Interaction of HIV-1 Tat with Puralpha in nuclei of human glial cells: characterization of RNA-mediated protein-protein binding.
A complex between the Tat protein, encoded by human immunodeficiency virus type 1 (HIV-1), and the cellular protein, Puralpha, has been implicated in activation of the late promoter of JC virus (JCV) and in enhancement of JCV DNA replication. JCV is the causative agent of progressive multifocal leukoencephalopathy (PML), an acquired immunodeficiency syndrome (AIDS) opportunistic infection of the brain. Puralpha also binds the HIV-1 TAR RNA element and activates HIV-1 transcription, suggesting a role for RNA binding in the action of this protein. Using immunoelectron microscopy, we find that in human glial cells expressing both proteins, Tat and Puralpha are colocalized in extranucleolar chromatin structural elements. The colocalized Puralpha and Tat are nearly exclusively nuclear, although individual proteins can be seen in both nucleus and cytoplasm, suggesting a preferential tropism of the complex for the nucleus. Analysis of the interaction between purified proteins indicates that the Tat-Puralpha interaction is strongly enhanced by the presence of RNA. Tat amino acids from 37-48 are essential for Tat binding. Residues 49-72, including the TAR RNA-binding domain, are critical for binding to Puralpha, while Cys(22), in the Tat transactivation domain, is responsible for an important global effect. Puralpha repeat II domains are involved in the interaction, and a polypeptide based on one such sequence inhibits binding. After RNase treatment of Puralpha enhancement of Tat binding can be partially restored by addition of a single-stranded JCV DNA PUR element, to which Tat does not bind. The results indicate that the Tat-Puralpha interaction is direct, rather than through an RNA link, and that RNA binding configures Puralpha for optimal interaction with Tat.
Wortman
M J
MJ
Department of Pathology and Brookdale Center for Molecular and Developmental Biology, Mount Sinai School of Medicine, New York, NY 10029, USA.
Krachmarov
C P
CP
Kim
J H
JH
Gordon
R G
RG
Chepenik
L G
LG
Brady
J N
JN
Gallia
G L
GL
Khalili
K
K
Johnson
E M
EM
D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004268:DNA-Binding Proteins; D015696:Gene Products, tat / Q000378:metabolism*; D015658:HIV Infections / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D009457:Neuroglia / Q000378:metabolism* / Q000473:pathology / Q000821:virology; D011485:Protein Binding; D012313:RNA / Q000378:metabolism*; D014157:Transcription Factors; D014407:Tumor Cells, Cultured; D054322:tat Gene Products, Human Immunodeficiency Virus
D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D015696:Gene Products, tat; C501130:PURA protein, human; D014157:Transcription Factors; D054322:tat Gene Products, Human Immunodeficiency Virus; D012313:RNA