| Budget Amount *help |
¥15,000,000 (Direct Cost: ¥15,000,000)
Fiscal Year 2005: ¥7,500,000 (Direct Cost: ¥7,500,000)
Fiscal Year 2004: ¥7,500,000 (Direct Cost: ¥7,500,000)
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| Research Abstract |
HIV-1Vpr, a virion-associated viral protein 96 amino-acids in length, has multiple biological functions including its nuclear localization activity, arrest at the G2/M phase of the cell cycle, positive and negative regulation of apoptosis.
Vpr is essential for the nuclear import of preintegration complex (PIC) in Macrophages, although the role of Vpr in the entry mechanism of PIC remains to be clarified. We firstly demonstrated that Vpr is targeted to the nuclear envelope and then transported into the nucleus by importin a alone in an importin 13-independent manner. Next, we demonstrated that the nuclear import of Vpr is strongly promoted by the addition of the cytoplasmic extract from macrophages but not from monocyte, and the nuclear import activity is lost by immunodepletion of importin q from the cytoplasmic extract. Immature monocytes express importin a at low level by immunoblot analysis and real-time PCR, while the expression of three major importin a isoforms markedly increases
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upon differentiation to macrophages, indicating requirement the expression of importin a for a nuclear import of Vpr. Furthermore, interaction between importin a and Vpr is indispensable not only for the nuclear import of Vpr but also for HIV-1 replication in macrophages. Finally, we demonstrated that the binding of Vpr to importin a that precedes a novel nuclear import process is potential target for therapeutic intervention.
On the other hand, we discovered a novel role for Vpr in the selective inhibition of cellular pre-mRNA splicing both in vivo and in vitro. Vpr exerted this effect via interactions not only with the essential splicing factor SAP145 but also with functional spliceosomal complexes. We also found that expression of Vpr selectively inhibit splicing of HIV-1 mRNA, resulting in an accumulation of the 4kb-form of viral mRNA and a decrease in the 2kb-form, and consequently altered the expression of corresponding proteins. Moreover, Vpr enhanced de novo synthesis of virion-associated proteins and increases the virus infectivity. This is the first report to demonstrate that Vpr enhances the virus infectivity via selective inhibition of viral pre-mRNA splicing. Less
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