaining the tertiary structure of the V3 domain. Monocytotropism was not simply dependent on either decreased positive charge in the V3 domain of NL4-3 or on mutation of lysine to glutamate at position 320, which is a characteristic amino acid of monocytotropic HIV-1. These findings contribute to our understanding of the significance of basic residues on the function of envelope glycoprotein.
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