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Journal of Virology, February 2001, p. 1522-1532, Vol. 75, No. 3
Gladstone Institute of Virology and
Immunology1 and Departments of
Medicine,2 Hematology and
Oncology,3 and Microbiology and
Immunology,4 University of California, San
Francisco, California
Received 8 August 2000/Accepted 26 September 2000
Human immunodeficiency virus type 1 (HIV-1) is capable of infecting
nondividing cells such as macrophages because the viral preintegration
complex is able to actively traverse the limiting nuclear pore due to
the redundant and possibly overlapping nuclear import signals present
in Vpr, matrix, and integrase. We have previously recognized the
presence of at least two distinct and novel nuclear import signals
residing within Vpr that, unlike matrix and integrase, bypass the
classical importin
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.3.1522-1532.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Nucleocytoplasmic Shuttling by Human
Immunodeficiency Virus Type 1 Vpr
/
-dependent signals and do not require energy
or a RanGTP gradient. We now report that the carboxy-terminal region of
Vpr (amino acids 73 to 96) contains a bipartite nuclear localization
signal (NLS) composed of multiple arginine residues. Surprisingly, when
the leucine-rich Vpr(1-71) fragment, previously shown to harbor an NLS, or full-length Vpr is fused to the C terminus of a green fluorescent protein-pyruvate kinase (GFP-PK) chimera, the resultant protein is almost exclusively detected in the cytoplasm. However, the
addition of leptomycin B (LMB), a potent inhibitor of CRM1-dependent nuclear export, produces a shift from a cytoplasmic localization to a
nuclear pattern, suggesting that these Vpr fusion proteins shuttle into
and out of the nucleus. Studies of nuclear import with GFP-PK-Vpr
fusion proteins in the presence of LMB reveals that both of the
leucine-rich
-helices are required for effective nuclear uptake and
thus define a unique NLS. Using a modified heterokaryon analysis, we
have localized the Vpr nuclear export signal to the second leucine-rich
helix, overlapping a portion of the amino-terminal nuclear import
signal. These studies thus define HIV-1 Vpr as a nucleocytoplasmic
shuttling protein.
*
Corresponding Author. Mailing address: Gladstone
Institute of Virology and Immunology, P.O. Box 419100, San Francisco,
CA 94141-9100. Phone: (415) 695-3800. Fax: (4150 826-1817. E-mail: wgreene{at}gladstone.ucsf.edu.
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