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Biomedical subjects

B R Cullen

Publications and source records attributed to B R Cullen.

At least 109 records · Page 6Linked to original sources
PubMed1992

The Bel-1 protein of human foamy virus activates human immunodeficiency virus type 1 gene expression via a novel DNA target site.

The Bel-1 protein of human foamy virus can activate transcription directed by the long terminal repeat (LTR) promoter of human immunodeficiency virus type 1 (HIV-1). The target sequence for Bel-1 is shown to lie within the HIV-1 LTR U3 region but does not coincide with any previously described factor-binding site. Gene expression directed by an HIV-1 LTR lacking functional sites for the inducible cellular transcription factor NF-kappa B was activated over 100-fold by coexpression of Bel-1. These observations suggest that Bel-1 has the potential to significantly enhance the level of HIV-1 gene expression in cells dually infected with HIV-1 and human foamy virus.

Base Sequence↗
PubMed1992

Comparative analysis of Rev function in human immunodeficiency virus types 1 and 2.

The Rev proteins of the related but distinct human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) display incomplete functional reciprocity. One possible explanation for this observation is that HIV-2 Rev is unable to interact with the HIV-1 Rev-response element (RRE1). However, an analysis of the biological activity of chimeric proteins derived from HIV-1 and HIV-2 Rev reveals that this target specificity does not map to the Rev RNA binding domain but is instead primarily determined by sequences known to mediate Rev multimerization. Both HIV-1 and HIV-2 Rev are shown to bind the RRE1 in vitro with identical RNA sequence specificity. The observation that HIV-2 Rev can inhibit RRE1-dependent HIV-1 Rev function in trans indicates that the direct interaction of HIV-2 Rev with the RRE1 also occurs in vivo. These data suggest that HIV-2 Rev forms a protein-RNA complex with the RRE1 that leads to only minimal Rev activity. It is hypothesized that this low level of Rev function results from the incomplete and/or aberrant multimerization of HIV-2 Rev on this heterologous RNA target sequence.

Amino Acid Sequence↗
PubMed1992

Mechanism of action of regulatory proteins encoded by complex retroviruses.

Complex retroviruses are distinguished by their ability to control the expression of their gene products through the action of virally encoded regulatory proteins. These viral gene products modulate both the quantity and the quality of viral gene expression through regulation at both the transcriptional and posttranscriptional levels. The most intensely studied retroviral regulatory proteins, termed Tat and Rev, are encoded by the prototypic complex retrovirus human immunodeficiency virus type 1. However, considerable information also exists on regulatory proteins encoded by human T-cell leukemia virus type I, as well as several other human and animal complex retroviruses. In general, these data demonstrate that retrovirally encoded transcriptional trans-activators can exert a similar effect by several very different mechanisms. In contrast, posttranscriptional regulation of retroviral gene expression appears to occur via a single pathway that is probably dependent on the recruitment of a highly conserved cellular cofactor. These two shared regulatory pathways are proposed to be critical to the ability of complex retroviruses to establish chronic infections in the face of an ongoing host immune response.

Base Sequence↗
PubMed1991

Productive human immunodeficiency virus type 1 (HIV-1) infection of nonproliferating human monocytes.

Human immunodeficiency virus type 1 (HIV-1) infection of T lymphocytes requires cellular proliferation and DNA synthesis. Human monocytes were shown to have low DNA synthesis rates, yet the monocytotropic BaL isolate of HIV-1 was able to infect these cells efficiently. Monocytes that were irradiated to assure no DNA synthesis could also be readily infected with HIV-1BaL. Such infections were associated with the integration of HIV-1BaL DNA into the high molecular weight, chromosomal DNA of monocytes. Thus, normal, nonproliferating monocytes differ from T lymphocytes in that a productive HIV-1 infection can occur independently of cellular DNA synthesis. These results suggest that normal nonproliferating mononuclear phagocytes, which are relatively resistant to the destructive effects of this virus, may serve as persistent and productive reservoirs for HIV-1 in vivo.

Cells, Cultured↗
PubMed1991

Molecular basis of latency in pathogenic human viruses.

Several human viruses are able to latently infect specific target cell populations in vivo. Analysis of the replication cycles of herpes simplex virus, Epstein-Barr virus, and human immunodeficiency virus suggests that the latent infections established by these human pathogens primarily result from a lack of host factors critical for the expression of viral early gene products. The subsequent activation of specific cellular transcription factors in response to extracellular stimuli can induce the expression of these viral regulatory proteins and lead to a burst of lytic viral replication. Latency in these eukaryotic viruses therefore contrasts with latency in bacteriophage, which is maintained primarily by the expression of virally encoded repressors of lytic replication.

HIV-1↗
PubMed1991

Identification of the envelope V3 loop as the primary determinant of cell tropism in HIV-1.

Cells of the monocyte-macrophage lineage are targets for human immunodeficiency virus-1 (HIV-1) infection in vivo. However, many laboratory strains of HIV-1 that efficiently infect transformed T cell lines replicate poorly in macrophages. A 20-amino acid sequence from the macrophage-tropic BaL isolate of HIV-1 was sufficient to confer macrophage tropism on HTLV-IIIB, a T cell line--tropic isolate. This small sequence element is in the V3 loop, the envelope domain that is the principal neutralizing determinant of HIV-1. Thus, the V3 loop not only serves as a target of the host immune response but is also pivotal in determining HIV-1 tissue tropism.

Amino Acid Sequence↗
PubMed1991

HIV-1 structural gene expression requires the binding of multiple Rev monomers to the viral RRE: implications for HIV-1 latency.

Expression of the structural proteins of HIV-1 requires the direct interaction of the viral Rev trans-activator with its cis-acting RNA target sequence, the Rev response element or RRE. Here, we demonstrate that this specific RNA-binding event is, as expected, mediated by the conserved arginine-rich motif of Rev. However, we also show that amino acid residues located proximal to this basic domain that are critical for in vivo Rev function are dispensable for sequence-specific binding to the RRE. Instead, these sequences are required for the multimerization of Rev on the viral RRE target sequence. The observation that Rev function requires the sequential binding of multiple Rev molecules to the RRE provides a biochemical explanation for the observed threshold effect for Rev function in vivo and suggests a molecular model for the high incidence of latent infection by HIV-1.

Cloning, Molecular↗
PubMed1991

The HIV-1 Rev protein: prototype of a novel class of eukaryotic post-transcriptional regulators.

Complex retroviruses, including Human Immunodeficiency Virus Type 1 (HIV-1), are characterized by the ordered temporal expression of the various viral gene products in infected cells. This effect is mediated by a novel class of RNA-sequence-specific regulatory proteins typified by the Rev trans-activator of HIV-1. Evidence suggests that Rev regulates HIV-1 gene expression by intervening in the normal pathway of eukaryotic mRNA processing and transport.

Base Sequence↗
PubMed1991

Regulation of HIV-1 gene expression.

The quantity and quality of HIV-1 gene expression is temporally controlled by a cascade of sequential regulatory interactions. Basal HIV-1 transcription is determined by interaction of cellular regulatory proteins with specific DNA target sequences within the HIV-1 long-terminal repeat. The most notable of these protein:DNA interactions involves NF-kappa B, a transcription factor that plays a pivotal role in the activation of genes important for cellular responses to infection and inflammation. A second level of control involves the virally encoded Tat trans-activator. Tat, in combination with as yet unidentified cellular proteins, activates HIV-1 gene expression through a specific interaction with the viral TAR RNA stem-loop target sequence. A final level of regulation is mediated by the viral Rev protein. Rev acts posttranscriptionally to induce the expression of HIV-1 structural proteins and thereby commits HIV-1 to the late, cytopathic phase of the viral replication cycle. Rev activity appears to require a critical, threshold level of Rev protein expression, thus preventing entry into this late phase in cells that are unable to support efficient HIV-1 gene expression. In total, this cascade of regulatory levels allows the HIV-1 provirus to respond appropriately to the intracellular milieu present in each infected cell. In activated cells, the combination of Tat and Rev can stimulate a very high level of viral gene expression and replication. In quiescent or resting cells, in contrast, these same regulatory proteins are predicted to maintain the HIV-1 provirus in a latent or nonproductive state.

Avian Leukosis Virus↗
PubMed1991

Effect of RNA secondary structure on polyadenylation site selection.

Functional polyadenylation [poly(A)] sites consist of two sequence elements, the AAUAAA and G/U box signals, that closely flank the site of mRNA 3'-end formation. In agreement with previous results, random sequence insertions between the AAUAAA and G/U box signals were observed to inhibit poly(A) site function. However, sequence insertions of similar size that were predicted to form RNA stem-loop structures were found to have little effect on the efficiency of polyadenylation and instead induced a 3' shift in the site of polyadenylation that was equal to the length of the inserted stem-loop. The in vivo utilization of a poly(A) site bearing an internal RNA stem-loop structure was inhibited by mutations that destabilized the predicted stem but was restored by compensatory mutations. These results strongly support the hypothesis that the appropriate spacing of the AAUAAA and G/U box signals is critical for poly(A) site function. Sequence insertions that are able to form RNA secondary structures that maintain the correct spacing of these two RNA target sequences are well tolerated, whereas sequence insertions that disturb this spacing inhibit poly(A) site recognition. It is proposed that the effect of sequence insertions on poly(A) site function may be sufficiently predictable to allow the development of an assay for in vivo RNA secondary structure that uses poly(A) site selection as a readout.

Animals↗
PubMed1991

Rev activates expression of the human immunodeficiency virus type 1 vif and vpr gene products.

The proteins encoded by human immunodeficiency virus type 1 (HIV-1) can be divided into two temporally regulated classes. Early gene products are encoded by multiply spliced mRNA species and are expressed constitutively. In contrast, late proteins are encoded by a class of unspliced or singly spliced viral transcripts whose cytoplasmic expression is induced by the viral Rev trans activator. Here, we demonstrate that the viral Vif and Vpr proteins are encoded by singly spliced viral mRNAs whose expression is activated by Rev. This activation is shown to result from the reduced utilization of splice sites adjacent to or within the vif and vpr coding sequences. Vif and Vpr therefore belong to the class of late HIV-1 gene products.

Animals↗
PubMed1991

Effects of chimeric mutants of human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex on nucleolar targeting signals.

Two chimeric mutant genes derived from rev of human immunodeficiency virus type 1 and rex of human T-cell leukemia virus type I were constructed to investigate the functions of the nucleolar-targeting signals (NOS) in Rev and Rex proteins. A chimeric Rex protein whose NOS region was substituted with the NOS of Rev was located predominantly in the cell nucleolus and functioned like the wild-type protein in the Rex assay system. However, a chimeric Rev with the NOS of Rex abolished Rev function despite its nucleolar localization. This nonfunctional nucleolar-targeting chimeric protein inhibited the function of both Rex and Rev. In the same experimental conditions, this mutant interfered with the localization of the functional Rex in the nucleolus.

Amino Acid Sequence↗
PubMed1991

Characterization of the transcriptional trans activator of human foamy retrovirus.

The human foamy viruses, or spumaviruses, a distinct subfamily of complex human retroviruses, remain poorly understood both in terms of their pathogenic potential and in terms of the regulatory mechanisms that govern their replication. Here, we demonstrate that the human spumaretrovirus shares with other complex human retroviruses the property of encoding a transcriptional trans activator of the homologous viral long terminal repeat. This regulatory protein is encoded by the viral Bel-1 open reading frame and is localized to the nucleus of expressing cells. The Bel-1 trans activator is shown to function effectively in cell lines derived from human, simian, murine, and avian sources. The viral target sequence for Bel-1 has been mapped 5' to the start of viral transcription and is therefore likely to be recognized as a DNA sequence. Our results further suggest that the mechanism of action of the Bel-1 protein may be distinct from those reported for the transcriptional trans activators encoded by members of the other human retroviral subfamilies.

Cell Line↗
PubMed1991

Efficient polyadenylation within the human immunodeficiency virus type 1 long terminal repeat requires flanking U3-specific sequences.

During transcription of the human immunodeficiency virus type 1 provirus, polyadenylation signals present in the 5' long terminal repeat (LTR) are disregarded while the identical polyadenylation signals present in the 3' LTR are utilized efficiently. As both transcribed LTR sequences contain all signals known to be required for efficient polyadenylation, the basis for this differential utilization has been unclear. Here, we describe experiments that suggest that transcribed sequences present within the human immunodeficiency virus type 1 LTR U3 region act in cis to enhance polyadenylation within the 3' LTR.

Base Sequence↗
PubMed1991

Conserved functional organization of the human immunodeficiency virus type 1 and visna virus Rev proteins.

Visna virus encodes a posttranscriptional regulatory protein that is functionally analogous to the Rev trans activator of human immunodeficiency virus type 1. Here, we demonstrate that the known functional organization of the human immunodeficiency virus type 1 Rev trans activator is shared by the distantly related visna virus Rev protein. In particular, both Rev proteins contain an N-terminal domain marked by a highly basic core motif that determines RNA sequence specificity, as well as a second C-terminal domain containing an essential leucine-rich motif that functions as an activation domain. Chimeric proteins consisting of the binding domain of one Rev protein fused to the activation domain of the other were fully functional in the viral sequence context cognate for the binding domain. We also describe derivatives of visna virus Rev bearing a defective activation domain that displayed a trans-dominant negative phenotype in transfected cells. These visna virus Rev mutants may prove useful in the derivation of transgenic animals resistant to this agriculturally important retroviral pathogen.

Amino Acid Sequence↗