scholarly journals Nuclear Localization of Human Immunodeficiency Virus Type 1 Integrase Expressed as a Fusion Protein with Green Fluorescent Protein

Virology ◽  
1999 ◽  
Vol 258 (2) ◽  
pp. 327-332 ◽  
Author(s):  
Wim Pluymers ◽  
Peter Cherepanov ◽  
Dominique Schols ◽  
Erik De Clercq ◽  
Zeger Debyser
Keyword(s):  
Human Immunodeficiency Virus ◽  
Green Fluorescent Protein ◽  
Fusion Protein ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Localization ◽  
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 Importin-α Promotes Passage through the Nuclear Pore Complex of Human Immunodeficiency Virus Type 1 Vpr

2005 ◽  
Vol 79 (6) ◽  
pp. 3557-3564 ◽  
Author(s):  
Masakazu Kamata ◽  
Yuko Nitahara-Kasahara ◽  
Yoichi Miyamoto ◽  
Yoshihiro Yoneda ◽  
Yoko Aida
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Localization ◽  
Fluorescent Protein ◽  
Nuclear Pore ◽  
Nuclear Entry ◽  
Immunodeficiency Virus ◽  
Importin Α

ABSTRACT Viral protein R (Vpr) of human immunodeficiency virus type 1 has potent karyophilic properties, but details of the mechanism by which it enters the nucleus remain to be clarified. We reported previously that two regions, located between residues 17 and 34 (αH1) and between residues 46 and 74 (αH2), are indispensable for the nuclear localization of Vpr. Here, we reveal that a chimeric protein composed of the nuclear localization signal of Vpr, glutathione S-transferase, and green fluorescent protein was localized at the nuclear envelope and then entered the nucleus upon addition of importin-α. An in vitro transport assay using a series of derivatives of importin-α demonstrated that the carboxyl terminus was required for this nuclear import process. We also showed that Vpr interacts with importin-α through αH1 and αH2; only the interaction via αH1 is indispensable for the nuclear entry of Vpr. These observations indicate that importin-α functions as a mediator for the nuclear entry of Vpr.

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.

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