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Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays.

ABSTRACT: The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFP enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs.

SUBMITTER: Costantini LM

PROVIDER: S-EPMC4323844 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays.

Costantini Lindsey M LM Irvin Susan C SC Kennedy Steven C SC Guo Feng F Goldstein Harris H Herold Betsy C BC Snapp Erik L EL

Virology 20141230

The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, q ...[more]

PMID: 25555152

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Project description:BACKGROUND: Binding of the viral envelope protein (Env), and particularly of its gp120 subunit, to the cellular CD4 receptor is the first essential step of the HIV-1 entry process. The CD4 binding site (CD4bs) of gp120, and especially a recessed cavity occupied by the CD4 Phe43 residue, are known to be highly conserved among the different circulating subtypes and therefore constitute particularly interesting targets for vaccine and drug design. The miniCD4 proteins are a promising class of CD4bs inhibitors. Studying virus evolution under pressure of CD4bs inhibitors could provide insight on the gp120-CD4 interaction and viral entry. RESULTS: The present study reports on the resistance induction of two subtype B HIV-1 against the most active miniCD4, M48U1, and its ancestor, M48, and how these mutated positions affect CD4bs recognition, entry efficiency, and sensitivity to other CD4bs inhibitors. Resistance against M48U1 was always associated with S375R/N substitution in both BaL and SF162; M48 resistance was associated with D474N substitution in SF162 and with H105Y substitution in BaL. In addition, some other mutations at position V255 and G471 were of importance for SF162 resistant viruses. Except for 474, all of these mutated positions are conserved, and introducing them into an SF162 Env expressing infectious molecular clone (pBRNL4.3 SF162) resulted in decreased entry efficiency. Furthermore, resistant mutants showed at least some cross-resistance towards other CD4bs inhibitors, the V3 monoclonal antibody 447-52D and some even against the monoclonal antibody 17b, of which the epitope overlaps the co-receptor binding site. CONCLUSIONS: The mutations H105Y, V255M, S375R/N, G471R/E, and D474N are found to be involved in resistance towards M48 and M48U1. All mutated positions are part of, or in close proximity to, the CD4bs; most are highly conserved, and all have an impact on the entry efficiency, suggesting their importance for optimal virus infectivity.

Dataset Information

Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays.

Publications

Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays.

Similar Datasets

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