THE HIV-1 TAT NUCLEAR-LOCALIZATION SEQUENCE CONFERS NOVEL NUCLEAR IMPORT PROPERTIES

The different classes of conventional nuclear localization sequences ( NLSs) resemble one another in that NLS-dependent nuclear protein impor t is energy-dependent and mediated by the cytosolic NLS-binding import in/ karyopherin subunits and monomeric GTP-binding protein Ran/TC4. Ba sed on analysis of the nuclear import kinetics mediated by the NLS of the human immunodeficiency virus accessory protein Tat using in vivo a nd in vitro nuclear transport assays and confocal laser scanning micro scopy, we report a novel nuclear import pathway. We demonstrate that t he Tat-NLS, not recognized by importin 58/97 subunits as shown using a n enzyme-linked immunosorbent assay-based binding assay, is sufficient to target the 476-kDa heterologous beta-galactosidase protein to the nucleus in ATP-dependent but cytosolic factor-independent fashion. Exc ess SV40 large tumor antigen (T-ag) NLS-containing peptide had no sign ificant effect on the nuclear import kinetics implying that the Tat-NL S was able to confer nuclear accumulation through a pathway distinct f rom conventional NLS-dependent pathways. Nucleoplasmic accumulation of the Tat-NLS-beta-galactosidase fusion protein, in contrast to that of a T-ag-NLS-containing fusion protein, also occurred in the absence of an intact nuclear envelope, implying that the Tat-NLS conferred bindi ng to nuclear components. This is in stark contrast to known NLSs such as those of T-ag which confer nuclear entry rather than retention. Si gnificantly, the ability to accumulate in the nucleus in the absence o f an intact nuclear envelope was blocked in the absence of ATP, as wel l as by nonhydrolyzable ATP and GTP analogs, demonstrating that ATP is required to effect release from a complex with insoluble cytoplasmic components. Taken together, the results demonstrate that, dependent on ATP for release from cytoplasmic retention, the Tat-NLS is able to co nfer nuclear entry and binding to nuclear components. These unique pro perties indicate that Tat accumulates in the nucleus through a novel i mport pathway.