Molecular and enzymatic characterization of XMRV protease by a cell-free proteolytic analysis.
Xenotropic murine leukemia virus-related virus (XMRV) is a virus generated under artificial conditions by the recombination of 2 murine leukemia virus (MLV) proviruses, PreXMRV-1 and PreXMRV-2, during the in vivo passage of human prostate cancer cells in athymic nude mice. The molecular etiology of XMRV infection has not been characterized and its implication in human prostate cancer progression remains equivocal. As a step toward resolving this issue we developed an in vitro enzymatic assay system to characterize XMRV protease (PR)-mediated cleavage of host-cell proteins. Enzymatically-active XMRV PR protein was synthesized using a wheat-germ cell-free system. By monitoring cleavage activity of XMRV PR by AlphaScreen and 2-color immunoblot analyses, we revealed that the catalytic activity of XMRV PR is selectively blocked by the HIV PR inhibitor, Amprenavir, and identified several human tumor suppressor proteins, including PTEN and BAX, to be substrates of XMRV PR. This system may provide an attractive means for analyzing the function of retrovirus proteases and provide a technology platform for drug screening.
Department of Microbiology, Yokohama City University School of Medicine, Yokohama 236-0004, Japan.
Satoko
S
Matsunaga
Tatsuya
T
Sawasaki
Hirotaka
H
Ode
Ryo
R
Morishita
Ayako
A
Furukawa
Ryuta
R
Sakuma
Wataru
W
Sugiura
Hironori
H
Sato
Masato
M
Katahira
Akifumi
A
Takaori-Kondo
Naoki
N
Yamamoto
Akihide
A
Ryo
D000818:Animals; D002219:Carbamates / Q000737:chemistry*; D045744:Cell Line, Tumor; D002474:Cell-Free System / Q000201:enzymology; D005663:Furans; D017320:HIV Protease Inhibitors / Q000737:chemistry*; D006801:Humans; D051379:Mice; D051059:PTEN Phosphohydrolase / Q000737:chemistry* / Q000378:metabolism; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000378:metabolism; D059748:Proteolysis; D013379:Substrate Specificity; D013449:Sulfonamides / Q000737:chemistry*; D014764:Viral Proteins; D058008:Xenotropic murine leukemia virus-related virus / Q000201:enzymology*; D051028:bcl-2-Associated X Protein / Q000737:chemistry* / Q000378:metabolism
C494826:BAX protein, human; D002219:Carbamates; D005663:Furans; D017320:HIV Protease Inhibitors; D013449:Sulfonamides; D014764:Viral Proteins; D051028:bcl-2-Associated X Protein; C095108:amprenavir; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human; D010447:Peptide Hydrolases