A sensitive assay using a native protein substrate for screening HIV-1 maturation inhibitors targeting the protease cleavage site between the matrix and capsid
Department of Biochemistry and Molecular Pharmacology
Medical Subject Headings
Capsid; Cell Line; Fluorescein; HIV Protease; HIV-1; Humans; Substrate Specificity; Virus Assembly; Virus Replication
The matrix/capsid processing site in the HIV-1 Gag precursor is likely the most sensitive target to inhibit HIV-1 replication. We have previously shown that modest incomplete processing at the site leads to a complete loss of virion infectivity. In the study presented here, a sensitive assay based on fluorescence polarization that can monitor cleavage at the MA/CA site in the context of the folded protein substrate is described. The substrate, an MA/CA fusion protein, was labeled with the fluorescein-based FlAsH (fluorescein arsenical hairpin) reagent that binds to a tetracysteine motif (CCGPCC) that was introduced within the N-terminal domain of CA. By limiting the size of CA and increasing the size of MA (with an N-terminal GST fusion), we were able to measure significant differences in polarization values as a function of HIV-1 protease cleavage. The sensitivity of the assay was tested in the presence of increasing amounts of an HIV-1 protease inhibitor, which resulted in a gradual decrease in the fluorescence polarization values demonstrating that the assay is sensitive in discerning changes in protease processing. The high-throughput screening assay validation in 384-well plates showed that the assay is reproducible and robust with an average Z' value of 0.79 and average coefficient of variation values of <3%. The robustness and reproducibility of the assay were further validated using the LOPAC(1280) compound library, demonstrating that the assay provides a sensitive high-throughput screening platform that can be used with large compound libraries for identifying novel maturation inhibitors targeting the MA/CA site of the HIV-1 Gag polyprotein.
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Citation: Biochemistry. 2013 Jul 23;52(29):4929-40. doi: 10.1021/bi4005232. Epub 2013 Jul 11. Link to article on publisher's site
Lee, Sook-Kyung; Cheng, Nancy; Hull-Ryde, Emily A.; Potempa, Marc; Schiffer, Celia A.; Janzen, William P.; and Swanstrom, Ronald I., "A sensitive assay using a native protein substrate for screening HIV-1 maturation inhibitors targeting the protease cleavage site between the matrix and capsid" (2013). Biochemistry and Molecular Pharmacology Publications and Presentations. 213.