Mutational analysis of cell cycle arrest, nuclear localization and virion packaging of human immunodeficiency virus type 1 Vpr

Abstract

Human immunodeficiency virus type 1 Vpr is a virion-associated, regulatory protein that is required for efficient viral replication in monocytes/macrophages. The protein is believed to act in conjunction with the Gag matrix protein to allow import of the viral preintegration complex in nondividing cells. In cells, Vpr localizes to the nucleus. Recently, we showed that Vpr prevents the activation of p34 cdc2-cyclin B. This results in arrest of Vpr-expressing cells in the G 2 /M phase of the cell cycle. Here, we use a panel of expression vectors encoding Vpr molecules mutated in the amino-terminal alpha-helical region, the central hydrophobic region, or the carboxy-terminal basic region to define the functional domains of the protein. The results showed cell cycle arrest was largely controlled by the carboxy-terminal basic domain of the protein. In contrast, the aminoterminal alpha-helical region of Vpr was required for nuclear localization and packaging into virions. The carboxy terminus appeared to be unnecessary for nuclear localization. In the alpha-helical region, mutation of Ala-30 to Pro resulted in a protein that localized to the cytoplasm. Surprisingly, fusion of Vpr to luciferase resulted in a molecule that failed to localize to the nucleus. In addition, we show that simian immunodeficiency virus Vpr, but not Vpx, induces G 2 arrest. We speculate that Vpr has two sites for interaction with cellular factors: one in the alpha-helical region that specifies nuclear localization and one in the carboxy-terminal domain that is required for Cdc2 inhibition.

A peptide derived from the N‐terminal region of HIV‐1 Vpr promotes nuclear import in permeabilized cells: elucidation of the NLS region of the Vpr

“…VprC-BSA conjugates were retained in the cytoplasm of the permeabilized cells. Our present work strengthens previous suggestions, based on genetic studies using Vpr mutants [13,14], that the N-terminal putative K-helical region of Vpr confers the karyophilic properties of this protein. The present use of VprN-BSA conjugates enables us to limit the active region to residues 17^34 and to prove for the ¢rst time that this region, and not VprC, possesses an NLS activity.…”

Micronuclei formation and aneuploidy induced byVpr, an accessory gene of human immunodeficiency virus type 1

“…Next we determined the functional domain of Vpr, which is necessary for inducing multinucleation and/or MIN formation. Since it has been reported that the carboxy-terminal region of Vpr is important for cell cycle abnormality (12,43), deletion mutants lacking carboxyl amino acids (Fig. 4A) were generated and expressed in HT1080.…”

Solution structure of human immunodeficiency virus type 1 Vpr(13-33) peptide in micelles

“…One may speculate, that the leucinezipper like sequence motif preceding Ala30 serves together with helical secondary structure as a basis for a variety of protein±protein interactions between Vpr and other viral and cellular factors. The importance of this secondary structure motif is supported by the finding that mutation of Ala30 to proline abolishes Vpr nuclear localization [23]. Also, mutations of either one of residues Asp17, Glu21, Glu24, Glu25 or Glu29 to proline dramatically reduces virion packaging of Vpr [25].…”

“…Some residues in Vpr(13±33) are known to be crucial for several Vpr activities. Mutation of Ala30 to leucine completely abolishes G 2 cell cycle arrest and virion packaging of Vpr [23]. Mutations of either Leu23 or Ala30 to phenylalanine dramatically reduce virion incorporation of Vpr but do not interfere with its nuclear localization [24].…”

“…We focused our structural studies on residues 13±33 of HIV-1 Vpr. This part of Vpr is known to be essential for ion chanel activity [15], cell cycle arrest [23], incorporation of Vpr into virus-like particles [24±26], and its nuclear localization [27]. Because a membranous or membranelike environment may be important for most of these activities, we carried out structural studies of Vpr(13±33) peptide in dodecylphosphocholine (dodecyl-PCho) containing micelles by CD and NMR spectroscopy.…”