Non-infectious in-cell HIV-1 protease assay utilizing translocalization of a fluorescent reporter protein and apoptosis induction.
This study describes a non-infectious in-cell imaging assay for HIV-1 protease inhibitor screening. It is based on re-distribution of a fluorescence reporter protein upon protease cleavage and the fact that HIV-infected cells undergo apoptosis. The in-cell assay utilizes fluorescent reporter proteins consisting of an intracellular translocation signal sequence, a caspase-3-specific cleavage sequence, and a fluorescent tagging protein. The reporter proteins are designed to change their intracellular localization upon cleavage, either from the cytosol to a subcellular organelle (type I) or from a subcellular organelle to the cytosol (type II). Inhibition of protease activity can be monitored at the single cell level. Interestingly, the expression of HIV-1 protease induced endogenous caspase-3 activation; thus, the fluorescence reporter protein containing the caspase-3 cleavage sequence translocalized upon cleavage. This is the first time that HIV-1 protease expression, not whole virus infection of the cell, was observed to trigger the apoptotic pathway, including caspse-3 activation. A validation of this assay was performed with a known HIV-1 protease inhibitor, Ac-Leu-Val-phenylalanine. The clear cellular change in fluorescence pattern makes this system an ideal tool for various types of life science and drug discovery research, including high throughput and high content screening applications.
R&D Center, Ahram Biosystems Inc., Seoul, 133-120, Korea.
Hyun Jin
HJ
Hwang
R&D Center, Ahram Biosystems Inc., Seoul, 133-120, Korea.
Sung Hee
SH
Kim
Department of Oral and Maxillofacial Pathology, Kyung Hee University, Seoul, 130-701, Korea.
Young-Ah
YA
Cho
Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul, 130-701, Korea. jhkimh@khu.ac.kr.|Department of Oral Biochemistry and Molecular Biology, Kyung Hee University, Seoul, 130-701, Korea. jhkimh@khu.ac.kr.
Jeong Hee
JH
Kim
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D016466:CHO Cells; D053148:Caspase 3 / Q000032:analysis / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000378:metabolism*; D016333:HIV Protease / Q000032:analysis / Q000096:biosynthesis*
D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D053148:Caspase 3; D016333:HIV Protease; C514803:p16 protease, Human immunodeficiency virus 1
Caspase-3; HIV-1 protease; In-cell assay; Trans-localization