High Levels of HIV-1 in Plasma During All Stages of Infection Determined By Competitive PCR

Piatak, Michael; Ms, Saag; Lc, Yang; Clark, Susan J.; Jc, Kappes; Luk, KC; Hahn, Beatrice H.; Shaw, George; Lifson, JD

doi:10.1126/science.8096089

Cited by 1,156 publications

(638 citation statements)

References 59 publications

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“…This is consistent with the very low ratio of infectious/noninfectious particles reported for HIV (Piatak et al, 1993;Gao and Goff, 1999). Our observation time (ϳ10 min) was limited by inactivation of fluorescently labeled virus caused by photobleaching after prolonged exposure to the laser beam and so our extent of fusion probably underestimates the actual extent.…”

Section: The Pseudoviral-cell Fusion Systemsupporting

confidence: 74%

Time-resolved Imaging of HIV-1 Env-mediated Lipid and Content Mixing between a Single Virion and Cell Membrane

Markosyan

Cohen

Melikyan

2005

MBoC

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A method has been developed to follow fusion of individual pseudotyped virus expressing HIV-1 Env to cells by time-resolved fluorescence microscopy. Viral envelopes were labeled with a fluorescent lipid dye (DiD) and virus content was rendered visible by incorporating a Gag-GFP chimera. The Gag-GFP is naturally cleaved to the much smaller NC-GFP fragment in the mature virions. NC-GFP was readily released upon permeabilization of the viral envelope, whereas the capsid was retained. The NC-GFP thus provides a relatively small and mobile aqueous marker to follow viral content transfer. In fusion experiments, virions were bound to cells at low temperature, and fusion was synchronously triggered by a temperature jump. DiD transferred from virions to cells without a significant lag after the temperature jump. Some virions released DiD but retained NC-GFP. Surprisingly, the fraction of lipid mixing events yielding NC-GFP transfer was dependent on the type of target cell: of three infectable cell lines, only one permitted NC-GFP transfer within minutes of raising temperature. NC-GFP release did not correlate with the level of CD4 or coreceptor expression in the target cells. The data indicate that fusion pores formed by HIV-1 Env can remain small for a relatively long time before they enlarge. INTRODUCTIONIn the process of membrane fusion, two continuities are created: separate membranes join and distinct aqueous compartments become one. To identify the mechanisms of membrane fusion, both continuities should be followed and the temporal order in which they occur identified. In the case of the HIV-1 fusion protein Env, mechanistic studies have largely relied on fusion of cells expressing the protein to cells expressing CD4 and cognate chemokine receptors (e.g., Munoz-Barroso et al., 1998;Melikyan et al., 2000;Reeves et al., 2002;Abrahamyan et al., 2003;Gallo et al., 2003;Markosyan et al., 2003). But monitoring lipid dye spread in HIV-1 Env-mediated cell-cell fusion has not proved to be very useful in following membrane continuity, for several reasons. The dye does not reliably move between cells, and when it does move, the time course is slow, in large part because the dye segregates nonuniformly over the cell membrane. Also, the dye enters intracellular pools, making it difficult to follow transfer between fused membranes. Furthermore, in cell-cell fusion, the two bound cells are in contact over an appreciable area, so fusion could potentially occur at many sites (Frolov et al., 2000;Leikina and Chernomordik, 2000); the sites of origin for the observed lipid and aqueous dye spread could well be different.By studying fusion of viral particles to cells, these problems can be overcome. The small size of virus minimizes the area of contact and accurately reflects the biological situation. Also, membrane retrieval processes that internalize lipid dye are absent in virions, and the viral envelope is simpler than a cell membrane. But until recently, suitable assays to follow both lipid and contents mixing and to time-order the...

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Section: The Pseudoviral-cell Fusion Systemsupporting

confidence: 74%

Time-resolved Imaging of HIV-1 Env-mediated Lipid and Content Mixing between a Single Virion and Cell Membrane

Markosyan

Cohen

Melikyan

2005

MBoC

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“…The selection of the PCR primers was pertative competitive PCR (QC-PCR) is now the method formed using the Oligo program (National Bioscience Plymof choice. [11][12][13] outh, MN). For each pair, primer sequences were chosen in Thus, in this study, we used a QC-PCR method to different exons to detect amplification products generated by assess ApoA-I and ApoB mRNA levels in alcoholic pa-any contaminating genomic DNA.…”

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confidence: 99%

Quantification of apolipoprotein A-I and B messenger RNA in heavy drinkers according to liver disease

Mathurin

Vidaud

et al. 1996

Hepatology

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It has previously been shown that, in heavy drinkers, Apolipoprotein A-I (ApoA-I), a major component of serum apolipoprotein A-I (ApoA-I) levels are closely re-high-density lipoprotein, is synthesized in equal prolated to the degree of liver injury: they are at a maximum portions by the liver and the intestine. Like the level in patients with steatosis, begin to decrease in patients of high-density lipoprotein the level of ApoA-I has been with fibrosis, and reach a minimum in patients with se-shown to be inversely correlated with the development vere cirrhosis. In contrast with serum ApoA-I variations, of coronary artery disease. Variations in serum ApoAserum apolipoprotein B (ApoB) levels are stable. The as-I are also correlated with alcohol consumption and the with fibrosis or nonsevere cirrhosis (group II), and 4 ApoA-I is increased in alcoholic patients with normal with severe cirrhosis (group III). For ApoA-I, group I liver or steatosis. 5-8 However, serum ApoA-I is dehad higher serum levels (208.4 { 37.6 mg/dL mean { SE) creased in alcoholic patients with liver fibrosis indeand mRNA levels (0.51 { 0.12) than group II (serum 116 pendently of nutritional parameters. 1,2 { 19 mg/dL, P õ .01, mRNA 0.40 { 0.11, P õ .09) or groupWe and others have validated a simple index referred III (serum 56.5 { 28.6 mg/dL, P õ .01, mRNA 0.27 { .02, to as PGA, which includes a specific test for severe liver P Å .008). For ApoB, the three groups had similar serum disease (prothrombin time), a sensitive test for alcohol- There was a significant correlation between serum and index varied from 0 to 12. When PGA was°2, the mRNA levels of ApoA-I (r Å .65, P Å .003) but no correla-probability of cirrhosis was 0% and the probability of tion between serum and mRNA levels of ApoB (r Å .19, normal liver or minimal changes 83%. Conversely, NS). We suggest that (1) steatosis is associated with in-when PGA was ¢9, the probability of cirrhosis was creased ApoAI mRNA; (2) fibrosis is associated with de-86%.

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“…Plasma levels of HIV-1 determined by PCR methods are 60,000-fold higher than virus titers determined by endpoint dilution culture (36), and the discrepancy is likely due to substantial proportions of defective or otherwise non-infectious virus (37).…”

Section: Discussionmentioning

confidence: 99%

Immune Reconstitution Prevents Continuous Equine Infectious Anemia Virus Replication in an Arabian Foal with Severe Combined Immunodeficiency: Lessons for Control of Lentiviruses

Mealey

Fraser

Oaks

et al. 2001

Clinical Immunology

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Acute infection with equine infectious anemia virus (EIAV), a lentivirus of horses, results in a persistent high-level viremia in Arabian foals affected with severe combined immunodeficiency (SCID). This observation argues against the idea that the transient nature of acute lentiviral viremia is solely a function of viral population dynamics. To extend these studies, EIAV-specific immune reconstitution was attempted prior to EIAV challenge in 2 SCID foals, using adoptively transferred virus-stimulated lymphocytes derived from persistently EIAV-infected half sibling donors. Following transfer, lymphocyte engraftment occurred in 1 foal, and EIAV-specific cytotoxic T lymphocytes as well as neutralizing antibody activity developed. Following a brief period of plasma viremia in this foal, EIAV replication was controlled and plasma virus could not be detected by RT-PCR or culture. These results provide further direct evidence that a specific immune response is required for termination of plasma viremia in acute lentiviral infections.

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High Levels of HIV-1 in Plasma During All Stages of Infection Determined By Competitive PCR

Cited by 1,156 publications

References 59 publications

Time-resolved Imaging of HIV-1 Env-mediated Lipid and Content Mixing between a Single Virion and Cell Membrane

Time-resolved Imaging of HIV-1 Env-mediated Lipid and Content Mixing between a Single Virion and Cell Membrane

Quantification of apolipoprotein A-I and B messenger RNA in heavy drinkers according to liver disease

Immune Reconstitution Prevents Continuous Equine Infectious Anemia Virus Replication in an Arabian Foal with Severe Combined Immunodeficiency: Lessons for Control of Lentiviruses

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