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Biomedical subjects

B R Cullen

Publications and source records attributed to B R Cullen.

At least 91 records · Page 5Linked to original sources
PubMed1993

Downregulation of cell-surface CD4 expression by simian immunodeficiency virus Nef prevents viral super infection.

The nef gene product encoded by the mac239 proviral clone of simian immunodeficiency virus (SIV) markedly enhances viral replication and pathogenesis in vivo. We have used this biologically active nef isolate to examine the phenotype of Nef in retrovirally transduced human T cells in culture. SIV Nef is shown to dramatically inhibit cell-surface expression of the CD4 glycoprotein without significantly affecting the total steady-state level of cellular CD4. This downregulation of the cell-surface CD4 receptor for human immunodeficiency virus type 1 (HIV-1) infection correlated with the acquisition of resistance to superinfection by HIV-1. However, SIV Nef did not affect the level of gene expression directed by the HIV-1 long terminal repeat. It is hypothesized that downregulation of cell-surface CD4 by Nef facilitates the efficient release of infectious progeny virions and, hence, viral spread in vivo.

CD4 Antigens↗
PubMed1993

Transcriptional trans activators of human and simian foamy viruses contain a small, highly conserved activation domain.

The Bel-1 protein of human foamy virus is a potent transcriptional trans activator of its homologous long terminal repeat promoter element. Here, we demonstrate that Bel-1 can also efficiently activate gene expression when targeted to a heterologous promoter by fusion to the DNA-binding motif of the yeast GAL4 protein. Analysis of a series of deletion mutants of Bel-1 generated in this hybrid protein context suggests the presence of a single transcription activation domain that is fully contained within a discrete, approximately 30-amino-acid segment located proximal to the Bel-1 carboxy terminus. Although this short motif can be shown to function effectively in eukaryotic cells of mammalian, avian, and fungal origin, it does not bear any evident sequence homology to the known classes of eukaryotic activation domain. However, this Bel-1 activation domain was found to be fully conserved, in terms of both biological activity and location, in the distantly related Taf trans activator of simian foamy virus type 1.

Chromosomal Proteins, Non-Histone↗
PubMed1993

Identification of the activation domain of equine infectious anemia virus rev.

Several members of the lentivirus family of complex retroviruses have been shown to encode proteins that are functionally equivalent to the Rev posttranscriptional regulatory protein of human immunodeficiency virus type 1 (HIV-1). Furthermore, the domain organization of HIV-1 Rev, featuring a highly basic N-terminal RNA binding domain and a leucin-rich C-terminal effector domain, has also been shown to be highly conserved among Rev proteins derived from not only the primate but also the ovine and caprine lentiviruses. Although it has therefore appeared highly probable that the lentivirus equine infectious anemia virus (EIAV) also encodes a Rev, the predicted amino acid sequence of this putative EIAV regulatory protein does not display any evident homology to the basic and leucine-rich motifs characteristic of other known Rev proteins. By fusion of different segments of the proposed EIAV Rev protein to the well-defined RNA binding domain of either HIV-1 or visna virus Rev, we have identified a segment of this EIAV protein that can efficiently substitute in cis for the otherwise essential activation motif. Interestingly, the minimal EIAV Rev activation motif identified in this study comprises approximately 18 amino acids located toward the protein N terminus that lack any evident similarity to the leucine-rich activation domains found in these other lentivirus Rev proteins. It therefore appears that the Rev protein of EIAV, while analogous in function to Rev proteins defined in lentiviruses of primate, ovine, and caprine origin, is nevertheless distinguished by an entirely novel domain organization.

Amino Acid Sequence↗
PubMed1993

Functional organization of the Bel-1 trans activator of human foamy virus.

Human foamy virus encodes a 300-amino-acid nuclear regulatory protein termed Bel-1 that is required for human foamy virus replication in culture. Bel-1 is a potent trans-activator of gene expression directed by the homologous HFV long terminal repeat as well as the long terminal repeat of human immunodeficiency virus type 1. We have used mutational analysis to define several discrete functional domains within Bel-1. The C-terminal approximately 50 amino acids of Bel-1 are shown to be essential for Bel-1 activity but can be effectively substituted by the C-terminal activation domain of VP16. We therefore conclude that the Bel-1 C terminus forms part of an activation domain. Mutations within a central, approximately 100-amino-acid segment of Bel-1 preclude trans-activation by either Bel-1 or the Bel-1/VP16 chimera. These sequences are therefore proposed to direct the interaction of Bel-1 with its viral DNA target sequences. A short Bel-1 segment located between the activation and binding domains is shown to mediate the nuclear localization of this regulatory protein. Although the functional organization of Bel-1 therefore appears comparable to that reported for other eukaryotic transcriptional activators, Bel-1 does not contain sequences homologous to known transcriptional activation or DNA-binding motifs.

Amino Acid Sequence↗
PubMed1993

Genetic analysis of the cofactor requirement for human immunodeficiency virus type 1 Tat function.

The Tat protein of human immunodeficiency virus type 1 is a potent transcriptional trans activator of the viral long terminal repeat promoter element. Tat function requires the direct interaction of Tat with a cis-acting viral RNA target sequence termed the trans-activation response (TAR) element and has also been proposed to require at least one cellular cofactor. We have used a genetic approach to attempt to experimentally define the role of the cellular cofactor in Tat function and TAR binding. Our data suggest that neither Tat nor the cellular cofactor binds to TAR alone in vivo and indicate, instead, that the interaction of Tat with its cellular cofactor is a prerequisite for TAR binding. The known species tropism of lentivirus Tat proteins appears to arise from the fact that not only Tat but also the cellular cofactor can markedly influence the RNA sequence specificity of the resultant protein complex. These data also suggest that the Tat cofactor is likely a cellular transcription factor that has been highly conserved during vertebrate evolution. We hypothesize that the primary function of Tat is to redirect this cellular factor to a novel viral RNA target site and to thereby induce activation of viral gene expression.

Amino Acid Sequence↗
PubMed1993

Genetic evidence that the Tat proteins of human immunodeficiency virus types 1 and 2 can multimerize in the eukaryotic cell nucleus.

The formation of dimers or higher-order multimers is critical to the biological activity of many eukaryotic regulatory proteins. However, biochemical analyses of the multimerization capacity of the Tat trans activator of human immunodeficiency virus types 1 (HIV-1) and 2 (HIV-2) have yielded contradictory results. We used the two-hybrid genetic assay for protein-protein interactions in the eukaryote Saccharomyces cerevisiae (S. Fields and O.-K. Song, Nature [London] 340:245-246, 1989) to examine the multimerization of Tat in vivo. Both HIV-1 and HIV-2 Tat are shown to form specific homo- but not heteromultimers in the yeast cell nucleus. Mutational analysis indicates a critical role for the essential core motif of Tat in mediating this interaction but demonstrates that efficient Tat multimerization does not require an intact cysteine motif. These data raise the possibility that the multimerization of Tat may be important for Tat function in higher eukaryotes.

Cell Nucleus↗
PubMed1993

Functional analysis of interactions between Tat and the trans-activation response element of human immunodeficiency virus type 1 in cells.

Transcriptional trans-activation of the human immunodeficiency virus type 1 long terminal repeat requires that the virally encoded Tat effector interacts with its target trans-activation response element (TAR) RNA stem-loop. Although the arginine-rich region of Tat from amino acids 49 to 59 is sufficient to bind to TAR RNA in vitro, the RNA-binding domain of Tat has not been defined in vivo. Human immunodeficiency virus type 1 also encodes the Rev protein, which acts through an RNA stem-loop called the Rev-response element to transport unspliced and singly spliced viral RNA species from the nucleus to the cytoplasm. To map the RNA-binding domain of Tat, we performed assays that relied on Rev function using the heterologous RNA-tethering mechanism of Tat and the TAR. By examining the effects of selected targeted mutations of Tat on the abilities of hybrid Tat/Rev proteins to rescue the expression of unspliced mRNA via the TAR, we demonstrated that residues throughout the N-terminal 59 amino acids of Tat are required for binding of Tat and TAR RNA in vivo.

Animals↗
PubMed1993

Rev and the fate of pre-mRNA in the nucleus: implications for the regulation of RNA processing in eukaryotes.

Although a great deal is known about the regulation of gene expression in terms of transcription, relatively little is known about the modulation of pre-mRNA processing. In this study, we exploited a genetically regulated system, human immunodeficiency virus type 1 (HIV-1) and its trans-activator Rev, to examine events that occur between the synthesis of pre-mRNA in the nucleus and the translation of mRNA in the cytoplasm. Unlike the majority of eukaryotic pre-mRNAs whose introns are efficiently recognized and spliced prior to nucleocytoplasmic transport, HIV-1 mRNAs containing functional introns must be exported to the cytoplasm for the expression of many viral proteins. Using human T cells containing stably integrated proviruses, we demonstrate that such incompletely spliced viral mRNAs are exported to the cytoplasm only in the presence of the Rev trans-activator. In the absence of Rev, these intron-containing RNAs are sequestered in the T-cell nucleus and either spliced or, more commonly, degraded. Because Rev does not inhibit the expression of fully spliced viral mRNA species in T cells, we propose that Rev, rather than inhibiting viral pre-mRNA splicing, is acting here both to prevent the nuclear degradation of HIV-1 pre-mRNAs and to induce their translocation to the cytoplasm. Taken together, these findings indicate that the cellular factors responsible for the nuclear retention of unspliced pre-mRNAs, although most probably splicing factors, do not invariably commit these RNAs to productive splicing and can, instead, program such transcripts for degradation.

Animals↗
PubMed1992

Stable expression of transdominant Rev protein in human T cells inhibits human immunodeficiency virus replication.

The human immunodeficiency virus (HIV) Rev protein is essential for viral structural protein expression (Gag, Pol, and Env) and, hence, for viral replication. In transient transfection assays, mutant forms of Rev have been identified that inhibit wild-type Rev activity and therefore suppress viral replication. To determine whether such transdominant Rev proteins could provide long-term protection against HIV infection without affecting T cell function, T leukemia cell lines were stably transduced with a retroviral vector encoding a transdominant mutant of the Rev protein, M10. While all the M10-expressing cell lines remained infectable by HIV-1, these same cells failed to support a productive replication cycle when infected with a cloned isolate of HIV-1. In addition, two out of three M10-expressing CEM clones were also resistant to highly productive infection by a heterogeneous HIV-1 pool. Expression of M10 did not affect induction of HIV transcription mediated by the kappa B regulatory element or Tat. Importantly, constitutive expression of Rev M10 did not alter the secretion of interleukin 2 in response to mitogen stimulation of EL-4 and Jurkat cells. The inhibition of HIV infection in cells stably expressing a transdominant Rev protein, in the absence of any deleterious effect on T cell function, suggests that such a strategy could provide a therapeutic effect in the T lymphocytes of acquired immunodeficiency syndrome patients.

Clone Cells↗
PubMed1992

Derivation of a biologically contained replication system for human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) proviral mutants that lack viral regulatory genes are unable to replicate unless rescued by complementation in trans. Structurally intact virus can be produced by infecting recombinant cell lines expressing the deficient genes. A HIV-1 mutant functionally defective in tat and rev (vIIIB delta Tat/Rev), which replicates only in a recombinant T-cell line expressing tat and rev (CEMTART), is described in this report. Infection of the CEMTART cell line with vIIIB delta Tat/Rev permits the complete HIV-1 life cycle, including cytopathology, decreased expression of CD4, and production of viral structural proteins, to be biologically contained. Culture supernatants from infected CEMTART contain virus that is able to replicate only in uninfected CEMTART. No reversion of vIIIB delta Tat/Rev to wild-type HIV-1 was observed as measured either by sequencing proviral vIIIB delta Tat/Rev or by detecting the ability of vIIIB delta Tat/Rev to replicate in CEM or activated CD4-bearing T lymphocytes. Defective HIV-1 mutants produced by trans complementation of essential genes permit infection and analysis of defined genotypes on cellular function and phenotype. Authentic HIV-1 structural proteins and infected cells can be prepared in mass, and agents that interfere with the HIV-1 life cycle can be studied on a large scale with minimum risk of exposing workers to virulent HIV-1.

3T3 Cells↗
PubMed1992

Identification of envelope V3 loop as the major determinant of CD4 neutralization sensitivity of HIV-1.

Laboratory isolates of human immunodeficiency virus type-1 (HIV-1) such as HTLV-IIIB are generally T cell line-tropic and highly sensitive to neutralization by soluble CD4 (sCD4), a potential antiviral agent that is undergoing clinical trial. However, many primary HIV-1 isolates are macrophage-tropic and sCD4-resistant. Envelope V3 loop sequences derived from primary HIV-1 isolates were sufficient to confer on HTLV-IIIB not only the tissue tropism but also the degree of sCD4 neutralization resistance characteristic of their HIV-1 strains of origin. Single amino acid changes in the V3 loop enhanced sCD4 resistance by up to tenfold. These observations suggest that the tissue tropism and sCD4 neutralization sensitivity of HIV-1 isolates are regulated by similar mechanisms.

Amino Acid Sequence↗
PubMed1992

Identification of a high-affinity RNA-binding site for the human immunodeficiency virus type 1 Rev protein.

Expression of the structural proteins of human immunodeficiency virus type 1 requires the direct interaction of multiple copies of the viral Rev protein with its highly structured RNA target sequence, the Rev response element (RRE). Nucleotides critical for Rev monomer binding have been mapped by chemical interference to a single site flanking the base of an RNA helix (stem IIB) located within the 234-nucleotide RRE. Binding of additional Rev molecules to an RRE probe did not require any RNA primary sequence information detectable by modification interference beyond that required for binding of a single Rev protein molecule. A synthetic 29-nucleotide RNA molecule designed to incorporate nucleotides identified as critical for Rev binding retained the ability to bind Rev specifically and, therefore, represents a minimal Rev-binding site. We propose that Rev binding to the RRE initiates with the direct interaction of a Rev monomer with a high-affinity binding site located at the base of the IIB stem of the RRE. The subsequent formation of Rev multimers on the RRE appears, in contrast, primarily driven by specific protein-protein interactions.

Base Sequence↗
PubMed1992

A comparison of regulatory features in primate lentiviruses.

Historically, research into the regulation of gene expression in primate lentiviruses has focused on human immunodeficiency virus type 1 (HIV-1), the primary cause of acquired immunodeficiency syndrome (AIDS) in humans. The increasing emergence of HIV-2 as a human pathogen, and the importance of the various simian immunodeficiency viruses (SIV) as models for the treatment and prevention of HIV-1-induced disease, suggest that an understanding of gene regulation in these related viruses will become increasingly important. Here, the present state of knowledge in this latter field is reviewed. In general, while the data support the hypothesis that viral gene expression is regulated by very similar mechanisms in all primate lentiviruses, it also is clear that differences in detail do exist. These differences may influence the pathogenic potential of the different strains of primate lentiviruses and must be considered in evaluating SIV as an appropriate in vivo model for HIV-1.

Amino Acid Sequence↗
PubMed1992

The VP16 transcription activation domain is functional when targeted to a promoter-proximal RNA sequence.

Among eukaryotic transcription trans-activators, the human immunodeficiency virus type 1 (HIV-1) Tat protein is exceptional in that its target site TAR is an RNA rather than a DNA sequence. Here, we confirm that fusion of Tat to the RNA-binding domain of the HIV-1 Rev protein permits the efficient activation of an HIV-1 long terminal repeat (LTR) promoter in which critical TAR sequences have been replaced by RNA sequences derived from the HIV-1 Rev response element (RRE). An RRE target sequence as small as 13 nucleotides is shown to form an effective in vivo target for Rev binding. More important, a fusion protein consisting of Rev attached to the VP16 transcription activation domain was also observed to efficiently activate the HIV-1 LTR from this nascent RNA target. These data demonstrate that trans-activation of transcription by acidic activation domains does not require a stable interaction with the promoter DNA and suggest that VP16, like Tat, can act on steps subsequent to the formation of the HIV-1 LTR preinitiation complex. The finding that the activation domains of VP16 and Tat are functionally interchangeable raises the possibility that these apparently disparate viral trans-activators may nevertheless act via similar mechanisms.

Base Sequence↗
PubMed1992

Specific binding of the human T-cell leukemia virus type I Rex protein to a short RNA sequence located within the Rex-response element.

Expression of the structural proteins of human T-cell leukemia virus type I is dependent upon the interaction of the viral Rex trans activator with its highly structured cis-acting RNA target sequence, the 254-nucleotide Rex-response element. Nucleotides critical for Rex binding in vitro have been mapped by modification interference analysis to a discrete 12-nucleotide RNA sequence that is predicted to form a stem-bulge-stem structure. This minimal RNA binding site was sufficient to mediate specific Rex binding in vitro when analyzed in the context of a short RNA probe. The critical importance of this short RNA sequence in mediating Rex function in vivo is supported by its complete conservation among all primate T-cell leukemia virus isolates.

Base Sequence↗
PubMed1992

Structural and functional analysis of the visna virus Rev-response element.

The distantly related lentiviruses human immunodeficiency virus type 1 (HIV-1) and visna virus each encode a posttranscriptional regulatory protein, termed Rev, that is critical for expression of the viral structural proteins. We genetically mapped the cis-acting target sequence for visna virus Rev, the visna virus Rev-response element or RRE-V, to a complex 176-nucleotide RNA stem-loop structure that coincides with sequences encoding the N terminus of the transmembrane component of envelope. The computer-predicted structure of the RRE-V was validated by in vitro analysis of structure-specific RNase cleavage patterns. The visna virus Rev protein was shown to interact specifically with the genetically defined RRE-V in vitro but was unable to bind the HIV-1 RRE. Similarly, HIV-1 Rev was also unable to bind the RRE-V specifically. We therefore conclude that the HIV-1 and visna virus Rev proteins, while functionally analogous, nevertheless display distinct RNA sequence specificities. These findings provide a biochemical explanation for the observation that these two viral regulatory proteins are functional only in the homologous viral system.

Base Sequence↗