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Novel Nuclear Import of Vpr Promoted by Importin Is Crucial for Human Immunodeficiency Virus Type 1 Replication in Macrophages

Yuko Nitahara-Kasahara,^1, Masakazu Kamata,^1, Takuya Yamamoto,² Xianfeng Zhang,¹ Yoichi Miyamoto,³ Koho Muneta,¹ Sayuki Iijima,^1, Yoshihiro Yoneda,³ Yasuko Tsunetsugu-Yokota,² and Yoko Aida^1*

Retrovirus Research Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan,¹ Department of Frontier Biosciences, Department of Immunology, National Institute of Infectious Diseases, Toyama, Shinjuku-ku, Tokyo 162-8640, Japan,² Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan³

Received 5 September 2006/ Accepted 19 February 2007

Monocytes/macrophages are major targets of human immunodeficiency virus type 1 (HIV-1) infection. The viral preintegration complex (PIC) of HIV-1 enters the nuclei of monocyte-derived macrophages, but very little PIC migrates into the nuclei of immature monocytes. Vpr, one of the accessory gene products of HIV-1, is essential for the nuclear import of PIC in these cells, although the role of Vpr in the entry mechanism of PIC remains to be clarified. We have shown previously that Vpr is targeted to the nuclear envelope and then transported into the nucleus by importin alone, in an importin ß-independent manner. Here we demonstrate that the nuclear import of Vpr is strongly promoted by the addition of cytoplasmic extract from macrophages but not of that from monocytes and that the nuclear import activity is lost with immunodepletion of importin from the cytoplasmic extract. Immunoblot analysis and real-time PCR demonstrate that immature monocytes express importin at low levels, whereas the expression of three major importin isoforms markedly increases upon their differentiation into macrophages, indicating that the expression of importin is required for nuclear import of Vpr. Furthermore, interaction between importin and the N-terminal -helical domain of Vpr is indispensable, not only for the nuclear import of Vpr but also for HIV-1 replication in macrophages. This study suggests the possibility that the binding of Vpr to importin , preceding a novel nuclear import process, is a potential target for therapeutic intervention.

Published ahead of print on 7 March 2007.

Present address: National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.

Present address: University of California, Los Angeles, CA 90095.

Present address: National Institute of Biomedical Innovation, Hachimandai 1-1, Tsukuba, Ibaraki 305-0843, Japan.