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.

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.

HIV-2 EHO isolate has a divergent envelope gene and induces single cell killing by apoptosis.

The human immunodeficiency virus type 2 (HIV-2)-related isolate, referred to as HIV-2 EHO, has been isolated from an Ivory Coast patient with acquired immunodeficiency syndrome (AIDS). Infection of CD4 expressing cells with this highly infectious virus mediates a cytopathic effect characterized by single-cell killing as a consequence of apoptosis. Nucleotide sequence analysis of the HIV-2 EHO genome revealed a significant degree of divergence of its envelope gene from that of other known HIV-2 strains.

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.

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.

Differential expression and distinct structure of 69- and 100-kDa forms of 2-5A synthetase in human cells treated with interferon.

Using specific monoclonal and polyclonal antibodies, the induction, synthesis and subcellular localization of 69- and 100-kDa forms of 2',5'-oligoadenylate (2-5A) synthetase (p69 and p100) were investigated in alpha-interferon-treated human HeLa and Daudi cells. Although both p69 and p100 were induced by interferon, there were significant differences in the interferon dose-response and the kinetics of synthesis of each protein in these cell lines. Both proteins are localized mainly in the cytoplasm.

Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon.

The double-stranded (ds) RNA-activated protein kinase from human cells is a 68 kd protein (p68 kinase) induced by interferon. On activation by dsRNA in the presence of ATP, the kinase becomes autophosphorylated and can catalyze the phosphorylation of the alpha subunit of eIF2, which leads to an inhibition of the initiation of protein synthesis. Here we report the molecular cloning and characterization of several related cDNAs from which can be deduced the full-length p68 kinase sequence.

Reduced activity of the interferon-induced double-stranded RNA-dependent protein kinase during a heat shock stress.

Previous studies have shown that the antiviral response induced by interferon in murine cells could be degraded after a heat shock. Here we have confirmed that a similar effect occurs also in interferon-treated human HeLa cells subjected to a heat shock. In addition, we have investigated the fate of the interferon-induced, double-stranded RNA-dependent protein kinase in heat-shocked cells.

Preparation and characterization of polyclonal antibodies specific for the 69 and 100 k-dalton forms of human 2-5A synthetase.

Recently, the existence of 40-, 46-, 69- and 100- kDa forms of 2',5'-oligoadenylate (2-5A) synthetase have been established in interferon-treated human cells. Using monoclonal antibodies specific for 69- and 100- kDa forms of 2-5A synthetase, we purified these proteins by immunoaffinity chromatography and raised murine polyclonal antibodies. All immunized mice developed antibodies (anti-69 or anti-100 kDa form) which were characterized by their capacity to immunoprecipitate [35S] cysteine labeled proteins from interferon-treated cells or identify these proteins by electrophoretic transfer immunoblot analysis of extracts from control and interferon-treated cells.

A type 1 phosphoprotein phosphatase active with phosphorylated Mr = 68,000 initiation factor 2 kinase.

A type 1 protein phosphatase from reticulocytes is shown to efficiently dephosphorylate the Mr = 68,000 phosphopeptide of the double-stranded RNA-dependent kinase that phosphorylates the alpha subunit of eukaryotic peptide initiation factor 2, eIF-2. The kinase, activated in the presence of double-stranded RNA with concomitant phosphorylation of the Mr = 68,000 peptide, causes inhibition of peptide initiation and thereby effects translational control of protein synthesis. The Mn2+-dependent phosphatase is classified as a type 1 enzyme in that it is inhibited by inhibitor 2 in nanomolar concentrations and appears to have a Mr = 35,000 catalytic subunit.

The binding of double-stranded RNA and adenovirus VAI RNA to the interferon-induced protein kinase.

The protein kinase from human cells dependent on double-stranded (ds) RNA is a 68-kDa protein (p68 kinase), the level of which is enhanced significantly in cells treated with interferon. When activated by low concentrations of dsRNA, the p68 kinase becomes phosphorylated and thereby catalyzes the phosphorylation of the protein-synthesis initiation factor, eIF2. Here, we have purified the p68 kinase to homogeneity using a specific monoclonal antibody to investigate its capacity to bind dsRNA, poly(I).

Interferon- and sarcolectin-dependent cellular regulatory interactions.

Interferons, via specific membrane-bound receptors, induce various cellular functions of which antiviral protection is the most extensively studied. We have previously reported the existence of interferon antagonists (referred to as sarcolectins) in various tissue extracts from placental blood, cartilage, brain, muscle, or from sarcomas. These sarcolectins have been fully characterized and purified to homogeneity.

Expression of interferon-induced genes in different tissues of mice.

In vivo responses to interferon (IFN) in mice were determined by measuring the steady-state levels of induced mRNAs following injection of IFN and poly(I)-poly(C). With cDNA probes for mouse 2'-5' oligoadenylate synthetase (2-5A synthetase) and 1-8, constitutive expression of the corresponding mRNA was detectable in different organs of normal C3H/He mice. These mRNA levels were increased by as much as 15-fold over control levels in various tissues, including the brain, after IFN and poly(I)-poly(C) treatment, coincident with increases in 2-5A synthetase enzyme activity.

Autophosphorylation of the protein kinase dependent on double-stranded RNA.

The double-stranded RNA (dsRNA)-dependent protein kinase (p68 kinase) from interferon-treated human cell is a Mr 68,000 protein induced by interferon. By the use of a specific monoclonal antibody, we have been able to study the two distinct protein kinase activities characteristic of purified p68 kinase. The first activity is functional for endogenous phosphorylation of the enzyme (p68 kinase), whereas the second one is responsible for the phosphorylation of exogenous substrates such as eukaryotic initiation factor 2 and histone.

The double-stranded RNA-dependent protein kinase is also activated by heparin.

The double-stranded(ds)-RNA dependent protein kinase from human cells is a Mr 68,000 protein (p68 kinase), the level of which is enhanced significantly in cells treated with interferon. When activated by dsRNA, the p68 kinase becomes autophosphorylated. The phosphorylated p68 kinase then can catalyze the phosphorylation of exogenous substrates, such as eIF2 and histone.

Rapid decrease in the levels of the double-stranded RNA-dependent protein kinase during virus infections.

The double-stranded RNA-dependent protein kinase from human cells is a 68,000 molecular weight protein (p68 kinase), the level of which is enhanced significantly in cells treated with interferon. With a monoclonal antibody specific for p68 kinase, here we show the phosphorylation and steady-state levels of p68 kinase during virus infection. The p68 kinase is phosphorylated in interferon-treated cells during infection with encephalomyocarditis virus (EMCV), vesicular stomatitis virus (VSV), and vaccinia virus, thus indicating activation of p68 kinase during these virus infections, an essential step required for autophosphorylation of p68 kinase.

Identification of 69-kd and 100-kd forms of 2-5A synthetase in interferon-treated human cells by specific monoclonal antibodies.

Recently, the existence of 40-kd and 46-kd 2-5A synthetases in interferon-treated cells has been confirmed by cloning and characterization of cDNA corresponding to these small size enzymes. By the use of specific monoclonal antibodies, we describe here two forms of high mol. wt 2-5A synthetases of 69 and 100 kd in human cells.

Four different forms of interferon-induced 2',5'-oligo(A) synthetase identified by immunoblotting in human cells.

Antibodies against synthetic peptides derived from the cDNA sequence of interferon-induced 2',5'-oligo(A) synthetase, and which immunoprecipitate the native enzyme activity, were found to detect multiple enzyme forms in denaturing electrophoretic immunoblots. In some human cell lines, four different interferon-induced proteins of 40, 46, 67, and 100 kDa were found to react with the same peptide antibodies. Each isolated form was shown to have 2',5'-oligo(A) synthetase activity, but the dependence on double-stranded RNA was markedly different for activation of the individual enzymes.

Adenovirus VAI RNA complexes with the 68 000 Mr protein kinase to regulate its autophosphorylation and activity.

We have investigated the interaction of VAI RNA with the interferon-induced, double-stranded (ds) RNA-activated protein kinase, P68, both of which regulate protein synthesis in adenovirus-infected cells. Previous work has shown that during infection by the VAI RNA-negative mutant, dl331, both viral and cellular protein synthesis are inhibited due to phosphorylation of the alpha-subunit of the eukaryotic initiation factor, eIF-2, by the P68 protein kinase. Utilizing monoclonal antibodies specific for P68, we demonstrated that the physical levels of P68 in dl331-infected, wild-type Ad2-infected and uninfected cells were all comparable suggesting that the elevated kinase activity detected during mutant infection was not due to increased P68 synthesis.

Neutralization of interferon produced early during rabies virus infection in mice.

Inoculation of mice (strain C3H/He) with a purified preparation of fixed rabies virus led to the production of interferon with two different peaks of activity detectable in the plasma: an early peak 24 h after inoculation followed by another peak on the 7th day after infection. The level of splenic 2-5A synthetase was enhanced in parallel with the pattern of interferon activity. Neutralization of the first peak of interferon activity by anti-mouse alpha/beta interferon globulin blocked the induction of splenic 2-5A synthetase and modified the development of disease.

Two interferon-induced proteins are involved in the protein kinase complex dependent on double-stranded RNA.

The double-stranded (ds) RNA-dependent protein kinase is a 100,000-110,000 Mr complex of two interferon-induced subunits each having ATP binding sites: a 48,000 Mr protein (p48) which appears to be responsible for the phosphorylation of a 68,000 Mr protein (p68) in the presence of dsRNA. The p68 subunit once phosphorylated is converted to an active protein kinase capable of phosphorylating exogenous substrates such as the alpha subunit of protein synthesis initiation factor eIF2 or calf thymus histone. The phosphorylation of exogenous substrates is highly correlated with the degree of phosphate saturation of p68 and does not require the presence of dsRNA.

Monoclonal antibodies to an interferon-induced Mr 68,000 protein and their use for the detection of double-stranded RNA-dependent protein kinase in human cells.

Extracts from interferon-treated human cells show an enhanced level of a double-stranded RNA-dependent protein kinase activity that is manifested by the phosphorylation of an endogenous Mr 69,000-72,000 protein in its phosphate-saturated state. By using a highly purified protein kinase fraction from interferon-treated human Daudi cells, we can now describe the preparation of murine monoclonal antibodies directed against this phosphoprotein, the Mr of which in its native state is found to be 68,000. These monoclonal antibodies (class IgG1) can identify the electrophoresed protein (p68) in polyacrylamide gels by the electrophoretic transfer blotting technique.