Splicing variability in HIV type 1 revealed by quantitative RNA polymerase chain reaction.

A quantitative RNA-polymerase chain reaction (PCR) method able to detect the majority of mRNAs produced by human immunodeficiency virus type 1 (HIV-1) was developed and used to study expression of different HIV-1 clones in human cells. Amplified mRNAs were compared to known cDNA standards. This comparison permitted the optimization of PCR conditions and eliminated the generation of artifactual PCR bands.

Elements distinct from human immunodeficiency virus type 1 splice sites are responsible for the Rev dependence of env mRNA.

In the absence of the viral regulatory protein Rev, the human immunodeficiency virus type 1 gag/pol and env mRNAs are inefficiently expressed, since nucleocytoplasmic transport, stability, and polysomal loading are impaired. It has been suggested that splicing is necessary for Rev function and that the low expression of the unspliced and intermediate spliced mRNAs in the absence of Rev is associated with specific splice sites. Previous studies identified distinct RNA elements within the gag/pol region responsible for low expression that are not associated with splice sites.

Identification of in vivo phosphorylation sites of SET, a nuclear phosphoprotein encoded by the translocation breakpoint in acute undifferentiated leukemia.

SET, the translocation breakpoint-encoded protein in acute undifferentiated leukemia (AUL), is identified as a 39-kDa phosphoprotein found predominantly in the cell nuclei [1994, J. Biol. Chem.

Identification of the caprine arthritis encephalitis virus Rev protein and its cis-acting Rev-responsive element.

Caprine Arthritis Encephalitis Virus (CAEV) is a lentivirus closely related to visna virus of sheep and more distantly related to the human lentivirus HIV-1. The genomes of lentiviruses contain additional genes that regulate the lentivirus gene expression; one of these is Rev, a protein that regulates the expression of viral proteins via post-transcriptional mechanisms. A cDNA clone was isolated from CAEV infected cells and shown to encode the 18-kDa Rev protein of CAEV.

Identification and characterization of SET, a nuclear phosphoprotein encoded by the translocation break point in acute undifferentiated leukemia.

The translocation (6;9) in acute nonlymphocytic leukemia results in the formation of a dek-can fusion gene. In a case of acute undifferentiated leukemia, the oncogene can is fused to a different gene, named set, instead of dek and is assumed to be activated. Transcripts of set encode a putative SET protein with a predicted molecular mass of 32 kDa.

Inhibition of Rev-mediated HIV-1 expression by an RNA binding protein encoded by the interferon-inducible 9-27 gene.

Interferon inhibits expression of human immunodeficiency virus type-1 (HIV-1) through unknown mechanisms. A gene inducible by interferon-alpha (IFN-alpha) and interferon-gamma (IFN-gamma) was isolated by screening of a human complementary DNA library for proteins binding to the Rev-responsive element (RRE) of HIV-1. The product of this gene, RBP9-27, was shown to bind RNA in vitro and to inhibit HIV-1 expression after transfection into human cells.

Mutational inactivation of an inhibitory sequence in human immunodeficiency virus type 1 results in Rev-independent gag expression.

We have characterized an inhibitory RNA element in the human immunodeficiency virus type 1 (HIV-1) gag coding sequence that prevents gag expression. The inhibition exerted by this element could be overcome by the presence of the Rev-responsive element in cis and of Rev protein in trans. To understand the mechanism of function, we inactivated the inhibitory element by mutagenesis while maintaining an intact gag coding region.

The Rev protein of human immunodeficiency virus type 1 promotes polysomal association and translation of gag/pol and vpu/env mRNAs.

Biochemical examination of the Rev-dependent expression of gag mRNAs produced from gag-Rev-responsive element (RRE) expression plasmids showed a large discrepancy between the level of cytoplasmic gag mRNA and the produced Gag protein. Significant levels of the mRNA produced in the absence of Rev were localized in the cytoplasm, while very low levels of Gag protein were produced. In the presence of Rev, the levels of mRNA increased by 4- to 16-fold, while the Gag protein production increased by 800-fold.

Complex splicing in the human T-cell leukemia virus (HTLV) family of retroviruses: novel mRNAs and proteins produced by HTLV type I.

Novel cytoplasmic mRNA species produced by human T-cell leukemia virus type I (HTLV-I) were cloned by using the polymerase chain reaction technique. Five novel 3' splice sites located in the X region and upstream of the env gene were identified. Splicing to the 3' splice sites in the X region generates mRNAs that express two previously unidentified viral proteins, named Rof and Tof.

Sulfonic acid dyes: inhibition of the human immunodeficiency virus and mechanism of action.

Over 50 different commercially available sulfonic acid-containing dyes were analyzed for their ability to prevent HIV-1-induced cell killing and in inhibiting HIV-1 replication. Compounds of remarkably similar structure, but with differing patterns of sulfonic acid group substitutions, had a wide range of potency in inhibiting HIV-1. Chicago sky blue (CSB) was highly effective in the inhibition of HIV-1 with less toxicity to CEM-SS cells than most of the other sulfonated dyes tested.

Mechanism of translation of monocistronic and multicistronic human immunodeficiency virus type 1 mRNAs.

We have used a panel of cDNA clones expressing wild-type and mutant human immunodeficiency virus type 1 (HIV-1) mRNAs to study translation of these mRNAs in eucaryotic cells. The tat open reading frame (ORF) has a strong signal for translation initiation, while rev and vpu ORFs have weaker signals. The expression of downstream ORFs is inhibited in mRNAs that contain the tat ORF as the first ORF.

Distinct RNA sequences in the gag region of human immunodeficiency virus type 1 decrease RNA stability and inhibit expression in the absence of Rev protein.

The expression of Gag, Pol, Vif, Vpr, Vpu, and Env proteins from unspliced and partially spliced human immunodeficiency virus type 1 (HIV-1) mRNAs depends on the viral protein Rev, while the production of Tat, Rev, and Nef from multiply spliced mRNAs does not require Rev. To investigate the difference between gag and tat mRNAs, we generated plasmids expressing tat-gag hybrid mRNAs. Insertion of the gag gene downstream of the tat open reading frame in the tat cDNA resulted in the inhibition of Tat production.

The Rex regulatory protein of human T-cell lymphotropic virus type I binds specifically to its target site within the viral RNA.

The Rex protein of human T-cell leukemia virus type I (HTLV-I) was expressed in bacteria and partially purified. Rex was shown to bind in vitro specifically to an RNA sequence located in the 3' long terminal repeat of HTLV-I, named Rex-responsive element (RXRE). Rex also bound in vitro to the human immunodeficiency virus type 1 (HIV-1) Rev-responsive element (RRE), while purified HIV-1 Rev protein did not bind to the RXRE.

Expression of human immunodeficiency virus type 1 vif and vpr mRNAs is Rev-dependent and regulated by splicing.

We have analyzed the structure and expression of the HIV-1 vif and vpr mRNAs. The results revealed that the predominant vif and vpr mRNAs belong to the intermediate size class of HIV-1 mRNAs and that their expression is dependent on the presence of Rev protein. In addition, low levels of a small multiply spliced vpr mRNA were produced by HIV-1.

Rapid activation and subsequent down-regulation of the human immunodeficiency virus type 1 promoter in the presence of Tat: possible mechanisms contributing to latency.

The mechanism of induction of gene expression of the human immunodeficiency virus type 1 long terminal repeat (LTR) by the Tat transactivator protein was studied in a cell fusion assay. Tat causes a rapid activation of both transcription from the LTR and accumulation of hybrid LTR-chloramphenicol acetyltransferase mRNAs. Approximately 4 h after induction by Tat, expression from the LTR promoter is down-regulated, resulting in a decrease in the accumulation of LTR mRNA.

Different sites of interaction for Rev, Tev, and Rex proteins within the Rev-responsive element of human immunodeficiency virus type 1.

We have analyzed the action of the Rev and Tev proteins of human immunodeficiency virus type 1 (HIV-1) and of the Rex protein of human T-cell leukemia virus type I (HTLV-I) on a series of Rev-responsive element (RRE) mutants. The minimum continuous RRE region necessary and sufficient for Rev function was determined to be 204 nucleotides. Interestingly, this region was not sufficient for Tev or Rex function.

Binding of trans-dominant mutant Rev protein of human immunodeficiency virus type 1 to the cis-acting Rev-responsive element does not affect the fate of viral mRNA.

The binding of Rev protein of human immunodeficiency virus type 1 (HIV-1) to the cis-acting Rev-responsive element (RRE) was compared to the binding of a trans-dominant Rev mutant. RevBL, which inhibits Rev function. Rev and RevBL expressed in bacteria were purified and shown to bind in vitro to the RRE with similar affinities.

Env and Vpu proteins of human immunodeficiency virus type 1 are produced from multiple bicistronic mRNAs.

Three size classes of human immunodeficiency virus type 1 (HIV-1) mRNAs are produced in infected cells: full-length, intermediate, and small. Here we report that the intermediate-size class of viral mRNAs is heterogeneous, consisting of at least 12 differentially spliced species. This group contains nine bicistronic mRNAs producing Env and Vpu and three mRNAs expressing only the first exon of tat.

A bioassay for HIV-1 based on Env-CD4 interaction.

The binding of human immunodeficiency virus type 1 (HIV-1) gp120env to CD4 is the first event leading to infection and represents an important target for possible therapeutic intervention. To provide a tool for screening and quantitation of the effects of drugs inhibiting the Env-CD4 interaction, we developed a simple, fast and quantitative bioassay measuring the fusion between two cell lines generated by stable transfection: one expressing high levels of HIV-1 proteins but no infectious virus (HL2/3), and the other expressing the CD4 receptor and containing an inducible chloramphenicol acetyltransferase (CAT) gene linked to the HIV-1 long terminal repeat (HLCD4-CAT). Upon cocultivation of HL2/3 and HLCD4-CAT cells, efficient cell fusion is observed within 8 h.

Conservative mutations in the putative metal-binding region of human immunodeficiency virus tat disrupt virus replication.

The tat trans-activator proteins of the primate immunodeficiency viruses contain a highly conserved cysteine-rich domain. In human immunodeficiency virus type 1 tat there are seven cysteines located between residues 22 and 37 that are thought to form a metal-nucleic acid-binding structure. Most of the previous mutagenesis studies had demonstrated that these residues are essential for tat activity and virus expression.

NIH conference. Antiretroviral therapy in AIDS.

Objective: To review recent developments of antiretroviral therapy for the acquired immunodeficiency syndrome (AIDS) and related disorders.

Data Sources, Study Selection, And Data Extraction: An edited and updated summary of a Clinical Staff Conference held 26 October 1988 at the National Institutes of Health. The speakers discussed their own work as well as related work from other groups.