Use of a Green Fluorescent Protein as a Marker for Human Immunodeficiency Virus Type 1 Infection

1997 ◽  
Vol 13 (13) ◽  
pp. 1077-1081 ◽  
Author(s):  
KATHLEEN A. PAGE ◽  
TERI LIEGLER ◽  
MARK B. FEINBERG
Keyword(s):  
Human Immunodeficiency Virus ◽  
Green Fluorescent Protein ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Fluorescent Protein ◽  
Immunodeficiency Virus ◽  
Green Fluorescent

scholarly journals Replication-Competent Rhabdoviruses with Human Immunodeficiency Virus Type 1 Coats and Green Fluorescent Protein: Entry by a pH-Independent Pathway

1999 ◽  
Vol 73 (8) ◽  
pp. 6937-6945 ◽  
Author(s):  
Eli Boritz ◽  
Jennifer Gerlach ◽  
J. Erik Johnson ◽  
John K. Rose
Keyword(s):  
Human Immunodeficiency Virus ◽  
Green Fluorescent Protein ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Neutralizing Antibodies ◽  
Fluorescent Protein ◽  
Gene Encoding ◽  
Immunodeficiency Virus ◽  
Green Fluorescent

ABSTRACT We describe a replication-competent, recombinant vesicular stomatitis virus (VSV) in which the gene encoding the single transmembrane glycoprotein (G) was deleted and replaced by anenv-G hybrid gene encoding the extracellular and transmembrane domains of a human immunodeficiency virus type 1 (HIV-1) envelope protein fused to the cytoplasmic domain of VSV G. An additional gene encoding a green fluorescent protein was added to permit rapid detection of infection. This novel surrogate virus infected and propagated on cells expressing the HIV receptor CD4 and coreceptor CXCR4. Infection was blocked by SDF-1, the ligand for CXCR4, by antibody to CD4 and by HIV-neutralizing antibody. This virus, unlike VSV, entered cells by a pH-independent pathway and thus supports a pH-independent pathway of HIV entry. Additional recombinants carrying hybrid env-G genes derived from R5 or X4R5 HIV strains also showed the coreceptor specificities of the HIV strains from which they were derived. These surrogate viruses provide a simple and rapid assay for HIV-neutralizing antibodies as well as a rapid screen for molecules that would interfere with any stage of HIV binding or entry. The viruses might also be useful as HIV vaccines. Our results suggest wide applications of other surrogate viruses based on VSV.

scholarly journals Mobility of Human Immunodeficiency Virus Type 1 Pr55Gag in Living Cells

2006 ◽  
Vol 80 (17) ◽  
pp. 8796-8806 ◽  
Author(s):  
Candace Y. Gomez ◽  
Thomas J. Hope
Keyword(s):  
Human Immunodeficiency Virus ◽  
Plasma Membrane ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Fluorescent Protein ◽  
Live Cells ◽  
Particle Assembly ◽  
Immunodeficiency Virus ◽  
Green Fluorescent

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) assembly requires the converging of thousands of structural proteins on cellular membranes to form a tightly packed immature virion. The Gag polyprotein contains all of the determinants important for viral assembly and must move around in the cell in order to form particles. This work has focused on Gag mobility in order to provide more insights into the dynamics of particle assembly. Key to these studies was the use of several fluorescently labeled Gag derivatives. We used fluorescence recovery after photobleaching as well as photoactivation to determine Gag mobility. Upon expression, Gag can be localized diffusely in the cytoplasm, associated with the plasma membrane, or in virus-like particles (VLPs). Here we show that Gag VLPs are primarily localized in the plasma membrane and do not colocalize with CD63. We have shown using full-length Gag as well as truncation mutants fused to green fluorescent protein that Gag is highly mobile in live cells when it is not assembled into VLPs. Results also showed that this mobility is highly dependent upon cholesterol. When cholesterol is depleted from cells expressing Gag, mobility is significantly decreased. Once cholesterol was replenished, Gag mobility returned to wild-type levels. Taken together, results from these mobility studies suggest that Gag is highly mobile and that as the assembly process proceeds, mobility decreases. These studies also suggest that Gag assembly must occur in cholesterol-rich domains in the plasma membrane.

scholarly journals Cell-Based Fluorescence Assay for Human Immunodeficiency Virus Type 1 Protease Activity

2001 ◽  
Vol 45 (9) ◽  
pp. 2616-2622 ◽  
Author(s):  
Kristina Lindsten ◽  
Tat'ána Uhlı́ková ◽  
Jan Konvalinka ◽  
Maria G. Masucci ◽  
Nico P. Dantuma
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Fluorescent Protein ◽  
Autocatalytic Cleavage ◽  
Immunodeficiency Virus ◽  
Green Fluorescent ◽  
Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) protease is essential for production of infectious virus and is therefore a major target for the development of drugs against AIDS. Cellular proteins are also cleaved by the protease, which explains its cytotoxic activity and the consequent failure to establish convenient cell-based protease assays. We have exploited this toxicity to develop a new protease assay that relies on transient expression of an artificial protease precursor harboring the green fluorescent protein (GFP-PR). The precursor is activated in vivo by autocatalytic cleavage, resulting in rapid elimination of protease-expressing cells. Treatment with therapeutic doses of HIV-1 protease inhibitors results in a dose-dependent accumulation of the fluorescent precursor that can be easily detected and quantified by flow cytometric and fluorimetric assays. The precursor provides a convenient and noninfectious model for high-throughput screenings of substances that can interfere with the activity of the protease in living cells.

scholarly journals Genetic Recombination between Human Immunodeficiency Virus Type 1 (HIV-1) and HIV-2, Two Distinct Human Lentiviruses

2007 ◽  
Vol 82 (4) ◽  
pp. 1923-1933 ◽  
Author(s):  
Kazushi Motomura ◽  
Jianbo Chen ◽  
Wei-Shau Hu
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Fluorescent Protein ◽  
Genetic Recombination ◽  
Green Fluorescent Protein Gene ◽  
Low Frequencies ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) and HIV-2 are genetically distinct viruses that each can cause AIDS. Approximately 1 million people are infected with both HIV-1 and HIV-2. Additionally, these two viruses use the same receptor and coreceptors and can therefore infect the same target cell populations. To explore potential genetic interactions, we first examined whether RNAs from HIV-1 and HIV-2 can be copackaged into the same virion. We used modified near-full-length viruses that each contained a green fluorescent protein gene (gfp) with a different inactivating mutation. Thus, a functional gfp could be reconstituted via recombination, which was used to detect the copackaging of HIV-1 and HIV-2 RNAs. The GFP-positive (GFP+) phenotype was detected in approximately 0.2% of the infection events, which was 35-fold lower than the intrasubtype HIV-1 rates. We isolated and characterized 54 GFP+ single-cell clones and determined that all of them contained proviruses with reconstituted gfp. We then mapped the general structures of the recombinant viruses and characterized the recombination junctions by DNA sequencing. We observed several different recombination patterns, including those that had crossovers only in gfp. The most common hybrid genomes had heterologous long terminal repeats. Although infrequent, crossovers in the viral sequences were also identified. Taken together, our study demonstrates that HIV-1 and HIV-2 can recombine, albeit at low frequencies. These observations indicate that multiple factors are likely to restrict the generation of viable hybrid HIV-1 and HIV-2 viruses. However, considering the large coinfected human population and the high viral load in patients, these rare events could provide the basis for the generation of novel human immunodeficiency viruses.

scholarly journals Macrophages in Vaginal but Not Intestinal Mucosa Are Monocyte-Like and Permissive to Human Immunodeficiency Virus Type 1 Infection

2009 ◽  
Vol 83 (7) ◽  
pp. 3258-3267 ◽  
Author(s):  
Ruizhong Shen ◽  
Holly E. Richter ◽  
Ronald H. Clements ◽  
Lea Novak ◽  
Kayci Huff ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Fluorescent Protein ◽  
Female Genital Tract ◽  
Vaginal Mucosa ◽  
Innate Response ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Mucosal surfaces play a major role in human immunodeficiency virus type 1 (HIV-1) transmission and pathogenesis, and yet the role of lamina propria macrophages in mucosal HIV-1 infection has received little investigative attention. We report here that vaginal and intestinal macrophages display distinct phenotype and HIV-1 permissiveness profiles. Vaginal macrophages expressed the innate response receptors CD14, CD89, CD16, CD32, and CD64 and the HIV-1 receptor/coreceptors CD4, CCR5, and CXCR4, similar to monocytes. Consistent with this phenotype, green fluorescent protein-tagged R5 HIV-1 entered macrophages in explanted vaginal mucosa as early as 30 min after inoculation of virus onto the epithelium, and purified vaginal macrophages supported substantial levels of HIV-1 replication by a panel of highly macrophage-tropic R5 viruses. In sharp contrast, intestinal macrophages expressed no detectable, or very low levels of, innate response receptors and HIV-1 receptor/coreceptors and did not support HIV-1 replication, although virus occasionally entered macrophages in intestinal tissue explants. Thus, vaginal, but not intestinal, macrophages are monocyte-like and permissive to R5 HIV-1 after the virus has translocated across the epithelium. These findings suggest that genital and gut macrophages have different roles in mucosal HIV-1 pathogenesis and that vaginal macrophages play a previously underappreciated but potentially important role in mucosal HIV-1 infection in the female genital tract.

scholarly journals Virological Consequences of Early Events following Cell-Cell Contact between Human Immunodeficiency Virus Type 1-Infected and Uninfected CD4+ Cells

2008 ◽  
Vol 82 (16) ◽  
pp. 7773-7789 ◽  
Author(s):  
Eliana Ruggiero ◽  
Roberta Bona ◽  
Claudia Muratori ◽  
Maurizio Federico
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Fluorescent Protein ◽  
Cell Contact ◽  
Target Cells ◽  
Immunodeficiency Virus ◽  
Virus Expression ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1)-infected cells transmit viral products to uninfected CD4+ cells very rapidly. However, the natures of the transmitted viral products and the mechanism of transmission, as well as the relative virological consequences, have not yet been fully clarified. We studied the virological events occurring a few hours after contact between HIV-1-infected and uninfected CD4+ cells using a coculture cell system in which the virus expression in target cells could be monitored through the induction of a green fluorescent protein reporter gene driven by HIV-1 long terminal repeats. Within 16 h of coculture, we observed two phenomena not related to the cell-free virus infection, i.e., the formation of donor-target cell fusions and a fusion-independent internalization of viral particles likely occurring at least in part through intercellular connections. Both events depended on the expression of Env and CD4 in donor and target cells, respectively, whereas the HIV-1 internalization required clathrin activity in target cells. Importantly, both phenomena were also observed in cocultures of primary CD4+ lymphocytes, while primary macrophages supported only HIV-1 endocytosis. By investigating the virological consequences of these events, we noticed that while fused cells released infectious HIV-1 particles, albeit with reduced efficiency compared with donor cells, no virus expression was detectable upon HIV-1 endocytosis in target cells. In sum, the HIV-1 transmission following contact between an HIV-1-infected and an uninfected CD4+ cell can occur through different mechanisms, leading to distinguishable virological outcomes.

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