Biomedical subjects

J Hauber

Publications and source records attributed to J Hauber.

At least 55 records · Page 3Linked to original sources

PubMed1992

Expression of chimeric neo-Rev response element sequences interferes with Rev-dependent HIV-1 Gag expression.

Recombinant plasmids containing reiterated human immunodeficiency virus type 1 (HIV-1) Rev response element (RRE) sequences were constructed to suppress Rev-dependent HIV-1 Gag expression. The mammalian expression vectors pMAMneo containing one, three, or six repeats of the RRE sequence were cotransfected with a HIV-1 HTLV-IIIB proviral DNA into HeLa cells. All three RRE expression plasmids reduced replication of HIV-1 with similar efficacy. Furthermore, the chimeric expression vector pCMV neoRRE6 x ----(containing six copies of the RRE sequence) was used to establish HeLa cell lines constitutively expressing RRE. A plasmid encoding a Rev-dependent HIV-1 p24 Gag protein was cotransfected with the wild-type Rev expression plasmid into three different RRE-expressing HeLa cell lines. p24 Gag protein production in the culture supernatants of the HeLaneoRRE cells was compared with two neo-expressing cell lines. Although all cell lines (HeLaneoRRE, HeLaneo) displayed similar transfection efficiencies, p24 Gag protein synthesis was markedly reduced in the RRE-expressing cell lines in comparison to the control cells.

Base Sequence↗

PubMed1992

Definition of the human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex protein activation domain by functional exchange.

The human retroviruses human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type I (HTLV-I) are characterized by complex regulation of gene expression. Each virus encodes a posttranscriptional regulator, the 19-kDa HIV-1 Rev protein and the 27-kDa HTLV-I Rex protein, which is required for viral replication. Expression of these trans activators results in the cytoplasmic accumulation of unspliced or singly spliced viral mRNA which encode the gag, pol, and env gene products. The finding that the HTLV-I Rex protein is able to functionally substitute for the Rev protein of HIV-1 indicates that HIV-1 Rev and HTLV-I Rex may interact with the same component of a cellular pathway involved in either mRNA splicing or transport. In this study, we have generated functional Rev/Rex hybrid proteins by domain exchange. We have defined, using in vivo and in vitro analyses, the activation domains of Rev and Rex which are the putative targets of a common host cell factor(s) required for Rev and Rex function.

Amino Acid Sequence↗

PubMed1992

Dominant-negative mutants are clustered in a domain of the human T-cell leukemia virus type I Rex protein: implications for trans dominance.

The 27-kDa Rex trans-acting protein appears to be essential for replication of human T-cell leukemia virus type I. Mutations introduced outside of the Rex RNA-binding domain-nucleolar localization signal display either wild-type activity or, conversely, yield dominant-negative proteins. We generated missense mutations in a particular domain of the Rex protein (amino acid residues 54 to 69) which is characterized by a cluster of dominant-negative mutants. Our results indicate that amino acids 57 to 67 are critically important for Rex function mediated through the RxRE cis-acting RNA sequence. Within this domain, only amino acids 61 to 63 could be mutated without loss of function. All other missense and deletion mutants yielded dominant-negative proteins. In vitro RNA-binding studies performed with glutathione S-transferase-Rex fusion proteins demonstrated that all of the mutant Rex proteins interacted specifically with RxRE RNA. Analysis of chimeric Rex-Rev proteins suggests that this Rex domain is important for oligomerization.

Amino Acid Sequence↗

PubMed1991

Characterization of HIV-1 REV protein: binding stoichiometry and minimal RNA substrate.

The HIV-1 REV protein binds to the stem II region of the REV-responsive element (RNA). Studies to further define the RNA sequence and structure specifically bound by REV protein identify a minimal RNA element of 40 nucleotides. Analysis of RNA fragments by gel retardation and filter binding suggest that a core element composed of one particular stem with flanking sequences capable of forming a second double stranded region is essential for specific recognition by REV protein. Stable REV-RNA complexes are formed in a stoichiometry of 1 REV: 1 RNA. The minimal RNA element binds 1 REV molecule while the stem II saturates at 3 REV molecules per RNA. These results establish that REV recognizes a primary binding site within the RRE and support the notion that the initial viral transcript binding event involves a monomeric REV protein.

Autoradiography↗

PubMed1991

trans-activation of the HIV-1 LTR by the HIV-1 Tat and HTLV-I Tax proteins is mediated by different cis-acting sequences.

Human immunodeficiency virus type 1 (HIV-1) gene expression is regulated by viral and cellular factors interacting with cis-elements located in the retroviral long terminal repeat (LTR). In this report we analyzed HIV-1 LTR-specific regulatory sequences responsive to the HIV-1 Tat and HTLV-I Tax trans-activator proteins. Our results indicate that the Sp1 binding sites in the HIV-1 LTR are crucially involved in Tat-mediated gene expression in human Jurkat T-cells whereas they are dispensable for HTLV-I Tax-induced activation. In contrast, the NF-kB binding sites within the HIV-1 LTR are essential for Tax-mediated transcription but had only marginal effect on Tat-induced reporter gene expression.

Animals↗

PubMed1991

Mutational analysis of functional domains in the HIV-1 Rev trans-regulatory protein.

HIV-1 replication depends on the expression of trans-regulatory genes (tat, rev) encoded in the 3' part of the retroviral genome. HIV-1 Rev trans-activator protein allows the cytoplasmic translocation of incompletely spliced retroviral mRNA which is required for the translational switch from regulatory (Tat, Rev, Nef) to structural proteins (Gag, Pol, Env). The HIV-1 Rev regulatory protein comprises an activation domain (RAD) and a RNA binding domain (RBD). Both functional domains are not well defined and the RBD appears to overlap with the nuclear localization signal (NLS). Our mutational analysis localized the Rev protein domain important for RRE (nucleotide 7781 to 8000) binding in vitro to amino acid residues 31 to 50. Mutations in this domain always resulted in exclusion from the nucleoli. Furthermore, these mutants did not support Rev-dependent p24 Gag production in vivo. Sequences immediately upstream of this domain (RevM4, RevM19) were attenuated in their in vivo activity possibly indicating a role in Rev protein oligomerization. The observed tight correlation between subcellular localization and RNA binding in vitro indicates that this short stretch of amino acids supports two essential functions required for HIV-1 replication.

Amino Acid Sequence↗

PubMed1991

Transdominant repressors for human T-cell leukemia virus type I rex and human immunodeficiency virus type 1 rev function.

Human T-cell leukemia virus type I (HTLV-I) encodes a 27-kDa trans-acting gene product (Rex) which is involved in the regulated expression of transcripts coding for the viral structural proteins. We used oligonucleotide-directed mutagenesis to generate a series of mutant HTLV-I rex genes. Transient expression experiments demonstrated that 3 of 28 mutant proteins are functionally inactive on the homologous HTLV-I rex response element, whereas an additional 2 mutant proteins are functionally inactive on the heterologous human immunodeficiency virus type 1 rev response element. One of these mutants is able to suppress the function of the wild-type HTLV-I Rex protein in trans on the homologous rex response element sequence. Furthermore, all of these mutants are able to inhibit Rex function on the heterologous rev response element sequence. Intriguingly, only three of these mutants are able to inhibit the human immunodeficiency virus type 1 Rev protein in a dominant-negative manner.

Amino Acid Sequence↗

PubMed1991

Functional mapping of the human immunodeficiency virus type 1 Rev RNA binding domain: new insights into the domain structure of Rev and Rex.

Expression of human immunodeficiency virus type 1 (HIV-1) structural proteins requires the direct interaction of the viral trans-activator protein Rev with its cis-acting RNA sequence (Rev-response element [RRE]). A stretch of 14 amino acid residues of the 116-amino-acid Rev protein is sufficient to impose nucleolar localization onto a heterologous protein. Our results demonstrated that these same amino acid residues confer Rev-specific RRE binding to the heterologous human T-cell leukemia virus type I Rex protein. In addition, our results indicated that amino acids distinct from the nuclear localization signal are important for Rex-specific RRE RNA binding.

Amino Acid Sequence↗

PubMed1991

Mutational analysis of the human T-cell leukemia virus type I trans-acting rex gene product.

Expression of the human T-cell leukemia virus type I (HTLV-I) rex gene is a prerequisite for the expression of the retroviral structural proteins. We have generated internal deletion mutants of this 27-kDa nucleolar trans-acting gene product to define functional domains in the Rex protein. The phenotype of the various mutant proteins was tested on the homologous HTLV-I rex response element sequence and the heterologous human immunodeficiency virus type 1 (HIV-1) rev response element sequence. Our results indicate that a region between amino acid residues 55 and 132 in the 189-amino-acid Rex protein is required for Rex-mediated trans activation on both retroviral response element sequences. In addition, substitution of the Rex nuclear localization signal by a sequence of the HIV-1 rev gene product targets the Rex protein to the correct subcellular compartment required for Rex function.

Amino Acid Sequence↗

PubMed1991

Functional analysis of human T-cell leukemia virus type I rex-response element: direct RNA binding of Rex protein correlates with in vivo activity.

The human T-cell leukemia virus type I rex gene product plays a critical role in the expression of the retroviral structural proteins Gag and Env from incompletely spliced mRNAs. Rex protein acts through a cis element (rex-response element [RxRE]) which is located in the U3/R region of the 3' long terminal repeat and is present on all human T-cell leukemia virus type I-specific mRNAs. Two domains of the predicted secondary structure of the RxRE are crucially important for Rex action in vivo as measured by two assay systems. In vitro studies using highly purified recombinant Rex protein revealed a specific and direct interaction with radiolabeled RxRE sequences. The correlation between our in vivo results and the direct binding of Rex protein to mutant and wild-type RxRE sequences supports both the existence of the predicted secondary structure and the importance of this direct interaction with the cis-acting RNA sequence for Rex function in vivo.

Base Sequence↗

PubMed1990

HIV-1 structural gene expression requires binding of the Rev trans-activator to its RNA target sequence.

Expression of human immunodeficiency virus type 1 structural proteins requires both the viral Rev trans-activator and its cis-acting RNA target sequence, the Rev response element (RRE). The RRE has been mapped to a conserved region of the HIV-1 env gene and is predicted to form a complex, highly stable RNA stem-loop structure. Site-directed mutagenesis was used to define a small subdomain of the RRE, termed stem-loop II, that is essential for biological activity. Gel retardation assays demonstrated that the Rev trans-activator is a sequence-specific RNA binding protein. The RRE stem-loop II subdomain was found to be both necessary and sufficient for the binding of Rev by the RRE. We propose that the HIV-1 Rev trans-activator belongs to a new class of sequence-specific RNA binding proteins characterized by the presence of an arginine-rich binding motif.

Animals↗

PubMed1989

Functional dissection of the HIV-1 Rev trans-activator--derivation of a trans-dominant repressor of Rev function.

Human immunodeficiency virus type 1 (HIV-1) encodes a nuclear trans-activator, termed Rev, that is required for the expression of the viral structural proteins and, hence, for viral replication. The Rev protein acts posttranscriptionally to induce the sequence-specific nuclear export of unspliced HIV-1 mRNA species that are otherwise excluded from the cell cytoplasm. We have used site-directed mutagenesis to identify two distinct regions of the HIV-1 Rev protein that are required for in vivo biological activity. The larger and more N-terminal of these two regions includes, but extends beyond, an arginine-rich sequence element required for nuclear localization. Mutation of a second, more C-terminal Rev protein sequence element was found to yield defective Rev proteins that act as trans-dominant inhibitors of Rev function. These Rev mutants are shown to inhibit HIV-1 replication when expressed in transfected cells and may have potential application in the treatment of HIV-1 related disease.

Amino Acid Sequence↗

PubMed1989

The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA.

Human immunodeficiency virus type 1 (HIV-1) replication requires the expression of two classes of viral mRNA. The early class of HIV-1 transcripts is fully spliced and encodes viral regulatory gene products. The functional expression of one of these nuclear regulatory proteins, termed Rev (formerly Art or Trs), induces the cytoplasmic expression of the incompletely spliced, late class of HIV-1 mRNAs that encode the viral structural proteins, including Gag and Env. Here, we provide evidence that this induction reflects the export from the cell nucleus to the cytoplasm of a pool of unspliced viral RNA constitutively expressed in the nucleus. The hypothesis that Rev acts on RNA transport, rather than splicing, is further supported by the observation that the cytoplasmic expression of a non-spliceable HIV-1 env gene sequence is also subject to Rev regulation. Here we show that this Rev response requires a specific target sequence which coincides with a complex RNA secondary structure present in the env gene. The response to Rev is fully maintained when this sequence is relocated to other exonic or intronic locations within env but is ablated by inversion. These results indicate that the HIV-1 rev gene product induces HIV-1 structural gene expression by activating the sequence-specific nuclear export of incompletely spliced HIV-1 RNA species.

Base Sequence↗

PubMed1989

Comparative analysis of the HTLV-I Rex and HIV-1 Rev trans-regulatory proteins and their RNA response elements.

The Rex proteins of types I and II human T-cell leukemia viruses (HTLV-I, HTLV-II) are required for expression of the viral structural gene products, gag and env and, thus, are essential for the replication of these pathogenic retroviruses. The action of Rex is sequence specific, requiring the presence of a cis-acting Rex response element located in the 3' long terminal repeat. This element corresponds to a predicted RNA secondary structure and functions in an orientation-dependent but position-independent manner. Rex acts through this response element to stimulate the nuclear export of the unspliced or singly spliced viral mRNA species encoding the virion structural proteins that are normally excluded from the cytoplasm. Although the Rex proteins of HTLV-I and HTLV-II can also function via the related Rev response element present in the env gene of the type I human immunodeficiency virus (HIV-1), the analogous HIV-1 Rev protein is unable to act on the HTLV-I Rex response element. This nonreciprocal pattern of genetic complementation by Rex and Rev suggests that these viral trans-regulators may interact directly with their RNA response elements.

Base Sequence↗

PubMed1989

Identification of a U5-specific sequence required for efficient polyadenylation within the human immunodeficiency virus long terminal repeat.

Retrovirus mRNAs are normally polyadenylated within the proviral 3' long terminal repeat (LTR). The site of retrovirus transcript polyadenylation is flanked 3' by an LTR-specific sequence termed the U5 region, but the role of U5 in the determination of polyadenylation efficiency has not been addressed. We have used site-directed mutagenesis of a human immunodeficiency virus LTR to map U5 sequences which are required for efficient polyadenylation within the LTR. These LTR U5 region sequences display homology to a motif termed the G-T cluster, which is known to facilitate the efficient polyadenylation of mRNAs encoded by several cellular and viral genes. These results suggest that the LTR U5 region functions in vivo to permit efficient polyadenylation within the proviral 3' LTR.

Base Sequence↗

PubMed1989

Functional analysis of the Tat trans activator of human immunodeficiency virus type 2.

The trans-activator (Tat) proteins of the related but distinct type 1 and type 2 human immunodeficiency viruses (HIV-1 and HIV-2) display incomplete functional reciprocity. One possible explanation of this observation, suggested by computer analysis of potential RNA secondary structures within the viral trans-activation response (TAR) elements, is that HIV-2 Tat requires the presentation of two viral RNA stem-loop sequences for full activity whereas HIV-1 Tat is maximally active upon presentation of a single stem-loop structure. Here, we demonstrate that the HIV-2 long terminal repeat indeed contains two functionally independent TAR elements. However, the second (3') TAR element of HIV-2 is significantly less active than the 5' TAR element and is functionally masked in the context of an intact HIV-2 long terminal repeat. Evidence is presented suggesting that the activities of these two HIV-2 TAR elements reflect, at least in part, their relative distances from the site of transcription initiation. Although the HIV-2 TAR element proximal to the viral mRNA cap site appears to be sufficient for effective trans activation by HIV-2 Tat in vitro, this functional redundancy may nevertheless serve to enhance HIV-2 replication in infected cells in vivo.

Animals↗

PubMed1989

Mutational analysis of the conserved basic domain of human immunodeficiency virus tat protein.

The tat trans-activators encoded by the known strains of primate immunodeficiency virus share a conserved, highly basic protein domain. Mutagenesis of this sequence in the tat gene of human immunodeficiency virus type 1 is shown here to reduce, but not eliminate, the trans-activation of human immunodeficiency virus type 1-specific gene expression. The degree of inhibition is shown to vary in a dose-dependent manner and is most marked at low levels of tat expression. Multiple mutations of the basic domain of tat were found to impair both the in vivo stability and the nuclear localization of the tat protein. It is proposed that this protein domain serves to efficiently target the tat gene product to its appropriate site or substrate within the nucleus of expressing cells.

Cell Compartmentation↗

PubMed1989

Functional characterization of a complex protein-DNA-binding domain located within the human immunodeficiency virus type 1 long terminal repeat leader region.

Transcriptional trans activation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) by the viral tat trans activator is mediated by an LTR-specific sequence located immediately 3' to the start of transcription initiation. We have used a range of molecular techniques to examine DNA-protein interactions that occur in the vicinity of this cis-acting sequence. Our results demonstrate the existence of a sequence-specific DNA-protein interaction involving the HIV-1 leader DNA and map this binding event to between -2 and +21 base pairs relative to the HIV-1 LTR transcription start site. Evidence suggesting that this interaction involves three distinct protein-DNA contact sites extending along one side of the DNA helix is presented. Mutation of these sites was found to ablate protein-DNA binding yet was observed to have no effect on either the basal or tat trans-activated level of HIV-1 LTR-specific gene expression. We therefore conclude that this DNA-protein interaction has a function distinct from the regulation of HIV-1 LTR-specific gene expression.

Base Sequence↗