Studies with GFP-Vpr fusion proteins: induction of apoptosis but ablation of cell-cycle arrest despite nuclear membrane or nuclear localization.
The human immunodeficiency virus type 1 (HIV-1) Vpr protein is known to arrest the cell cycle in G(2)/M and induce apoptosis following arrest. The functions of Vpr relative to its location in the cell remain unresolved. We now demonstrate that the location and function of Vpr are dependent on the makeup of fusion proteins and that the functions of G(2)/M arrest and apoptosis are separable. Using green fluorescence protein mutants (EGFP or EYFP), we found that fusion at either the N- or C-terminus compromised the ability of Vpr to arrest cell cycling, relative to that of His-Vpr or wild-type protein. Additionally, utilizing the ability to specifically identify cells expressing the fusion proteins, we confirm that Vpr can induce apoptosis, but appears to be independent of cell-cycle arrest in G(2)/M. Both N- and C-terminal Vpr/EYFP fusion proteins induced apoptosis but caused minimal G(2)/M arrest. These studies with Vpr fusion proteins indicate that the functions of Vpr leading to G(2)/M arrest and apoptosis are separable and that fusion of Vpr to EGFP or EYFP affected the localization of the protein. Our findings suggest that nuclear membrane localization and nuclear import and export are strongly governed by modification of the N-terminus of Vpr.
Waldhuber
Megan G
MG
Department of Microbiology, Monash University, Clayton, Victoria, 3168, Australia.
Bateson
Michael
M
Tan
Judith
J
Greenway
Alison L
AL
McPhee
Dale A
DA
D017209:Apoptosis; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology*; D002467:Cell Nucleus / Q000378:metabolism*; D005434:Flow Cytometry; D016195:G2 Phase; D016336:Gene Products, vpr / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D049452:Green Fluorescent Proteins; D015497:HIV-1; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D008938:Mitosis; D009685:Nuclear Envelope / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D054325:vpr Gene Products, Human Immunodeficiency Virus
D016336:Gene Products, vpr; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D054325:vpr Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins