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B R Cullen

Publications and source records attributed to B R Cullen.

At least 19 recordsLinked to original sources
PubMed1997

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.

Amino Acid Sequence↗
PubMed1997

Identification of regions in HIV-1 Nef required for efficient downregulation of cell surface CD4.

Downregulation of cell surface CD4 is a characteristic property of all lentiviral Nef proteins. We have used mutational analysis to define regions within HIV-1 Nef that are critical for this biological activity. Two discontinuous regions in Nef, extending approximately from residues 96 to 144 and from residues 175 to 186, are reported to be essential for efficient CD4 downregulation. Interestingly, these sequences coincide with two conserved regions of the Nef protein that are juxtaposed to form a single surface on the known structure of Nef. A third, more amino terminal conserved region in Nef, previously reported to be important for Nef enhancement of virion infectivity, was found to be largely dispensable for CD4 downregulation. These data raise the possibility that Nef may contain two structurally distinct functional domains, only one of which contributes to the CD4 downregulation phenotype.

Amino Acid Sequence↗
PubMed1997

A yeast TATA-binding protein mutant that selectively enhances gene expression from weak RNA polymerase II promoters.

We describe a unique gain-of-function mutant of the TATA-binding protein (TBP) subunit of Saccharomyces cerevisiae TFIID that, at least in part, renders transcriptional transactivators dispensable for efficient mRNA expression. The yTBPN69S mutant enhances transcription from weaker yeast promoter elements by up to 50-fold yet does not significantly increase gene expression directed by highly active promoters. Therefore, this TBP mutant and transcriptional transactivators appear to affect a common rate-limiting step in transcription initiation. Consistent with the hypothesis that this step is TFIID recruitment, tethering of TBP to a target promoter via a heterologous DNA binding domain, which is known to bypass the need for transcriptional transactivators, also nullifies the enhancing effect exerted by the N69S mutation. These data provide genetic support for the hypothesis that TFIID recruitment represents a rate-limiting step in the initiation of mRNA transcription that is specifically enhanced by transcriptional transactivators.

Binding Sites↗
PubMed1997

Nuclear import of hnRNP A1 is mediated by a novel cellular cofactor related to karyopherin-beta.

Heterogeneous nuclear ribonucleoprotein A1 contains a sequence, termed M9, that functions as a potent nuclear localization signal (NLS) yet bears no similarity to the well-defined basic class of NLSs. Here, we report the identification of a novel human protein, termed MIP, that binds M9 specifically both in vivo and in vitro yet fails to interact with non-functional M9 point mutants. Of note, the 101 kDa MIP protein bears significant homology to human karyopherin/importin-beta, a protein known to mediate the function of basic NLSs. The in vitro nuclear import of a protein substrate containing the M9 NLS was found to be dependent on provision of the MIP protein in trans. Cytoplasmic microinjection of a truncated form of MIP that retains the M9 binding site blocked the in vivo nuclear import of a substrate containing the M9 NLS yet failed to affect the import of a similar substrate bearing a basic NLS. These data indicate that nuclear import of hnRNP A1 is mediated by a novel cellular import pathway that is distinct from, yet evolutionarily related to, the pathway utilized by basic NLS sequences.

Amino Acid Sequence↗
PubMed1996

HIV-1: is Nef a PAK animal?

HIV-1 Nef is clearly essential for efficient viral replication in vivo, but it has been difficult to determine why. Recent evidence that Nef specifically activates a PAK-dependent signalling cascade may be the first step in defining the mechanism of action of this enigmatic viral protein.

Animals↗
PubMed1996

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).

Amino Acid Sequence↗
PubMed1996

Functional consequences of natural sequence variation in the activation domain of HIV-1 Rev.

Initial infection with an attenuated form of human immunodeficiency virus type 1 (HIV-1) may give rise to some of the rare asymptomatic infections that have been observed. Recently, data have been presented suggesting that a persistent mutation in the essential activation domain of the HIV-1 Rev regulatory protein might have contributed to the maintenance of the asymptomatic state in one individual. Here, we have used a range of assays for in vivo Rev function to examine whether natural sequence variation in the normally highly conserved Rev activation domain can indeed affect Rev function. Analysis of five distinct natural sequence variants of the Rev domain demonstrated that each produced a two- to fourfold drop in Rev function when compared to the consensus activation domain sequence A sixth sequence, reported for the MN isolate of HIV-1, proved entirely inactive. However, resequencing of this region of the MN genome revealed that this isolate actually encodes a consensus Rev activation domain. Overall, these data reveal that even natural sequence variation in the essential Rev activation domain can result in significantly reduced Rev function and suggest that isolates containing such sequence variation are likely to replicate less effectively.

Amino Acid Sequence↗
PubMed1996

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

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.

Amino Acid Sequence↗
PubMed1996

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

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.

Amino Acid Sequence↗
PubMed1996

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.

Animals↗
PubMed1996

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

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.

Base Sequence↗
PubMed1996

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.

Amino Acid Sequence↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1995

Identification of a novel human zinc finger protein that specifically interacts with the activation domain of lentiviral Tat proteins.

Transcriptional activation of HIV-1 gene expression by the viral Tat protein requires the interaction of a cellular cofactor with the Tat activation domain. This domain has been shown to consist of the cysteine-rich and core motifs of HIV-1 Tat and is functionally conserved in the distantly related Tat proteins of HIV-2 and EIAV. Using the yeast two-hybrid system, we have identified a novel human gene product, termed HT2A, that specifically and precisely binds to the activation domain of HIV-1 Tat and that can also interact with the HIV-2 and EIAV Tat proteins in vivo. We present data further demonstrating that the interaction between the activation domain of HIV-1 Tat and the HT2A protein can be readily detected in the mammalian cell nucleus. Sequence analysis demonstrates that HT2A is a novel member of the C3HC4 or ring finger family of zinc finger proteins that includes several known oncogenes and transcription factors. Overall, these data suggest that HT2A may play a significant role in mediating the biological activity of the HIV-1 Tat protein in vivo.

Amino Acid Sequence↗
PubMed1995

Functional similarities between HIV-1 Tat and DNA sequence-specific transcriptional activators.

The Tat regulatory protein encoded by human immunodeficiency virus type 1 (HIV-1) induces high levels of transcription from the viral long terminal repeat (LTR) promoter element after interacting with a promoter proximal RNA target sequence. In the wild-type HIV-1 LTR, this activation is facilitated by the synergistic interaction of Tat with the NF-kappa B and, particularly, SP1 regulatory proteins that bind to DNA sequences within the LTR promoter element. Using a synthetic Tat responsive indicator construct, we here demonstrate that NF-kappa B and SP1 are not uniquely or even unusually competent to synergize with HIV-1 Tat. Instead, these proteins can be functionally replaced by several, but not all, of the heterologous cellular and viral transcriptional activators tested. Tat therefore shares the ability to functionally synergize with a range of transcriptional activators, which is characteristic of DNA-sequence-specific regulatory proteins.

Animals↗