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Journal of Virology, November 2004, p. 11563-11573, Vol. 78, No. 21
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.21.11563-11573.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Evaluation of the Functional Involvement of Human Immunodeficiency Virus Type 1 Integrase in Nuclear Import of Viral cDNA during Acute Infection

Tamako Ikeda,1,2 Hironori Nishitsuji,1 Xin Zhou,1 Nobuo Nara,2 Takashi Ohashi,1 Mari Kannagi,1 and Takao Masuda1*

Departments of Immunotherapeutics,1 Laboratory Medicine, Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, Tokyo, Japan2

Received 23 April 2004/ Accepted 18 June 2004

Nuclear import of viral cDNA is a critical step for establishing the proviral state of human immunodeficiency virus type 1 (HIV-1). The contribution of HIV-1 integrase (IN) to the nuclear import of viral cDNA is controversial, partly due to a lack of identification of its bona fide nuclear localization signal. In this study, to address this putative function of HIV-1 IN, the effects of mutations at key residues for viral cDNA recognition (PYNP at positions 142 to 145, K156, K159, and K160) were evaluated in the context of viral replication. During acute infection, some mutations (N144Q, PYNP>KL, and KKK>AAA) severely reduced viral gene expression to less than 1% the wild-type (WT) level. None of the mutations affected the synthesis of viral cDNA. Meanwhile, the levels of integrated viral cDNA produced by N144Q, PYNP>KL, and KKK>AAA mutants were severely reduced to less than 1% the WT level. Quantitative PCR analysis of viral cDNA in nuclei and fluorescence in situ hybridization analysis showed that these mutations significantly reduced the level of viral cDNA accumulation in nuclei. Further analysis revealed that IN proteins carrying the N144Q, PYNP>KL, and KKK>AAA mutations showed severely reduced binding to viral cDNA but kept their karyophilic properties. Taken together, these results indicate that mutations that reduced the binding of IN to viral cDNA resulted in severe impairment of virus infectivity, most likely by affecting the nuclear import of viral cDNA that proceeds integration. These results suggest that HIV-1 IN may be one of the critical constituents for the efficient nuclear import of viral cDNA.

* Corresponding author. Mailing address: Department of Immunotherapeutics, Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. Phone: 81 (3) 5803-5799. Fax: 81 (3) 5803-0235. E-mail: tmasu.impt{at}tmd.ac.jp.

Journal of Virology, November 2004, p. 11563-11573, Vol. 78, No. 21
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.21.11563-11573.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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