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A G Hovanessian

Publications and source records attributed to A G Hovanessian.

At least 19 recordsLinked to original sources
PubMed1995

HIV-1 Tat directly interacts with the interferon-induced, double-stranded RNA-dependent kinase, PKR.

We present evidence that the HIV-1 Tat protein and the RNA-dependent cellular protein kinase, PKR, interact with each other both in vitro and in vivo. Using GST fusion chromatography, we demonstrate that PKR, interacts directly with the HIV-1 Tat protein. The region in Tat sufficient for binding PKR maps within amino acids 20 to 72. In in vitro assays, the two-exon form of Tat (Tat 86) was phosphorylated by PKR, while the one exon form of Tat (Tat 72) inhibited PKR autophosphorylation and substrate phosphorylation. The ability of Tat to interact with PKR was demonstrated in both yeast and mammalian cells. Expression of PKR in yeast results in a growth suppressor phenotype which was reversed by coexpression of a one exon form of Tat. Expression of Tat 72 in HeLa cells resulted in direct interaction with PKR as detected by coimmunprecipitation with a Tat antibody. Tat and PKR also form a coimmunoprecipitable complex in cell-free extracts prepared from productively infected T lymphocytes. The interaction of Tat with PKR provides a potential mechanism by which HIV could suppress the interferon system.

Cell Line↗
PubMed1995

Molecular mechanisms responsible for malignant transformation by regulatory and catalytic domain variants of the interferon-induced enzyme RNA-dependent protein kinase.

Double-stranded RNA-dependent protein kinase (PKR) is suggested to play an important role in both the antiviral and antiproliferative arms of the interferon response. To gain insights into the molecular mechanisms underlying PKR's growth regulatory properties, we examined the biological and biochemical properties of PKR variants containing either a mutation in catalytic domain II (PKR-M1) or a deletion of RNA binding domain I (PKR-M7) in both reticulocyte translation extracts and in vitro kinase assays with purified reagents and compared these results with those using the same mutants stably expressed in vivo. While wild-type PKR (PKR-WT) efficiently inhibited mRNA translation in a reticulocyte extract, the inactive PKR-M1 had no effect. The PKR-M7 mutant was modestly inhibitory in this assay. The PKR-M1 variant was able to reverse the translational inhibitory effects and increased eukaryotic initiation factor (eIF)-2 alpha phosphorylation levels caused by addition of double-stranded RNA to reticulocyte extract, whereas PKR-M7 could not. Both PKR-M1 and PKR-M7 functioned as transdominant inhibitors of PKR-WT in our in vitro kinase assays. While the inhibition by PKR-M1 required a vast excess of mutant to shut down PKR function, PKR-M7 inhibited PKR-WT at approximately stoichiometric levels. To complement these experiments, we compared growth rates and alpha phosphorylation levels in transformed cell lines overexpressing either PKR-M1 or PKR-M7. Levels of endogenous eIF-2 alpha phosphorylation were significantly more diminished in PKR-M7 overexpressing cells compared with PKR-M1. These paradoxical data will be discussed in terms of the potential molecular mechanisms underlying malignant transformation caused by the PKR variants.

3T3 Cells↗
PubMed1995

Nuclear localization of the interferon-inducible protein kinase PKR in human cells and transfected mouse cells.

The levels and subcellular distribution of the interferon-inducible double-stranded RNA-dependent protein kinase PKR have been measured in human Daudi cells and stably transfected mouse NIH 3T3 cells expressing the human protein kinase. Immunofluorescence of intact cells and quantitative immunoblotting of cell extracts indicate that PKR occurs in both the cytoplasm and the cell nucleus, with staining specifically in the nucleolus. The ratio of cytoplasmic to nuclear PKR is approximately 5:1 in control cells; in response to interferon treatment the protein kinase is induced severalfold in the cytoplasm whereas the level in the nucleus does not increase significantly. Analysis of individual transfected cells by confocal microscopy reveals a pattern of distribution of PKR similar to that in Daudi cells, with immunostaining of cytoplasm and nucleoli. Similar results are observed whether cells expressing wild-type PKR or a catalytically inactive mutant form of the kinase are analyzed, but untransfected 3T3 cells are not stained by the antibody used. Two-dimensional isoelectric focusing analysis of PKR in whole cell extracts reveals the presence of multiple forms with different pI values whereas similar analysis of the nuclear fraction indicates only one predominant species with a relatively basic pI. These results suggest that PKR may have a role in the cell nucleus as well as the cytoplasm and that the subcellular distribution of the protein kinase may be related to post-translational modifications.

3T3 Cells↗
PubMed1994

HIV-1 TAR RNA has an intrinsic ability to activate interferon-inducible enzymes.

The TAR sequence at the 5'-termini of all HIV-1 mRNA species forms a stable structure that is responsible for both transcriptional and translational regulation of HIV-1. Previously we and others reported that purified TAR RNA synthesized by in vitro transcription could activate two interferon-induced enzymes, the protein kinase (PKR) and 2-5A-synthetase. Because the PKR- and 2-5A-systems block protein synthesis initiation and induce RNA decay, respectively, these findings suggested mechanisms for the control of HIV-1 replication by the interferon system. To determine if contaminating dsRNA from in vitro transcription reactions was responsible for this effect, as suggested by Gunnery et al. 1990, (Proc., Natl. Acad. Sci. USA 87, 8687), we have reexamined these findings using chemically synthesized TAR (nucleotides +1 to +57). TAR RNA is shown here to have an intrinsic ability to activate PKR and 2-5A-synthetase. In contrast, a mutant form of TAR designed to have a disrupted secondary structure did not stimulate either enzyme. Chemically synthesized TAR mimicked other dsRNA species in its ability to activate and inhibit PKR at low and high RNA concentrations, respectively. HIV-1 TAT protein inhibited activation of PKR by HIV-1 TAR RNA suggesting an escape mechanism for the virus.

2',5'-Oligoadenylate Synthetase↗
PubMed1994

Specific inhibition of viral protein synthesis in HIV-infected cells in response to interferon treatment.

The mechanism of action of different types of interferons (IFN-alpha, -beta, and -gamma) against human immunodeficiency virus (HIV)-1 infection was investigated in chronically infected monocytoid U937 cells and during an acute infection of the T lymphoblastoid CEM cells. Two chronically infected U937 cell populations, obtained independently (referred to as type A and B cells), were analyzed for their response to IFNs. In type A cells, IFNs mainly inhibited virus particle release, whereas in type B cells, the anti-HIV effect of IFNs cells was found to be largely due to a specific inhibition of viral protein synthesis without any apparent effect on total cellular protein synthesis. Interestingly, such a differential inhibition of HIV protein synthesis could also be demonstrated in acutely infected CEM cells in response to treatment with IFN-alpha. Both in chronically infected U937 type B and acutely infected CEM cells, equivalent amounts of nuclear and cytoplasmic HIV-1 mRNA were detected in control and IFN-treated cells in spite of at least 80% inhibition of HIV protein synthesis. Analysis of the distribution of cellular and viral mRNAs on polysomes in HIV-1-infected cells demonstrated that IFN treatment induces a specific block on viral mRNA translation. These results indicate that the antiviral mechanism of IFN on later stages of HIV replication cycle may be partly due to the inhibition of HIV mRNA translation, besides an effect on virus budding or release.

HIV-1↗
PubMed1994

Characterization of an interferon-induced 48-kD protein immunologically related to the double-stranded RNA-activated protein kinase PKR.

Polyclonal antibodies raised against purified and urea-denatured double-stranded protein kinase (PKR) from human origin cross-reacted by immunoblotting with a 48-kD protein (p48) induced by the three types of interferon (IFN), alpha, beta, and gamma. The induction of p48 is IFN dose dependent and its accumulation occurs a few hours after the addition of IFN. The induction of p48 is blocked by actinomycin D. Analysis by two-dimensional gel isoelectric-focusing, revealed p48 as a single spot with an isoelectric point (pI) of 6.8. In the same experiment the PKR was revealed as several subspecies with pI values in the pH range of 7.4-8.0. Cell fractionation experiments indicated that PKR and p48 have different subcellular localizations: PKR was found to be associated with the microsomal pellet as shown previously whereas p48 was recovered in the microsomal supernatant fraction. In addition to these differences, PKR and p48 were found to be differentially expressed in some human cells treated with the three types of IFN. For example, in HeLa cells, IFN-alpha or IFN-beta induced similarly both PKR and p48 whereas IFN-gamma induced mainly p48. In U937 cells in which PKR was not expressed with or without IFN treatment, p48 was strongly induced by all three types of IFN. These results suggest different mechanisms for the induction of PKR and p48. In view of its presence in different types of human cells and its induction by different types of IFN, it is possible to suggest that p48 might play an important role in mediating some of the action of IFN.

Enzyme Activation↗
PubMed1993

T cell activation antigen, CD26, as a cofactor for entry of HIV in CD4+ cells.

The CD4 molecule is essential for binding HIV particles, but is not sufficient for efficient viral entry and infection. The cofactor was shown to be dipeptidyl peptidase IV (DPP IV), also known as CD26. This serine protease cleaves its substrates at specific motifs; such motifs area also highly conserved in the V3 loops of HIV-1, HIV-2, and related simian isolates. Entry of HIV-1 or HIV-2 into T lymphoblastoid and monocytoid cell lines was inhibited by a specific monoclonal antibody against DPP IV or specific peptide inhibitors of this protease. Coexpression of human CD4 and CD26 in murine NIH 3T3 cells rendered them permissive to infection by HIV-1 and HIV-2. These observations could provide the basis for developing simple and specific inhibitors of HIV and open a possibility for vaccine development.

3T3 Cells↗
PubMed1993

Molecular cloning of two new interferon-induced, highly related nuclear phosphoproteins.

During the molecular cloning of the human dsRNA activated-p68 kinase (PKR), polyclonal antibodies against PKR selected, in addition to cDNAs corresponding to PKR, another cDNA presenting only slight homology with PKR cDNA. This cDNA recognized an mRNA species of 2 kilobases induced by both alpha- and gamma-interferons. Its transcription did not require protein synthesis. On further library screening, it selected two highly related cDNAs, referred to as 75 and 41, displaying perfect homology over 612 base pairs and divergent at both ends. In addition, cDNA 75 presents an insertion of 150 base pairs highly homologous to a region common to both sequences. The 75 and 41 peptidic sequences are very hydrophilic, rich in basic amino acid residues, and contain several potential phosphorylation sites for different serine/threonine kinases. Furthermore, they present two protamine- and histone-like nuclear targeting sequences as well as some homology with helix-loop-helix motifs of some DNA-binding proteins. The 75-encoded product, which resolved as a 52-kDa protein after in vitro expression in rabbit reticulocyte lysates, was found to migrate as a 65-67-kDa protein after in vivo expression in insect cells. In accord with sequence data, this 65-67-kDa protein was found to be phosphorylated in vivo in the insect cells and was recovered from the membrane/nuclear pellet. In contrast, the 41-encoded product (30-kDa protein in reticulocyte lysates) could not be expressed in vivo, as it provoked a rapid and severe shut-off of protein synthesis in insect cells. The function of the 75 and 41 proteins and their relation to PKR remains to be determined. However, the presence of nuclear targeting sequences, phosphorylation sites, and helix-loop-helix motif is consistent with a role of these proteins in the mechanism of transduction of the interferon action.

Amino Acid Sequence↗
PubMed1993

Tumor suppressor function of the interferon-induced double-stranded RNA-activated protein kinase.

RNA-dependent protein kinase is a M(r) 68,000 protein in human cells (p68 kinase) or a M(r) 65,000 protein in murine cells (p65 kinase). p65/p68 is a serine/threonine kinase induced by interferon treatment and generally activated by double-stranded RNAs. Once activated, the known function of this kinase is inhibition of protein synthesis through phosphorylation of the eukaryotic initiation factor 2. Here we have investigated the potential for tumorigenicity in mice of murine NIH 3T3 clones expressing human p68 kinase, either the wild-type or a mutant inactive kinase with a single amino acid substitution in the invariant lysine-296 in the catalytic domain II. Expression of the mutant p68 kinase was correlated with a malignant transformation phenotype, giving rise to the production of large tumors of at least 1 cm in diameter within 7-12 days in all inoculated mice. In contrast, no tumor growth was observed for several weeks in mice inoculated with NIH 3T3 cell clones expressing either the wild-type recombinant p68 kinase or only the endogenous p65 kinase, the murine analogue of the p68 kinase. These results suggest that functional p65/p68 kinase (recently called PKR), by a still undefined mechanism, may also act as a tumor suppressor. Consequently, one of the pathways by which interferon inhibits tumor growth might be through its capacity to induce the enhanced expression of this kinase.

3T3 Cells↗
PubMed1993

Localization of the human interferon-induced, ds-RNA activated p68 kinase gene (PRKR) to chromosome 2p21-p22.

The interferon-induced dsRNA-activated protein kinase (PRKR) belongs to a subclass of serine/threonine kinases, involved in the regulation of protein synthesis by phosphorylation of the alpha subunit of initiation factor eIF2. Somatic cell hybrids segregating human chromosomes were used to assign this kinase to human chromosome 2. Fluorescence in situ hybridization confirmed this assignment and further localized the gene (PRKR) to the boundary region of bands p21 and 22.

Chromosome Banding↗
PubMed1993

The interferon-induced double-stranded RNA-activated human p68 protein kinase potently inhibits protein synthesis in cultured cells.

The role of the interferon-induced double-stranded RNA (dsRNA)-activated human p68 protein kinase as an inhibitor of protein synthesis has been inferred from work with cell-free systems, but direct proof in animal cells is lacking. To document the action of p68 protein kinase in vivo, we have used an infection-transfection system where expression of p68 is driven by a vaccinia virus promoter regulated by the lacl repressor/operator controlling elements. In cultured cells infected with vaccinia virus and transfected with a plasmid containing the p68 gene, there is synthesis of p68 when lacl repressor is inhibited with isopropyl-beta-D-thiogalactoside. When infection-transfections are carried out with the p68 gene together with the luciferase (LUC) reporter gene, a strong inhibition of LUC expression developed with time postinfection. This inhibition was not observed with a mutant form of the kinase (Lys-->Arg at position 296) and it was reversed by antisense expression of the p68 gene. During inhibition of LUC expression the protein kinase was phosphorylated, possibly as a result of autophosphorylation activated by the dsRNA forms which are known to accumulate in vaccinia virus-infected cells. Inhibition of LUC expression was at the level of translation. Our findings demonstrate that expression and activation of the human p68 protein kinase in vivo potently inhibits protein synthesis.

Cells, Cultured↗
PubMed1992

Characterization and regulation of the 58,000-dalton cellular inhibitor of the interferon-induced, dsRNA-activated protein kinase.

The P68 protein kinase is a serine/threonine kinase induced by interferon treatment and activated by double-stranded RNAs (dsRNAs). Once activated, the kinase phosphorylates its natural substrate, the alpha subunit of eukaryotic initiation factor 2 (eIF-2) leading to potential limitations in functional eIF-2 and decreases in protein synthesis initiation. We have recently purified from influenza virus-infected cells a P68 kinase inhibitor, found to be a 58-kDa cellular protein. We have now investigated the mechanisms by which the 58-kDa inhibitor regulates P68 kinase activity and how the inhibitor itself is controlled. The 58-kDa inhibitor did not function by degrading or sequestering the dsRNA activator of P68 but could repress phosphorylation of eIF-2 alpha by an already activated protein kinase. Utilizing antibody prepared against a 58-kDa-specific peptide, we showed that the 58-kDa proteins from infected and uninfected cells were present in equivalent amounts. Although kinase inhibitory activity could not be detected in crude uninfected cell extracts, ammonium sulfate treatment unmasked this activity and allowed purification of the cellular inhibitor with identical chromatographic properties as that from influenza virus-infected cells. Finally, we have identified and partially purified a specific inhibitor of the 58-kDa protein which we refer to as an "anti-inhibitor." Based on these data, we present a model depicting the complex regulation of the interferon-induced protein kinase in eukaryotic cells.

Amino Acid Sequence↗
PubMed1992

The 69-kDa 2-5A synthetase is composed of two homologous and adjacent functional domains.

The existence of three distinct forms of 2-5A synthetase, p40-p46, p69, and p100, has been established in interferon-treated human cells. The expression of these enzymes varies according to the cell type studied, and their properties are relatively different. By the use of polyclonal antibodies specific to p69, we have cloned several cDNAs which identify four interferon-induced RNAs of 5.7, 4.5, 3.7, and 3.2 kilobases (kb). Analysis of the nucleotide sequence of three full-length cDNAs (5.6, 3.1, and 2.9 kb) revealed that they have a common open reading frame of 683 amino acids with different 3' termini; cDNAs 5.6 and 3.1 have an extension of 4 amino acids, whereas cDNA 2.9 has an extension of 44 amino acids. In vitro transcription-translation of cDNAs 3.1 and 2.9 kb generated proteins of 69 and 71 kDa, respectively. Both proteins bind a monoclonal antibody specific for p69 and can synthesize 2-5A. The deduced amino acid sequence of p69 revealed that it can be divided into two homologous and adjacent domains, each sharing strong homology to the first 346 amino acids common to the two isoforms of the small 2-5A synthetase (p40, p46). These results suggest that p69 might have two functional catalytic domains required for 2-5A synthetase activity and favor the hypothesis that its gene might have derived from the fusion of two ancestral genes analogous to the small 2-5A synthetase gene.

2',5'-Oligoadenylate Synthetase↗
PubMed1992

Human p68 kinase exhibits growth suppression in yeast and homology to the translational regulator GCN2.

The human p68 kinase is an interferon-regulated enzyme that inhibits protein synthesis when activated by double-stranded RNA. We show here that when expressed in Saccharomyces cerevisiae, the p68 kinase produced a growth suppressing phenotype resulting from an inhibition of polypeptide chain initiation consistent with functional protein kinase activity. This slow growth phenotype was reverted in yeast by two different mechanisms: expression of the p68 kinase N-terminus, shown to bind double-stranded RNA in vitro and expression of a mutant form of the alpha-subunit of yeast initiation factor 2, altered at a single phosphorylatable site. These results provide the first direct in vivo evidence that the p68 kinase interacts with the alpha-subunit of eukaryotic initiation factor 2. Sequence similarity with a yeast translational regulator, GCN2, further suggests that this enzyme may be a functional homolog in higher eukaryotes, where its normal function is to regulate protein synthesis through initiation factor 2 phosphorylation.

Amino Acid Sequence↗
PubMed1992

Antiviral action of polyadenylic-polyuridylic acid against HIV in cell cultures.

Polyadenylic-polyuridylic acid referred to as poly(A).poly(U) is a synthetic double-stranded RNA which has been shown to manifest both antitumoral and immunomodulatory activities. Here we used this agent to demonstrate its antiviral activity against the human immunodeficiency virus (HIV-1 and HIV-2). Treatment of cells with poly(A).poly(U) resulted in a significant delay in the development of the HIV-specific cytopathic effect characterized by the formation of syncytia and cell lysis. Furthermore, the production of virus measured by the concentration of the HIV major core protein was reduced by 90-95%. Under these experimental conditions, the synthesis of HIV proteins was reduced at least tenfold whereas the metabolism and proliferation of cells apparently were not affected. The inhibitory action of poly(A).poly(U) seems to be at the level of viral entry into cells. Combined treatment of infected cells with poly(A).poly(U) and azidothymidine (AZT) resulted in a 4-5-fold synergistic inhibitory effect. Previously, no toxicity has been observed in cancer patients with long-term treatment with poly(A).poly(U). In view of this and the significant anti-HIV effect, poly(A).poly(U) provides a potential candidate as a therapeutic drug in AIDS disease.

Antiviral Agents↗
PubMed1992

Constitutive expression of human double-stranded RNA-activated p68 kinase in murine cells mediates phosphorylation of eukaryotic initiation factor 2 and partial resistance to encephalomyocarditis virus growth.

The cDNA encoding interferon-induced human double-stranded RNA-activated p68 kinase was expressed in murine NIH 3T3 cells by using the pcDNA1/neo vector. Several stable clones were selected which expressed either the wild-type kinase or an inactive mutant possessing a single amino acid substitution in the invariant lysine 296 in the catalytic domain II. The transfected wild-type kinase showed properties similar to those of the natural kinase, such as subcellular ribosomal localization and dependence on double-stranded RNA for autophosphorylation. Upon infection with encephalomyocarditis virus (EMCV), wild-type- but not mutant-expressing clones were found to partially resist virus growth. Such natural antiviral activity was virus specific, since no inhibition was observed in the case of vesicular stomatitis virus infection. In accord with EMCV inhibition, the wild-type p68 kinase was found to be highly phosphorylated during infection. Furthermore, its natural substrate, the small subunit of protein synthesis initiation factor eIF2, was phosphorylated. These results demonstrate that p68 kinase is activated during EMCV infection, leading to reduced virus production.

3T3 Cells↗
PubMed1991

Functional expression and characterization of the interferon-induced double-stranded RNA activated P68 protein kinase from Escherichia coli.

The P68 protein (referred to as P68 on the basis of its molecular weight of 68,000 in human cells) is a serine/threonine kinase induced by interferon treatment and activated by double-stranded (ds) RNAs. Although extensively studied, little is currently known about the regulation of kinase function at the molecular level. What is known is that activation of this enzyme triggers a series of events which lead to an inhibition of protein synthesis initiation and may, in turn, play an integral role in the antiviral response to interferon. To begin to understand P68 and its biological functions in the eukaryotic cell, we have expressed the protein kinase in Escherichia coli under control of the bacteriophage T7 promoter. In rifampicin-treated cells, metabolically labeled with [35S]methionine and induced by IPTG, the P68 kinase was the predominant labeled product. Further, P68 was recovered from extracts as a fully functional enzyme, shown by its ability to become activated and phosphorylate its natural substrate, the alpha subunit of eukaryotic protein synthesis initiation factor 2 (eIF-2). Moreover, P68 was phosphorylated in vivo in E. coli, providing conclusive evidence that the kinase has the capacity to phosphorylate and activate itself in the absence of other eukaryotic proteins. In contrast, a mutant P68 protein, containing a single amino acid substitution in the invariant lysine in catalytic domain II, was completely inactive. Interestingly, both the mutant and wild-type protein kinases efficiently bound activator dsRNAs despite the fact that only the latter was activated by these RNAs. Finally, the expressed kinase could be isolated from contaminating E. coli proteins in an active form by immunoaffinity chromatography with a monoclonal antibody specific for P68.

Enzyme Activation↗