Screening for HIV-1 Vpr inhibitors.


Vpr is a 96 amino acid, 14 kD nucleophilic protein that is incorporated into mature virions of HIV-1. Vpr aids efficient translocation of the proviral DNA into the nucleus and is required for the HIV-1 infection of non-dividing cells such as macrophages. Vpr is also involved in activation of viral transcription, induction of cell cycle G2 arrest and apoptosis of the host cells upon the HIV-1 infection. Although specific roles of these Vpr activities in viral pathogenesis and disease progression have not been elucidated, the effects of Vpr mutations found in AIDS patients suggest that Vpr is an important cytotoxic component of HIV-1 infection. However, no Vpr targeted small molecule that can be used for AIDS therapy has yet been developed. We have established a screening system to isolate Vpr inhibitors using budding yeast cells. A multidrug sensitive yeast strain (MLC30) cells with Vpr expression plasmids were cultured to log phase in the expression silencing media at 30 oC, washed, suspended in expression inducing medium at OD600= 0.5 and cultured for an additional 30 min. Then, the culture was mixed with 9 volumes of expression inducing medium containing 2 % agar (Phytagar, GIBCO) and 0.001% SDS, poured into plastic plates and solidified at room temperature. Paper filters (diameter = 6 mm) containing compounds to be tested were put on the plates and the plates were incubated at 30 oC for several days.

Screening system to isolate Vpr inhibitors.
(A) Schematic presentation of Vpr screening system. Budding yeast cells expressing Vpr were embedded in agar plates containing inducer (copper). Paper filters with broths or compounds to be tested were put on the plates. Only the yeasts surrounding filters that contain Vpr inhibitors were able to grow. (B,C) Growing yeasts surrounding filters containing 10 ul of extract from the culture broth of a fungus with Vpr inhibitory activity (B) or purified fumagillin (C; 2 mg/ml, 10 ul). Plates were incubated for 4 days at 30 oC. (D) Chemical structures of fumagillin and TNP470. (Ref:1)


  1. N. Watanabe, Y. Nishihara, T. Yamaguchi, A. Koito, H. Miyoshi, H. Kakeya, H. Osada.
    Fumagillin suppresses HIV-1 infection of macrophages through the inhibition of Vpr activity.
    FEBS Lett., 580, 2598-2602 (2006).

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