Tat mRNA Research Articles

Analysis of the transcription pattern and mapping of the putative rev and env splice junctions of bovine immunodeficiency-like virus.

The bovine immunodeficiency-like virus (BIV) genome contains the obligatory structural genes of all retroviruses and, in addition, the complex central region of lentiviruses; this novel region may code for at least five nonstructural/regulatory genes in BIV (K.J.Garvey, M.S. Oberste, J.E. Elser, M.J. Braun, and M.A. Gonda, Virology 175:391-409, 1990). As a prelude to determining the function of these novel open reading frames, the transcriptional pattern of BIV was studied by Northern analysis of RNA from BIV-infected cells. Five size classes of BIV-specific RNAs of 8.5, 4.1, 3.8, 1.7, and 1.4 kb were detected. The 8.5-kb RNA contains sequences from all regions of the genome; it is the virion RNA and probably serves as the gag-pol transcript as well. By using gene-specific probes, subgenomic viral RNAs of 3.8, 1.7, and 1.4 kb were tentatively identified as the env, tat, and rev spliced messages, respectively. The 4.1-kb RNA could not be unambiguously identified but may encode vif. The hybridization patterns of the putative tat and rev mRNAs suggest that they are the products of multiple splicing events. Discrete transcripts for the BIV W and Y central region open reading frames were not defined. The characterization of partial cDNA clones has permitted the mapping of the env and putative rev splice junctions.

Trial of Antisense RNA Inhibition of HIV Replication and Gene Expression

We examined the feasibility of antisense RNA inhibition of human immunodeficiency virus (HIV) replication. In the first experiment, we established CD4+ T-cell lines constitutively expressing various antisense HIV sequences using the retrovirus-mediated gene transfer technique. These cell lines were tested for their ability to withstand HIV de novo infection. In this challenge assay, however, we could not detect any significant difference in the survival rate between these genetically engineered cell lines and control T cells. In the second approach, the effects of antisense sequences on Tat expression were studied by monitoring the activities of reporter enzymes. A functional Tat expression vector and the antisense sequence expression vector were co-introduced into HeLa cells stably transfected with either the HIV-long terminal repeat (LTR) directed chloramphenicol acetyltransferase (CAT) or luciferase. Although the concentration of the antisense RNA was at least 10-fold higher than that of the sense Tat mRNA in cells, these antisense sequences could not inhibit transactivation of HIV-LTR. Regulation of HIV gene expression has proven to be very complicated and Tat transactivation of the HIV-LTR is extraordinarily strong. Consequently, it may be difficult to block HIV replication by the antisense strategy.

Simian immunodeficiency virus (SIVmac) exhibits complex splicing for tat, rev, and env mRNA

The simian immunodeficiency virus (SIV) is a T-lymphotropic lentivirus associated with a fatal AIDS-like disease in rhesus macaques. SIV has a complex genome encoding virion structural proteins, transactivators, and accessory genes. From lymphoid cells chronically infected with a biologically active molecular clone of SIV, SIVmac1A11, the polymerase chain reaction technique has been used to selectively amplify transcripts for viral transactivators and the envelope gene. Three species of mRNA encoding only rev, and three mRNA encoding both rev and tat were identified by nucleotide sequence analysis. They differed in the splice acceptor sites utilized upstream of the first coding exon, in the presence or the absence of noncoding exons between the major splice donor at the LTR and the splice acceptor at the first coding exons, and in the splicing pattern between the coding exons. Alternate splice acceptors were utilized between the coding exons of tat and rev, but the altered tat proteins did not differ in their ability to transactivate the SIV-LTR. The splicing for env mRNA is more complex than previously reported. Both singly and multiply spliced transcripts exist for env mRNA, and the same splice acceptor site is utilized by both rev and env mRNA.

Feedback regulation of human immunodeficiency virus type 1 expression by the Rev protein

Rev is an essential regulatory protein of the human immunodeficiency virus type 1 (HIV-1) that affects the transport and half-life of certain viral mRNAs. Rev exerts its function via a unique element, the Rev-responsive element (RRE), located within the env region of HIV-1. It has been previously demonstrated that Rev affects the relative levels of RRE-containing and RRE-lacking mRNAs. We have studied the effects of Rev on the expression of the three different groups of small, multiply spliced mRNAs that lack the RRE sequence and encode the regulatory proteins Tat, Rev, and Nef. To monitor the tat, rev, and nef mRNAs we generated specific S1 nuclease mapping probes that distinguish among them. Analysis of all the mRNA species producing Tat, Rev, and Nef revealed that their levels are coordinately regulated by Rev. They are increased in the absence of Rev protein and are down regulated in the presence of Rev. The corresponding proteins were measured by immunoprecipitations, and their levels are in agreement with the RNA levels. These results verify the model proposing that Rev is a general regulator indirectly affecting all the multiply spliced mRNAs to a similar extent. Therefore, Rev down regulates its own expression and the expression of Tat and Nef.

Cloning and functional analysis of multiply spliced mRNA species of human immunodeficiency virus type 1

We have used the polymerase chain reaction technique to clone the small multiply spliced mRNA species produced after infection of human cells by a molecular clone of human immunodeficiency virus type 1 (HIV-1). We identified six Rev-expressing mRNAs, which were generated by the use of two splice acceptors located immediately upstream of the rev AUG. The class of small mRNAs included 12 mRNAs expressing Tat, Rev, and Nef. In addition, HIV-1 produced other multiply spliced mRNAs that used alternative splice sites identified by cloning and sequencing. All of these mRNAs were found in the cytoplasm and should be able to produce additional proteins. The coding capacity of the tat, rev, and nef mRNAs was analyzed by transfection of the cloned cDNAs into human cells. The tat mRNAs produced high levels of Tat, but very low levels of Rev and Nef. All the rev mRNAs expressed high levels of both Rev and Nef and were essential for the production of sufficient amounts of Rev. Therefore, HIV-1 uses both alternatively spliced and bicistronic mRNAs for the production of Tat, Rev, and Nef proteins.

Equine infectious anemia virus tat: insights into the structure, function, and evolution of lentivirus trans-activator proteins

Equine infectious anemia virus (EIAV) contains a tat gene which is closely related to the trans-activator genes of the human and simian immunodeficiency viruses. Nucleotide sequence analysis of EIAV cDNA clones revealed that the tat mRNA is composed of three exons; the first two encode Tat and the third may encode a Rev protein. Interestingly, EIAV Tat translation is initiated at a non-AUG codon in exon 1 of the mRNA, perhaps allowing an additional level of gene regulation. The deduced amino acid sequence of EIAV tat, combined with functional analyses of tat cDNAs in transfected cells, has provided some unique insights into the domain structure of Tat. EIAV Tat has a C-terminal basic domain and a highly conserved 16-amino-acid core domain, but not the cysteine-rich region, that are present in the primate immunodeficiency virus Tat proteins. Thus, EIAV encodes a relatively simple version of this kind of trans activator.

Chemical synthesis and expression of a gene encoding HIV-1 TAT protein

A gene encoding the human immunodeficiency virus-1 (HIV-1) TAT protein was chemically synthesized and expressed in HeLa cells and in a cell-free system. To facilitate both the assembly of the synthetic gene and further mutagenesis and gene fusion studies, several unique restriction endonuclease cleavage sites were included in the coding sequence without altering the encoded protein sequence. The synthetic TAT coding sequence was fused to a translation start signal and placed under SV40 early transcriptional control. Co-transfection of the TAT-encoding synthetic gene together with a reporter gene (chloramphenical acetyl transferase or beta-galactosidase) linked to an HIV LTR confirmed that the synthetic gene product exhibits similar activity to TAT expressed from HIV genomic DNA in the transactivation of the LTR. TAT mRNA prepared by cell-free transcription of the synthetic TAT coding sequence was also shown to produce functional TAT following microinjection into HeLa-derived cells containing an integrated reporter gene with the HIV LTR linked to beta-galactosidase.

Deletions near the albino locus on chromosome 7 of the mouse affect the level of tyrosine aminotransferase mRNA.

Overlapping chromosomal deletions at the albino locus on chromosome 7 of the mouse affect the expression of several liver enzymes, including tyrosine aminotransferase (TAT; L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5). With cloned TAT DNA the integrity of the TAT structural gene and its expression and inducibility by glucocorticoids and cAMP were examined in deletion homozygous mice. No difference in the structure of the gene between normal and mutant mice was detected by Southern blotting. Severely reduced amounts of TAT mRNA were detected in homozygous mutants. The residual mRNA levels could not be modulated by glucocorticoids or cAMP. We conclude that a trans-acting control function required for expression and inducibility of mouse TAT can be assigned to the chromosomal region near the albino locus.

Isolation and characterization of the rat tyrosine aminotransferase gene.

Tyrosine aminotransferase (TAT; L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) from rat liver is subject to glucocorticoid, cAMP, and developmental control. To study the underlying regulatory mechanisms, the TAT structural gene was isolated from a lambda bacteriophage rat DNA library. Heteroduplex analysis revealed that the 2.4-kilobase-long TAT mRNA is encoded by a gene that extends over 11 kilobases and is interrupted by 11 introns. To characterize the presumptive control region, the DNA sequence around the 5' end of the gene was determined and the start site of transcription was identified by nuclease S1 protection experiments. A short sequence homology in an equivalent position relative to the cap site was detected between TAT and tryptophan oxygenase, another glucocorticoid-controlled gene from rat liver. This sequence is related to the sequence 5' T-G-T-T-C-T 3' found in regions of the long terminal repeat of mouse mammary tumor virus, which has been shown to interact with the glucocorticoid receptor [Scheidereit, C., Geisse, J., Westphal, H. M. & Beato, M. (1983) Nature (London) 304, 749-752].

A post-transcriptional site of induction of tyrosine aminotransferase by dexamethasone in reuber H35 hepatoma cells

L-Tyrosine:2_oxoglutarate aminotransferase (EC 2.6.1 S, TAT) activity can be induced by adenosine 315kyclic AMP analogs [l-4] and corticosteroids [2,5,6] in rat liver and several hepatoma cell lines. The mechanism of the induction of TAT by DBcAMP has been extensively investigated [l-4]. Actinomycin D does not inhibit the induction of TAT activity by DBcAMP which indicates a target for DBcAMP at a post-transcriptional level [ 1,4]. We concluded that DBcAMP induces the TAT synthesis by an increase in the rate of initiation on the TAT mRNA [7]. However, under other experimental conditions evidence has been obtained that the elongation rate of the ribosomes along the TAT mRNA increased after DBcAMP induction [8]. Also, translational experimentsin several cell free systems with mRNA isolated from DBcAMPtreated rat liver indicate an increase in the amount of functional TAT mRNA [9,10]. It seems that dependent on the experimental conditions DBcAMP exerts a pleiotropic effect on TAT synthesis at different levels during protein synthesis. Here, we report a hitherto undescribed effect of DBcAMP on TAT synthesis in Reuber H35 hepatoma cells. The induction of TAT synthesis by dexamethasone is completely inhibited by actinomycin D. However, after preinduction with DBcAMP, simultaneous addition of actinomycin D and dexamethasone results in a dexamethasone-dependent increase in TAT synthesis. Apparently, DBcAMP causes a modulation of a regulatory site for TAT synthesis at a post-transcriptional level which is sensitive for dexamethasone in this modulated form.

Glucocorticoid requirement for tyrosine aminotransferase induction by adenosine 3′:5′-cyclic phosphate analogs in somatic cell hybrids

The ability of adenosine 3′:5′-cyclic phosphate (cyclic AMP) analogs to induce l-tyrosine:2-oxoglutarate aminotransferase (EC 2.6.1.5; TAT) in a rat hepatoma (H35)-rat liver cell (BRL) hybrid (BF5) and a subclone which has lost 29 chromosomes (BF5-1-1) has been analyzed. Cyclic AMP analogs alone were unable to increase TAT activity in either hybrid cell line or in the “normal” liver cells despite three- to fivefold induction of this enzyme in the hepatoma parental cells. In contrast, dexamethasone by itself reproducibly increased TAT activity both in BF5-1-1 cells and in the parental H35 hepatoma cells. Pretreatment of the hybrid cells with dexamethasone revealed a synergistic increase in TAT activity when a cyclic AMP analog was added. From studies of the thermal stability and immunological inhibition of TAT activity, it is concluded that the low basal activity in BRL, BF5, and BF5-1-1 cells represents tyrosine transamination catalyzed by a different aminotransferase, whereas all the induced activity does represent bona fide TAT. The results suggest that functional TAT mRNA may not be present in significant quantities in the hybrid cells in the absence of adrenal steroids and that this could account for the inability of cyclic AMP analogs to exert their presumably translational effect on TAT synthesis.