Biochemical mechanism of HIV-I Vpr function. Specific interaction with a cellular protein.
vpr is an accessory gene of human immunodeficiency virus I (HIV-I). Although unnecessary for viral replication in T cell lines, growing evidence suggests that it is essential for virus replication in monocytes/macrophages and for replication in vivo. We expressed HIV-I vpr in Escherichia coli and purified Vpr by affinity chromatography. In a coprecipitation assay, the purified Vpr interacted specifically with a cellular protein designated as Vpr-interacting protein, or RIP. Mutational analysis suggested that this interaction required a domain rich in leucine/isoleucine residues and highly conserved between HIV-I and SIVmac Vprs. During transient expression in mammalian cells, HIV-I Vpr was localized in the nucleus. However, mutational analysis failed to identify in Vpr a typical nuclear localization signal rich in basic amino acid residues. Instead, Vpr nuclear localization seemed to correlate with Vpr interaction with RIP. Mutations in the C-terminal 20-amino acid region containing a cryptic nuclear localization signal did not abolish Vpr nuclear localization or interaction with RIP, whereas point mutations in the leucine/isoleucine-rich domain abolished Vpr interaction with RIP and rendered Vpr unstable during transient expression. These results suggest that RIP may be involved in Vpr function.
Zhao
L J
LJ
Department of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center, Kansas City 66160-7424.
Mukherjee
S
S
Narayan
O
O
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000378:metabolism*; D016336:Gene Products, vpr / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D016334:Genes, vpr; D016679:Genome, Viral; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016376:Oligonucleotides, Antisense; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D054325:vpr Gene Products, Human Immunodeficiency Virus
D002352:Carrier Proteins; D017931:DNA Primers; D016336:Gene Products, vpr; D016376:Oligonucleotides, Antisense; D011994:Recombinant Proteins; D054325:vpr Gene Products, Human Immunodeficiency Virus