Showing papers by "Bryan R. Cullen published in 1991"

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

Abstract: 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.

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

Abstract: 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.

Regulation of HIV-1 gene expression.

Abstract: 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.

Mutational definition of the human immunodeficiency virus type 1 Rev activation domain.

Abstract: Replication of human immunodeficiency virus type 1 requires the functional expression of the virally encoded Rev protein. The binding of this nuclear trans activator to its viral target sequence, the Rev-response element, induces the cytoplasmic expression of unspliced viral mRNAs. Mutation of the activation domain of Rev generates inactive proteins with normal RNA binding capabilities that inhibit wild-type Rev function in a trans-dominant manner. Here, we report that the activation domain comprises a minimum of nine amino acids, four of which are critically spaced leucines. The preservation of this essential sequence in other primate and nonprimate lentivirus Rev proteins indicates that this leucine-rich motif has been highly conserved during evolution. This conclusion, taken together with the observed permissiveness of a variety of eukaryotic cell types for Rev function, suggests that the target for the activation domain of Rev is likely to be a highly conserved cellular protein(s) intrinsic to nuclear mRNA transport or splicing.

Human immunodeficiency virus as a prototypic complex retrovirus.

Abstract: Retroviruses are defined by their ability to reverse the normal flow of genetic information from genomic DNA to mRNA (19). Although not unique (pararetroviruses such as hepatitis B virus also share this property), retroviruses nevertheless form a clearly defined viral genus. Distinguishing characteristics include the morphology and composition of retroviral virions and the possession of a single-stranded, positive-sense RNA genome. While retroviruses form a relatively homogeneous viral family, they have customarily been subdivided into three taxonomic groupings primarily on the basis of the in vivo and in vitro consequences of infection (16, 19). The oncovirus subgroup includes retroviruses able to cause neoplastic disease in the infected host animal but also includes some related, relatively benign viruses. The lentivirus subgroup includes retroviruses that cause slow, chronic diseases that generally, but not always, lack a neoplastic component. Members of the spumavirus subgroup cause a foamy cytopathic effect in tissue culture and have yet to be clearly associated with any disease. Retroviruses were the first oncogenic viruses to be identified, and it is this ability to transform cells which first attracted scientific attention. Interest in the mechanisms of retroviral transformation led to considerable research into the life cycle of animal retroviruses belonging to the oncovirus subgroup, focusing particularly on the prototypic murine and avian leukemia viruses (MLV and ALV) (19). Retroviral replication is initiated by the intracytoplasmic penetration of the virion core, a process mediated by the specific interaction of the viral envelope glycoprotein with a host cell surface receptor. Subsequently, the virion-associated reverse transcriptase transcribes the single-stranded viral RNA genome into a double-stranded linear DNA proviral intermediate. This proviral intermediate then migrates to the nucleus where the viral integrase enzyme acts to covalently link the retroviral genome to the host chromosomal DNA, thereby forming the retroviral provirus. In its simplest form, as seen for example in MLV, retroviral replication requires only three distinct virus-encoded genes (19) (Fig. 1). These are the gag gene, which encodes the virion structural proteins, the pol gene, which encodes the various virion-associated enzymes, and env, which encodes the envelope glycoprotein. (A viral protease, required for the posttranslational processing of the Gag and Gag-Pol polyproteins, may be encoded in pol or may form part of gag.) In the integrated DNA provirus, these three genes are invariably arranged in the same order (5'-gag-pol-env-3') and are flanked by the characteristic long terminal repeats (LTRs) generated during the process of reverse transcription. The LTRs contain enhancer and promoter elements required for efficient transcription of the retroviral genome and also contain sequences important for efficient mRNA polyadenylation within the 3' LTR. In the case of MLV and the majority of other animal oncoviruses, the integrated provirus encodes only two distinct transcripts. These are the genomic RNA, which also functions as the mRNA for Gag and Pol synthesis, and a singly-spliced mRNA species that encodes Env. It therefore appeared that the life cycle of MLV, and by extension of retroviruses in general, was both simple and efficient. Viral gene products served structural or enzymatic functions, while regulation of viral gene expression at both the transcriptional and posttranscriptional levels was controlled by the interplay of cis-acting viral DNA or RNA sequences with trans-acting factors encoded entirely by the host cell. The discovery of directly pathogenic human retroviruses, particularly the emergence of human immunodeficiency vi-

Molecular basis of latency in pathogenic human viruses

Abstract: 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.

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

Abstract: 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.

Characterization of the transcriptional trans activator of human foamy retrovirus.

Abstract: 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.

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

Abstract: 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.

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

Abstract: 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.

Effect of RNA secondary structure on polyadenylation site selection

Abstract: 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.

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

Abstract: 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.

Characterization of human interferon-gamma and human interleukin-2 from recombinant mammalian cell lines and peripheral blood lymphocytes

Abstract: Human interleukin-2 (IL-2) and human interferon-gamma (IF-gamma) isolated from transfected heterologous cell lines were structurally compared with their natural counterparts isolated from peripheral blood lymphocytes (PBL). Several forms of IL-2 and IFN-gamma that were primarily due to differential glycosylation were produced by all cell types studied. Comparable molecular weights and sugar compositions were found for variants of natural and mammalian-derived recombinant IL-2, and variants of natural and mammalian-derived recombinant IFN-gamma. Furthermore, the specific biological activities of the natural and mammalian-derived recombinant lymphokines were similar and comparable to the specific activity of the Escherichia coli-derived recombinant proteins.

Regulation of gene expression in the human immunodeficiency virus type 1.

Abstract: Publisher Summary This chapter outlines the current understanding of gene regulation in HIV-1 In particular, it focuses on the various cellular and viral trans-activators that together serve to modulate both the quantity and quality of HIV-1-specific gene expression The pathogenic retrovirus human immunodeficiency virus type 1 (HIV-1) is the prototype of a group of primate lentiviruses, that also includes HIV-2 and a number of related but distinct simian immunodeficiency viruses In addition, HIV-1 is distantly related to several nonprimate lentiviruses, including visna-maedi virus, equine infectious anemia virus, bovine immunodeficiency virus, caprine encephalitis virus, and feline immunodeficiency virus Lentiviruses exhibit a tropism for T lymphocytes and/or cells of the monocyte/macrophage lineage, and share the ability to induce long-term chronic disease states in their hosts These retroviruses also display a level of genetic complexity that distinguishes them from the simpler type C family of oncogenic retroviruses