Analysis of intracellular trafficking and interactions of cytoplasmic HIV-1 Rev mutants in living cells.
The HIV-1 Rev protein is an essential nuclear regulatory viral protein. Rev mutants that are able to block wild-type (WT) Rev activity in trans have been reported and used in antiviral approaches. Not only nuclear but also cytoplasmic Rev mutants were described and suspected to be transdominant by retaining WT Rev in the cytoplasm. To investigate their potential for cytoplasmic retention, we studied the localization, trafficking, and interactions of cytoplasmic Rev mutants containing mutations in the N-terminal multifunctional domain. Using a novel dual-color autofluorescent protein-tagging system, we found that coexpression of the nucleolar blue-tagged WT Rev protein together with green-labeled cytoplasmic Rev mutants did not result in the retention of WT Rev in the cytoplasm but, on the contrary, in colocalization of the mutants to the nucleolus. A combination of mutations abolished the interaction with WT Rev, defining two domains important for Rev protein interaction. The identified domains were also essential for specific Rev responsive element (RRE) RNA binding and nuclear retention. Inactivation of the nuclear export signal shifted the steady-state distribution of the mutants from the cytoplasm to the nucleus, indicating their capability for nucleo-cytoplasmic shuttling. The cytoplasmic mutants were not transdominant compared to the nuclear mutant RevM10BL. These results emphasize that efficient oligomerization with WT Rev combined with RRE-specific RNA binding are prerequisites for effective transdominance.
Stauber
R H
RH
ABL-Basic Research Program, NCI-FCRDC, Frederick, Maryland, 21702-1201, USA.
Afonina
E
E
Gulnik
S
S
Erickson
J
J
Pavlakis
G N
GN
D002460:Cell Line; D002466:Cell Nucleolus / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D019084:Fluorescent Antibody Technique, Indirect; D015694:Gene Products, rev / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis; D019913:Nuclear Localization Signals / Q000235:genetics; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D020218:Response Elements / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics*; D014162:Transfection; D054321:rev Gene Products, Human Immunodeficiency Virus
D015694:Gene Products, rev; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins