R A Fridell

Protein sequence requirements for function of the human T-cell leukemia virus type 1 Rex nuclear export signal delineated by a novel in vivo randomization-selection assay.

The Rex protein of human T-cell leukemia virus type 1, like the functionally equivalent Rev protein of human immunodeficiency virus type 1, contains a leucine-rich activation domain that specifically interacts with the human nucleoporin-like Rab/hRIP cofactor. Here, this Rex sequence is shown to function also as a protein nuclear export signal (NES). Rex sequence libraries containing randomized forms of the activation domain/NES were screened for retention of the ability to bind Rab/hRIP by using the yeast two-hybrid assay. While the selected sequences differed widely in primary sequence, all were functional as Rex activation domains. In contrast, randomized sequences that failed to bind Rab/hRIP lacked Rex activity. The selected sequences included one with homology to the Rev activation domain/NES and a second that was similar to the NES found in the cellular protein kinase inhibitor alpha. A highly variant, yet fully active, activation domain sequence selected on the basis of Rab/hRIP binding retained full NES function even though this sequence preserved only a single leucine residue. In contrast, nonfunctional activation domain mutants that were unable to bind Rab/hRIP had also lost NES function. These data demonstrate that NES activity is a defining characteristic of the activation domains found in the Rev/Rex class of retroviral regulatory proteins and strongly support the hypothesis that the Rab/hRIP cofactor plays a critical role in mediating the biological activity of these NESs. In addition, these data suggest a consensus sequence for NESs of the Rev/Rex class.

Identification of a novel cellular cofactor for the Rev/Rex class of retroviral regulatory proteins

HIV-1 Rev is the prototype of a class of retroviral regulatory proteins that induce the sequence-specific nuclear export of target RNAs. This function requires the Rev activation domain, which is believed to bind an essential cellular cofactor. We report the identification of a novel human gene product that binds to not only the HIV-1 Rev activation domain in vitro and in vivo but also to functionally equivalent domains in other Rev and Rex proteins. The Rev/Rex activation domain-binding (Rab) protein occupies a binding site on HIV-1 Rev that precisely matches that predicted by genetic analysis. Rab binds the Rev activation domain when Rev is assembled onto its RNA target and can significantly enhance Rev activity when overexpressed. We conclude that Rab is the predicted activation domain-specific cofactor for the Rev/Rex class of RNA export factors.

HIV-1-INDUCED CELL FUSION IS MEDIATED BY MULTIPLE REGIONS WITHIN BOTH THE VIRAL ENVELOPE AND THE CCR-5 CO-RECEPTOR

Although the human hCCR‐5 chemokine receptor can serve as a co‐receptor for both M‐tropic (ADA and BaL) and dual‐tropic (89.6) strains of human immunodeficiency virus type 1 (HIV‐1), the closely related mouse mCCR‐5 homolog is inactive. We used chimeric hCCR‐5–mCCR‐5 receptor molecules to examine the functional importance of the three extracellular domains of hCCR‐5 that differ in sequence from their mCCR‐5 equivalents. While this analysis revealed that all three of these extracellular domains could participate in the functional interaction with HIV‐1 envelope, clear differences were observed when different HIV‐1 strains were analyzed. Thus, while the ADA HIV‐1 isolate could effectively utilize chimeric human–mouse CCR‐5 chimeras containing any single human extracellular domain, the BaL isolate required any two human extracellular sequences while the 89.6 isolate would only interact effectively with chimeras containing all three human extracellular sequences. Further analysis using hybrid HIV‐1 envelope proteins showed that the difference in co‐receptor specificity displayed by the ADA and BaL isolates was due partly to a single amino acid change in the V3 loop, although this interaction was clearly also modulated by other envelope domains. Overall, these data indicate that the interaction between HIV‐1 envelope and CCR‐5 is not only complex but also subject to marked, HIV‐1 isolate‐dependent variation.

A nuclear role for the Fragile X mental retardation protein.

Fragile X syndrome results from lack of expression of a functional form of Fragile X mental retardation protein (FMRP), a cytoplasmic RNA‐binding protein of uncertain function. Here, we report that FMRP contains a nuclear export signal (NES) that is similar to the NES recently identified in the Rev regulatory protein of human immunodeficiency virus type 1 (HIV‐1). Mutation of this FMRP NES results in mis‐localization of FMRP to the cell nucleus. The FMRP NES is encoded within exon 14 of the FMR1 gene, thus explaining the aberrant nuclear localization of a natural isoform of FMRP that lacks this exon. The NES of FMRP can substitute fully for the Rev NES in mediating Rev‐dependent nuclear RNA export and specifically binds a nucleoporin‐like cellular cofactor that has been shown to mediate Rev NES function. Together, these findings demonstrate that the normal function of FMRP involves entry into the nucleus followed by export via a pathway that is identical to the one utilized by HIV‐1 Rev. In addition, these data raise the possibility that FMRP could play a role in mediating the nuclear export of its currently undefined cellular RNA target(s).

Identification and Functional Characterization of a Novel Nuclear Localization Signal Present in the Yeast Nab2 Poly(A)+ RNA Binding Protein

ABSTRACT The nuclear import of proteins bearing a basic nuclear localization signal (NLS) is dependent on karyopherin α/importin α, which acts as the NLS receptor, and karyopherin β1/importin β, which binds karyopherin α and mediates the nuclear import of the resultant ternary complex. Recently, a second nuclear import pathway that allows the rapid reentry into the nucleus of proteins that participate in the nuclear export of mature mRNAs has been identified. In mammalian cells, a single NLS specific for this alternate pathway, the M9 NLS of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), has been described. The M9 NLS binds a transport factor related to karyopherin β1, termed karyopherin β2 or transportin, and does not require a karyopherin α-like adapter protein. A yeast homolog of karyopherin β2, termed Kap104p, has also been described and proposed to play a role in the nuclear import of a yeast hnRNP-like protein termed Nab2p. Here, we define a Nab2p sequence that binds to Kap104p and that functions as an NLS in both human and yeast cells despite lacking any evident similarity to basic or M9 NLSs. Using an in vitro nuclear import assay, we demonstrate that Kap104p can direct the import into isolated human cell nuclei of a substrate containing a wild-type, but not a defective mutant, Nab2p NLS. In contrast, other NLSs, including the M9 NLS, could not function as substrates for Kap104p. Surprisingly, this in vitro assay also revealed that human karyopherin β1, but not the Kap104p homolog karyopherin β2, could direct the efficient nuclear import of a Nab2p NLS substrate in vitro in the absence of karyopherin α. These data therefore identify a novel NLS sequence, active in both yeast and mammalian cells, that is functionally distinct from both basic and M9 NLS sequences.

Synergistic enhancement of both initiation and elongation by acidic transcription activation domains.

The effects of activation domain synergy on transcription initiation and elongation have been examined utilizing a system that permits the targeting of a defined number of activation modules to promoter DNA. As predicted, incremental increases in targeted activation potential were found to result in corresponding increases in transcription initiation. Surprisingly, however, transcriptional processivity, and hence mRNA synthesis, required a threshold level of activation domain synergy that exceeded the level required for at least modest levels of transcription initiation. The degree to which transcriptional processivity was enhanced was shown to depend on the quantity of activation modules targeted to the promoter DNA, rather than the quality. While the RNA‐sequence specific HIV‐1 Tat trans‐activator was also shown to enhance processivity in this assay system, Tat differed from DNA‐sequence specific activation domains in exerting a more dramatic effect on the efficiency of transcript elongation.

Nudeocytoplasmic shuttling by protein nuclear import factors

Protein nuclear import factors are not, in general, believed to function in the nuclear export of macromolecules and their reutilization therefore requires their recycling from the nucleus to the cytoplasm. Two possible mechanisms for recycling have been proposed. On the one hand, protein import factors such as importin beta and transportin (Trn) could continuously shuttle between cytoplasm and nucleoplasm. On the other hand, these proteins could penetrate into the nucleus only as far as the inner surface of the nuclear pore complex and then directly return to the cytoplasm. In this manuscript, we have used microinjection analysis in human cells, and in vitro nuclear assays, to demonstrate that importin beta, transportin and importin alpha are all nucleocytoplasmic shuttle proteins that efficiently enter and exit the cell nucleoplasm. In the case of transportin, we have mapped sequences required for nucleocytoplasmic shuttling to the carboxy-terminal 270 amino acids of this 890 amino acid import factor, thus demonstrating that nuclear export is independent of the amino-terminal Ran-binding domain of Trn. We further show that Trn shuttling is independent of nuclear RNA transcription. Overall, these data suggest that nucleocytoplasmic shuttling is likely to be a general attribute of protein nuclear import factors.