Biomedical subjects

G Chinnadurai

Publications and source records attributed to G Chinnadurai.

At least 73 records · Page 4Linked to original sources

PubMed1992

Synergistic activation of the human immunodeficiency virus type 1 promoter by the viral Tat protein and cellular transcription factor Sp1.

We have previously shown that the human immunodeficiency virus type 1 (HIV-1) Tat protein can activate a synthetic promoter containing consensus-binding sites for the cellular transcription factor Sp1. In this report, we show that a GAL-Tat fusion protein targeted via GAL4 DNA-binding sites can also trans activate an HIV-1 LTR promoter independently of the trans-activation response region. To show that the trans activation of the promoter by Tat directly involves the Sp1 protein, we have targeted a GAL-Sp1 fusion protein to the long terminal repeat promoter via upstream GAL4-binding sites. In the presence of Tat and GAL-Sp1, the promoter is synergistically trans activated at the transcriptional level, indicating that Tat and Sp1 functionally interact to trans activate the HIV-1 promoter. The Sp1 synergism is relatively specific, since another chimeric transcriptional activator, GAL-VP16, does not appear to be significantly synergistic with Tat.

Base Sequence↗

PubMed1992

Adenovirus E1a as a tumor-suppressor gene.

The E1a gene of group C adenoviruses is one of the most studied transforming genes of DNA tumor viruses. These transforming genes have been conventionally considered as dominant oncogenes since they transform cells in vitro and many of the resulting transformed cell lines induce tumors in experimental animals. It now appears that, in addition to its well-known transforming activities, E1a possesses activities which suppress transformation, tumorigenesis and malignant progression (metastasis) of tumor cells. Thus, E1a appears to meet the definition of both a dominant oncogene and a tumor-suppressor gene.

Adenovirus Early Proteins↗

PubMed1991

Sp1-dependent activation of a synthetic promoter by human immunodeficiency virus type 1 Tat protein.

The Tat protein coded by human immunodeficiency virus (HIV) is a strong activator of viral gene expression from the long terminal repeat (LTR). It appears that Tat-mediated trans-activation of the HIV LTR is predominantly a transcriptional event. It has been reported that Tat acts at the level of both transcriptional initiation and elongation through interaction with a nascent RNA target sequence termed TAR (for trans-activation response element). However, the precise mechanism(s) by which Tat mediates TAR-dependent transcriptional activity is not known. To determine whether Tat functions similarly to other eukaryotic transcriptional activators through any of the conventional promoter elements, we tested Tat activity on synthetic promoters containing consensus sequences required for binding transcription factor Sp1 and a TATA box. Here, we report that a chimeric Tat protein targeted to the promoter region by the DNA-binding domain of yeast transcription factor GAL4 activates the synthetic promoter. Because this trans-activation depends on Sp1-binding sites, Tat can apparently mediate transcriptional activation through its interaction with Sp1. Mutational analysis of the gal4-tat chimeric gene reveals that the N-terminal 48-amino acid region of Tat constitutes the activation region for Sp1-dependent trans-activation. This region of Tat exhibits substantially more activity than the N-terminal 58 amino acids of Tat, which includes the arginine-rich basic region. Effects of specific mutations in the 48-amino acid Tat region of GAL4-Tat on trans-activation of the synthetic promoter mimic the effects of these specific mutations on Tat-mediated trans-activation of the HIV-1 LTR, suggesting that trans-activation of both the synthetic promoter and the intact LTR occurs by a common mechanism.

Base Sequence↗

PubMed1991

Distinct cis-acting regions in U3 regulate trans-activation of the human spumaretrovirus long terminal repeat by the viral bel1 gene product.

The human spumaretrovirus (HSRV) genome contains, in addition to coding information for the structural proteins, open reading frames (ORFs) for at least three additional genes termed bel1, bel2 and bel3. We report here the localization of the transcriptional activator of HSRV to the bel1 ORF. In reporter-based transient expression assays in COS cells utilizing the bacterial CAT gene linked to HSRV LTR sequences between -710 and +309 with respect to the transcriptional initiation site, co-expression of the bel1 gene product alone caused an over 100 to 300-fold increase in the level of LTR activity. High-level trans-activation by bel1 was specific for the HSRV LTR, as relatively minor positive and negative regulatory effects were observed on HIV-1 LTR and RSV LTR expression, respectively, whereas HTLV-1 LTR activity remained unaffected. Distinct regions of the HSRV LTR were found to be involved in bel1-induced trans-activation. Specifically, deletions between -500 and -389 and between -136 and -62 in the U3 region resulted in a 4- and 30 to 35-fold decline, respectively, in the response to bel1. Limited mutagenesis of the bel1 ORF indicated that most of the bel1 coding region, except for the carboxy-terminal 27 residues, is essential for the activation function.

Amino Acid Sequence↗

PubMed1991

Modulation of HIV-enhancer activity by heterologous agents: a minireview.

The enhancer region of the human immunodeficiency virus (HIV) long terminal repeat (LTR) contains two 10-bp (5'-GGGACTTTCC) repeats (core enhancers) which constitute the binding sites for the ubiquitous inducible cellular transcription factor, NF-kappa B. The NF-kappa B motifs of the LTR play a central role in transcriptional activation of the LTR by several heterologous viral proteins and various external chemical and physical stimuli. Activation of the HIV enhancer by these agents may lead to the onset of HIV gene expression resulting in active viral replication. Viral genes and chemical agents, which interfere with the activity of the enhancers may be useful in inhibiting HIV gene expression, thereby suppressing HIV replication.

Base Sequence↗

PubMed1991

Heterologous basic domain substitutions in the HIV-1 Tat protein reveal an arginine-rich motif required for transactivation.

The Tat protein coded by HIV-1 is a unique eukaryotic transactivator. It activates gene expression from the viral LTR by its interaction with a nascent RNA element (TAR) located at the 5' end of all HIV-1 transcripts. Tat appears to bind to its target RNA structure in a highly sequence-specific manner. The TAR-binding activity of Tat has been localized in an Arg-rich basic domain located between residues 49 and 57 of the Tat protein. We have carried out domain substitution studies with heterologous basic domains which are also implicated in RNA binding. Here, we report that a 19 or a 12 amino acid region from the N-terminus of HTLV-I Rex can functionally substitute for the Tat basic domain. In contrast, the Arg-rich domains of the N gene products of bacteriophages lambda and 21 do not functionally substitute for the Tat basic domain. The positive and negative effects of various domain substitution mutants have facilitated identification of a consensus sequence (Arg/Lys-X-X-Arg-Arg-X-Arg-Arg) in the basic domain required for Tat activity. Conversion of the functionally inactive basic domain of the lambda N protein to the consensus motif restored the transactivation function of the Tat-N chimeric protein. Similarly, the Rex basic domain containing scrambled sequences unrelated or partially related to the consensus motif were either totally defective in transactivation or exhibited reduced activity. Our results further suggest that the activity of the core Arg motif may be enhanced by the presence of Gln or Asn within the basic domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1991

Requirement of the C-terminal region of adenovirus E1a for cell transformation in cooperation with E1b.

We have previously reported that adenovirus E1a mutants lacking the C-terminal 61 or 67 amino acids were severely defective in immortalization, but cooperated more efficiently (than wt E1a) with activated T24 ras oncogene in transformation of primary rat kidney (BRK) cells (Subramanian et al., 1989; Oncogene, 4:415-420). Here, we show that in contrast to these previous results, transformation of BRK cells in cooperation with the Ad2 E1b region is dependent on the C-terminal region of E1a. Mutational analysis of the C-terminal region has revealed that a region located between residues 266 and 276 may be important for E1a/E1b cooperative transformation. Like E1a/T24 ras cooperative transformation, E1a/E1b cooperative transformation also requires two essential domains involved in the binding of two cellular proteins (300K and 105K) within the N-terminal half of E1a. E1a/E1b cooperative transformation therefore requires an additional E1a activity encoded within the C-terminal region of E1a.

Adenoviridae↗

PubMed1990

Functional domains of the HIV-1 rev gene required for trans-regulation and subcellular localization.

The rev gene of human immunodeficiency virus type 1 (HIV-1) encodes a 116 amino acid nuclear regulatory protein (Rev) that increases the cytoplasmic expression of viral mRNAs containing the Rev response element (RRE) and coding for the structural proteins, Gag and Env. To identify the functional domains of Rev, amino acid deletion and chain termination mutations were introduced in the Rev coding region. The ability of these mutants to increase the cytoplasmic expression of a Rev-test plasmid (pSV-AR), containing the RRE cloned into the 3' noncoding region of the CAT gene in plasmid pSV2CAT, was examined in transient expression assays in HeLa cells. Our results indicate that three distinct regions mapping within the N-terminal 98 amino acids of Rev are essential for its activity. The subcellular localization of the various Rev proteins was examined in COS cells by indirect immunofluorescence. Rev was found to localize predominantly in the nucleolus of transfected cells. All mutant Rev proteins, with the exception of a deletion mutant (rev delta 41-44) lacking four Arg residues of a highly basic domain, were found to localize in the nucleolus. Mutant rev delta 41-44 exhibited weak diffuse fluorescence in the nucleus with a tendency to accumulate in the cytoplasm. A 15 amino acid region encompassing this basic domain (38-52) when fused to the Escherichia coli beta-galactosidase gene efficiently directed the fusion gene product to the nucleus and nucleolus, suggesting a role for this domain in the nucleolar localization of Rev.

Amino Acid Sequence↗

PubMed1990

Functional substitution of the basic domain of the HIV-1 trans-activator, Tat, with the basic domain of the functionally heterologous Rev.

The tat gene of HIV is a strong activator of the viral LTR. The Tat protein contains a highly basic domain that is important for its transport to the nuclear/nucleolar locations. The Tat basic domain when fused to Escherichia coli beta-galactosidase directed the chimeric protein to the nucleus and nucleolus. Tat mutants lacking the entire basic domain were severely defective in trans-activation. Substitution of the basic domain of Tat with that of the functionally unrelated HIV-1 Rev protein targeted the chimeric protein to the nucleolus and restored the function of Tat. In contrast, substitution with the nuclear targeting signal (NLS) of SV40 T antigen targeted the chimeric protein to the nucleus and accumulation in the nucleolar region was excluded. The Tat-NLS chimeric protein did not restore the trans-activation function of Tat efficiently. These results indicate that the arginine-rich basic domain of the trans-activator, Tat, and post-transcriptional trans-regulator, Rev, are functionally similar with regard to trans-activation of HIV-1 LTR.

Amino Acid Sequence↗

PubMed1990

Mutants in a conserved region near the carboxy-terminus of HIV-1 Rev identify functionally important residues and exhibit a dominant negative phenotype.

The rev protein (Rev) of human immunodeficiency virus increases the cytoplasmic expression of viral structural gene mRNAs. We had previously reported the existence of a region (residues 73-98) near the carboxy-terminus in HIV-1 Rev essential for its function. To further define the structural elements in this region, we examined the effects of substitution mutations in highly conserved residues in this region, between amino acids 75-81, on Rev function. Mutations in Pro76-77 and Arg80 retained Rev function, whereas those in Leu75 and Leu81 abolished Rev activity and exhibited trans-dominant suppression of wt Rev function. The Leu81 mutation, in particular, exhibited an efficient dominant negative phenotype. Leu75 and Leu81 thus appear to define residues essential to the Rev "effector" function.

Amino Acid Sequence↗

PubMed1990

Selective induction of toxicity to human cells expressing human immunodeficiency virus type 1 Tat by a conditionally cytotoxic adenovirus vector.

The human immunodeficiency viruses (HIVs) primarily infect CD4+ T lymphocytes, leading eventually to the development of a systemic immune dysfunction termed acquired immunodeficiency syndrome (AIDS). An attractive strategy to combat HIV-mediated pathogenesis would be to eliminate the initial pool of infected cells and thus prevent disease progression. We have engineered a replication-defective, conditionally cytotoxic adenovirus vector, Ad-tk, whose action is dependent on the targeted expression of the herpes simplex virus type 1 thymidine kinase gene (tk), cloned downstream of the HIV-1 long terminal repeat, in human cells expressing the HIV-1 transcriptional activator Tat. Infection of Tat-expressing human HeLa or Jurkat cells with Ad-tk resulted in high-level tk expression, which was not deleterious to the viability of these cells. However, in the presence of the antiherpetic nucleoside analog ganciclovir, Ad-tk infection resulted in a massive reduction in the viability of these Tat-expressing cell lines. As adenoviruses are natural passengers of the human lymphoid system, our results suggest adenovirus vector-based strategies for the targeted expression, under the control of cis-responsive HIV regulatory elements, of cytotoxic agents in HIV-infected cells for the therapy of HIV-mediated pathogenesis.

Adenoviruses, Human↗

PubMed1990

Silencing of human immunodeficiency virus long terminal repeat expression by an adenovirus E1a mutant.

Gene expression from the human immunodeficiency virus (HIV) long terminal repeat (LTR) is strongly stimulated by the viral tat gene. The HIV LTR is also activated by several physical and chemical agents and heterologous viral genes, including adenovirus E1a. As E1a has separable transcriptional activation and repression functions, we examined the negative regulatory effects of E1a on the expression of the HIV LTR by using a trans-dominant E1a mutant. Mutant hr5 strongly suppressed the basal activity of the LTR as well as trans-activation of the LTR by heterologous agents such as the cytomegalovirus immediate early gene or DNA-damaging agents such as mitomycin C and UV irradiation. In addition, hr5 also caused significant suppression of tat gene-mediated trans-activation. The suppression of HIV LTR expression by hr5 appears to be mediated, at least in part, by the repression of the HIV enhancer, as the activity of an enhancer test system composed of the human T-cell leukemia virus I LTR containing an HIV-1 enhancer substitution was severely repressed by hr5. Cotransfection of HIV-1 proviral DNA with hr5 DNA resulted in a significant reduction of HIV production.

Adenovirus Early Proteins↗

PubMed1989

Localization of a transcription start point within the human Hinf element.

The Hinf family is a repetitive nucleotide sequence of the human genome. Certain structural features of Hinf DNA resemble the eukaryotic RNA polymerase II promoters. Therefore, we studied the ability of the Hinf element to function as a transcriptional promoter in mammalian cells. We placed the Hinf element upstream from the thymidine kinase-encoding (tk) sequence in a plasmid construct, pAC401 and introduced it into Ltk- mouse cells. The Hinf-tk plasmid was able to transform Ltk- cells to Tk+ phenotype. In another plasmid construct, pAC Hinf-neo, the Hinf element was inserted upstream from the sequence encoding neomycin (Nm) resistance (neo), and this plasmid was able to confer Nm resistance to HeLa cells. The nature of transcription initiation of the tk gene in four of the Tk+ clones transformed by pAC401 was examined by S1 nuclease analysis, and the transcription start point (tsp) for the tk gene in these clones was mapped within the Hinf element. The same tsp in the Hinf element was found in HeLa cells. Our studies show that the Hinf element functions as a weak promoter.

Animals↗

PubMed1989

Multiple functional domains of Tat, the trans-activator of HIV-1, defined by mutational analysis.

The tat gene of HIV-1 is a potent trans-activator of gene expression from the HIV long terminal repeat (LTR). To define the functionally important regions of the product of the tat gene (Tat) of HIV-1, deletion, linker insertion and single amino acid substitution mutants within the Tat coding region of strain SF2 were constructed. The effect of these mutations on trans-activation was assessed by measuring the expression of the bacterial chloramphenicol acetyltransferase (CAT) reporter gene linked to the HIV-LTR. These studies have revealed that four different domains of the protein that map within the N-terminal 56 amino acid region are essential for Tat function. In addition to the essential domains, an auxiliary domain that enhances the activity of the essential region has also been mapped between amino acid residues 58 and 66. One of the essential domains maps in the N-terminal 20 amino acid region. The other three essential domains are highly conserved among the various strains of HIV-1 and HIV-2 as well as simian immunodeficiency virus (SIV). Of the conserved domains, one contains seven Cys residues and single amino acid substitutions for several Cys residues indicate that they are essential for Tat function. The second conserved domain contains a Lys X Leu Gly Ile X Tyr motif in which the Lys residue is essential for trans-activation and the other residues are partially essential. The third conserved domain is strongly basic and appears to play a dual role. Mutants lacking this domain are deficient in trans-activation and in efficient targeting of Tat to the nucleus and nucleolus. The combination of the four essential domains and the auxiliary domain contribute to the near full activity observed with the 101 amino acid Tat protein.

Amino Acid Sequence↗

PubMed1989

Enhanced ras oncogene mediated cell transformation and tumorigenesis by adenovirus 2 mutants lacking the C-terminal region of E1a protein.

Mutants of adenovirus 2 E1a defective in coding for the C-terminal 61 or 67 amino acids of a 243 amino acid (243R) protein are defective in immortalization of primary baby rat kidney (BRK) cells. However, they cooperate with T24 ras in oncogenic transformation more efficiently than wt. BRK cells transformed by the E1a C-terminal mutants and T24 ras induce rapidly growing tumors in syngeneic rats and athymic mice whereas cells transformed by the wt 243R and ras oncogene are not tumorigenic in syngeneic rats and can only induce slowly growing tumors in athymic mice. Cells transformed by the E1a mutants and ras oncogene also induce rapid metastatic tumors whereas cells transformed by the wt 243R and T24 ras can not do so. The increased tumorigenic ability exhibited by the 243R mutants does not appear to be due to differential levels of expression of p21 ras. Our results suggest that the C-terminal region of the 243R protein may have a novel function in suppression of cell transformation, tumorigenesis and tumor progression.

Adenoviridae↗

PubMed1988

An N-terminal region of adenovirus E1a essential for cell transformation and induction of an epithelial cell growth factor.

A new region of the adenovirus E1a protein essential for immortalization and transformation of primary rat kidney cells has been identified. This region is located between amino acid residues 18 to 20 in an N-terminal domain that is not conserved among the various adenovirus serotypes. The transformation defective mutant (18-0) mapping in this region is not impaired in its ability to trans-activate the viral E2 promoter and to repress the activity of certain enhancer elements. Mutant 18-0 appears to have only a partial defect in the induction of cellular DNA synthesis in quiescent primary cells suggesting that the N-terminal region plays a role in immortalization and transformation by a mechanism that may not fully depend on induction of cellular DNA synthesis. Mutant 18-0 and another transformation defective mutant (125-7) mapping between amino acid residues 125 to 127 in a conserved domain are defective in the induction of an epithelial cell growth factor, suggesting that growth factor induction may be important for some aspect of adenovirus mediated immortalization and transformation.

Adenovirus Early Proteins↗

PubMed1988

Cell type dependent transformation by adenovirus 5 E1a proteins.

The two major proteins of 243 amino acids (243R) and 289R encoded by the E1a region of adenovirus 5 are related, differing only by the presence of an internal conserved 46 amino acid segment in the 289R protein. We report here that these proteins have different cell transformation properties. Primary baby rat kidney (BRK) cells infected with an M-MuLV retrovirus vector that expresses the 243R protein are immortalized and the resulting foci consist predominantly of epithelial cells; a small fraction of the foci consist of fibroblast-like cells. In contrast, BRK cells infected with a retrovirus expressing the 289R protein are immortalized at a very low frequency and the foci induced consist of only fibroblast-like cells. The immortalizing properties of the 289R protein are dramatically changed by certain mutations within the unique 46 amino acid region: M-MuLV vectors with these mutations immortalize epithelial cells with high efficiency, as much as an order of magnitude higher than vectors that express the 243R protein. The enhanced immortalization property of the mutant forms of the 289R protein does not appear to be related to their transcriptional activation function. Our results suggest that the unique region of the 289R protein has the potential to inhibit immortalization of primary epithelial cells.

Adenovirus Early Proteins↗

PubMed1988

Separation of immortalization and T24-ras oncogene cooperative functions of adenovirus E1a.

An adenovirus 2 E1a gene coding for a protein of 243 (243R) amino acids can efficiently immortalize primary rat kidney (BRK) cells and cooperate with the activated cellular ras oncogene (T24 ras). A mutant (47-0) of the 243R gene that maps between amino acid residues 47-50 within a region that is highly conserved among the various adenovirus serotypes was found to be severely defective in immortalization. Despite the defect in immortalization, mutant 47-0 had the ability to cooperate with T24 ras in oncogenic transformation. These results suggest that the immortalization and the oncogene cooperation functions of the 243R are separable. Our results further suggest that the requirement for a separate immortalization function can be circumvented by oncogenic transformation and that the immortalization of cells transformed by E1a and T24 ras may be a secondary consequence of transformation by these two oncogenes.

Adenovirus Early Proteins↗