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 Previous Article

Journal of Virology, August 2000, p. 7179-7186, Vol. 74, No. 15
0022-538X/00/$04.00+0

Two Putative alpha -Helical Domains of Human Immunodeficiency Virus Type 1 Vpr Mediate Nuclear Localization by at Least Two Mechanisms

Masakazu Kamata and Yoko Aida*

RIKEN Tsukuba Institute, Tsukuba, Ibaraki 305-0074, Japan

Received 24 February 2000/Accepted 8 May 2000

To identify the domains of Vpr that are involved nuclear localization, we transfected HeLa cells with a panel of expression vectors that encode mutant Vpr protein with deletions or substitutions within putative domains. Immunofluorescence staining of transfected cells revealed that wild-type Vpr was localized predominantly in the nucleus and the nuclear envelope and certainly in the cytoplasm. Introduction of substitutions or deletions within alpha H1 or alpha H2 resulted, by contrast, in diffuse expression over the entire cell. In addition, double mutations within both of these alpha -helical domains led to the complete absence of Vpr from nuclei. Next, we prepared HeLa cells that express chimeric proteins which consist of the alpha H1 and alpha H2 domains fused individually with green fluorescent protein (GFP) and a Flag tag and extracted them with digitonin and Triton X-100 prior to fixation. Flag-alpha H1-GFP was detected in the nucleus but not in the cytoplasm, while Flag-alpha H2-GFP was retained predominantly in the nucleus and in a small amount in the cytoplasm. The immunostaining patterns were almost eliminated by substitutions in each chimeric protein. Thus, it appeared that the two alpha -helical domains might be involved in nuclear import by binding to certain cellular factors. Taken together, our data suggest that the two putative alpha -helical domains mediate the nuclear localization of Vpr by at least two mechanisms.

* Corresponding author. Mailing address: RIKEN Tsukuba Institute, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan. Phone: 81 298 36 3522. Fax: 81 298 36 9050. E-mail: aida{at}rtc.riken.go.jp.

Journal of Virology, August 2000, p. 7179-7186, Vol. 74, No. 15
0022-538X/00/$04.00+0


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