Showing papers by "Bryan R. Cullen published in 1996"

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.

Abstract: 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.

A nuclear role for the Fragile X mental retardation protein.

Abstract: 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).

Amphibian transcription factor iiia proteins contain a sequence element functionally equivalent to the nuclear export signal of human immunodeficiency virus type 1 rev

Abstract: The human immunodeficiency virus type 1 (HIV-1) Rev protein is required for nuclear export of late HIV-1 mRNAs. This function is dependent on the mutationally defined Rev activation domain, which also forms a potent nuclear export signal. Transcription factor IIIA (TFIIIA) binds to 5S rRNA transcripts and this interaction has been proposed to play a role in the efficient nuclear export of 5S rRNA in amphibian oocytes. Here it is reported that amphibian TFIIIA proteins contain a sequence element with homology to the Rev activation domain that effectively substitutes for this domain in inducing the nuclear export of late HIV-1 mRNAs. It is further demonstrated that this TFIIIA sequence element functions as a protein nuclear export signal in both human cells and frog oocytes. Thus, this shared protein motif may play an analogous role in mediating the nuclear export of both late HIV-1 RNAs and 5S rRNA transcripts.

Nuclear export of late HIV-1 mRNAs occurs via a cellular protein export pathway

Abstract: The Rev protein of HIV-1 is essential for the nuclear export of incompletely spliced viral mRNAs. This action depends on the mutationally defined Rev activation domain, which both binds the nucleoporin-like human cellular cofactor Rab/hRIP and also functions as a nuclear export signal. Protein kinase inhibitor alpha (PKI) also contains a potent nuclear export signal. However, PKI plays no role in nuclear RNA export and instead induces the nuclear export of a specific protein target, the catalytic subunit of cAMP-dependent protein kinase. Here, it is demonstrated that the nuclear export signal of PKI not only binds the Rab/hRIP cofactor specifically but also can effectively substitute for the Rev activation domain in mediating the nuclear export of HIV-1 mRNAs. We conclude that HIV-1 Rev and PKI act through an identical nuclear export pathway and that Rev, rather than using a dedicated RNA export pathway, is instead acting as an adaptor that allows viral mRNAs to access a cellular protein export pathway.

The human foamy virus Bel-1 transcription factor is a sequence-specific DNA binding protein.

Abstract: The Bel-1 transcriptional transactivator encoded by human foamy virus (HFV) can efficiently activate gene expression directed by both the HFV long terminal repeat (LTR) and internal (Int) promoter elements. By DNA footprinting and gel retardation analysis, we demonstrate that Bel-1 can specifically bind to discrete sites in both the LTR and Int promoter elements in vitro. However, transactivation of the HFV LTR by Bel-1 was observed to require not only the promoter-proximal Bel-1 binding site identified in vitro but also additional promoter-distal sequences. These data suggest that Bel-1 binding is necessary but not sufficient for efficient transactivation of Bel-1-responsive promoters in mammalian cells and therefore raise the possibility that Bel-1 function may require the action of a cellular DNA binding protein(s). Importantly, these data demonstrate that Bel-1 is unique among retroviral regulatory proteins in being a sequence-specific DNA binding protein.

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

Abstract: 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.