A new reporter cell line to monitor HIV infection and drug susceptibility in vitro
Alain Gervaix,Daniel West,Lorenzo M. Leoni,Douglas D. Richman,Flossie Wong-Staal,Jacques Corbeil +5 more
TLDR
The CEM-GFP reporter cell line provides a simple, rapid, and direct method for monitoring HIV infectivity titers and antiretroviral drug susceptibility of syncytium-inducing strains.Abstract:
Determination of HIV infectivity in vitro and its inhibition by antiretroviral drugs by monitoring reduction of production of p24 antigen is expensive and time consuming. Such assays also do not allow accurate quantitation of the number of infected cells over time. To develop a simple, rapid, and direct method for monitoring HIV infection, we generated a stable T-cell line (CEM) containing a plasmid encoding the green fluorescent protein (humanized S65T GFP) driven by the HIV-1 long terminal repeat. Clones were selected that displayed low constitutive background fluorescence, but a high level of GFP expression upon infection with HIV. HIV-1 infection induced a 100- to 1,000-fold increase in relative fluorescence of cells over 2 to 4 days as monitored by fluorescence microscopy, cytofluorimetry, and flow cytometry. Addition of inhibitors of reverse transcriptase, protease, and other targets at different multiplicities of infection permitted the accurate determination of drug susceptibility. This technique also permitted quantitation of infectivity of viral preparations by assessment of number of cells infected in the first round of infection. In conclusion, the CEM-GFP reporter cell line provides a simple, rapid, and direct method for monitoring HIV infectivity titers and antiretroviral drug susceptibility of syncytium-inducing strains.read more
Citations
More filters
Journal ArticleDOI
The green fluorescent protein
TL;DR: In just three years, the green fluorescent protein from the jellyfish Aequorea victoria has vaulted from obscurity to become one of the most widely studied and exploited proteins in biochemistry and cell biology.
Journal ArticleDOI
Solution structure and basis for functional activity of stromal cell-derived factor-1; dissociation of CXCR4 activation from binding and inhibition of HIV-1.
Matthew P. Crump,Jiang Hong Gong,Pius Loetscher,Krishna Rajarathnam,Ali Amara,Fernando Arenzana-Seisdedos,Jean Louis Virelizier,Marco Baggiolini,Brian D. Sykes,Ian Clark-Lewis +9 more
TL;DR: The three‐dimensional structure of stromal cell‐derived factor‐1 was determined by NMR spectroscopy and the RFFESH formed a receptor binding site, which is proposed to be an important initial docking site of SDF‐1 with its receptor.
Journal ArticleDOI
HIV-1 drug resistance in newly infected individuals.
Daniel Boden,Arlene Hurley,Linqi Zhang,Yunzhen Cao,Yong Guo,Elizabeth Jones,John Tsay,James E. Ip,Charles Farthing,Kay Limoli,Neil Parkin,Martin Markowitz +11 more
TL;DR: The data support expanded use of resistance testing in the setting of primary HIV-1 infection, and clinical trials should be initiated to establish whether therapy guided by resistance testing, compared with the use of empirical triple combination antiretroviral therapy, provides additional virological and immunological benefit.
Journal ArticleDOI
Applications of the green fluorescent protein in cell biology and biotechnology
Tom Misteli,David L. Spector +1 more
TL;DR: An overview of some of the major applications of GFP, namely its use in protein tagging and in monitoring gene expression as well as its potential in a variety of biological screens are presented.
Journal ArticleDOI
Vpu Directs the Degradation of the Human Immunodeficiency Virus Restriction Factor BST-2/Tetherin via a βTrCP-Dependent Mechanism
Janet L. Douglas,Kasinath Viswanathan,Matthew N. McCarroll,Jean K. Gustin,Klaus Früh,Ashlee V. Moses +5 more
TL;DR: Data support the hypothesis that, in similarity to its role in CD4 degradation, Vpu acts as an adapter molecule linking BST-2 to the cellular ubiquitination machinery via βTrCP, resulting in enhanced HIV-1 virion release.
References
More filters
Journal ArticleDOI
Green fluorescent protein as a marker for gene expression
TL;DR: A complementary DNA for the Aequorea victoria green fluorescent protein produces a fluorescent product when expressed in prokaryotic or eukaryotic cells, which can be used to monitor gene expression and protein localization in living organisms.
Journal ArticleDOI
HIV-1 Entry Cofactor: Functional cDNA Cloning of a Seven-Transmembrane, G Protein-Coupled Receptor
TL;DR: A cofactor for HIV-1 (human immunodeficiency virus-type 1) fusion and entry was identified with the use of a novel functional complementary DNA (cDNA) cloning strategy that is a putative G protein-coupled receptor with seven transmembrane segments.
Journal Article
HIV-1 Entry Cofactor: Functional cDNA Cloning of a Seven-Transmembrane, G Protein–Coupled Receptor
TL;DR: Fusin this article is a putative G protein-coupled receptor with seven transmembrane segments, which enabled CD4-expressing nonhuman cell types to support HIV-1 Env-mediated cell fusion and infection.
Journal ArticleDOI
Identification of a major co-receptor for primary isolates of HIV-1
Hongkui Deng,Rong Liu,Wilfried Ellmeier,S Choe,Derya Unutmaz,M Burkhart,P Di Marzio,Shoshana Marmon,R E Sutton,C M Hill,C B Davis,S C Peiper,T J Schall,Dan R. Littman,Nathaniel R. Landau +14 more
TL;DR: The principal cofactor for entry mediated by the envelope glycoproteins of primary macrophage-tropic strains of HIV-1 is CC-CKR-5, a receptor for the β-chemokines RANTES, Mip-1α and MIP-1β.