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.

Nucleotide sequence of the HIV-2 EHO genome, a divergent HIV-2 isolate.

PIP: The HIV-2 EHO isolate from Cote d'Ivoire has been characterized as a highly cytopathic HIV-2 strain, which can be differentiated from other isolates by the smaller size of its external envelope glycoprotein. The entire nucleotide sequence (10,352 bp) of the HIV-2 EHO genome is filed in the EMBL/Genbank Data Libraries under Accession No. U27200. Despite its high degree of variability, the genetic organization of HIV-2 EHO was found to be similar to other HIV-2 and simian immunodeficiency virus (SIV) isolates: it contains the long terminal repeats (LTR) and the 5' and 3' ends, the structural genes, and the genes encoding proteins which transactivate the HIV genome and those for accessory proteins. McClure et al. have demonstrated that HIV-2 EHO can infect Macaca nemestrina, resulting in progression to an AIDS-like disease in 50% of the macaques challenged. The researchers have more recently reported that passage of HIV-2 EHO in such macaques increases its virulence, resulting in a persistent infection and progression to AIDS in all inoculated animals. Given these observations, its unique biological and genetic properties, and the fact that it was isolated from a full-blown AIDS patient, it is plausible to suggest that HIV-2 EHO represents a distinct subtype of HIV-2 with an increased potential to trigger a rapid disease progression toward AIDS. The nucleotide sequence reported in this paper should be helpful in determining the prevalence of HIV-2 EHO in HIV-2 seropositive patients, and in evaluating its role in the evolution of AIDS.^ieng

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.

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. This divergence for the deduced amino acid sequence corresponding to the extracellular envelope glycoprotein was 26 to 30%. These unique genetic and biological properties suggest that the HIV-2 EHO isolate is a distinct prototype in the HIV-2 family.

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.

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.

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. However, immunoenzymatic staining analysis of interferon-treated cells indicated that a proportion of p69 is concentrated around the nuclei and the rest is distributed in a specific pattern in the cytoplasm whereas p100 is found in a diffuse state in the cytoplasm. In accord with its association to cell membranes, p69 is myristilated. The [35S]cysteine and [3H]myristate-labeled p69 preparations were resolved by two-dimensional gel isoelectric focusing with pI values in the pH range of 7.0-8.0 and 7.0-7.5, respectively. These observations suggested the existence of two forms of p69 which might be complexed together as a dimer. In favor with a dimeric form of p69, in gel filtration experiments the peak of p69 was routinely found in fractions corresponding to a molecular mass of 160 kDa whereas p100 was recovered as a monomer. Taken together, these results indicate that p69 and p100 have distinct characteristics and that their expression is a specific property of each cell type in response to interferon.

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. All of the cDNAs identify a 2.5 kb RNA that is strongly induced by interferon. The deduced amino acid sequence of the p68 kinase predicts a protein of 550 amino acids containing all of the conserved domains specific for members of the protein kinase family, including the catalytic domain characteristic of serine/threonine kinases. In vitro translation of a reconstructed full-length p68 kinase cDNA yields a protein of 68 kd that binds dsRNA, is recognized by a monoclonal antibody raised against the native p68 kinase, and is autophosphorylated.

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. This protein kinase is a Mr 68,000 protein (p68 kinase) which, when autophosphorylated, catalyzes phosphorylation of the protein synthesis eukaryotic initiation factor-2, thus mediating inhibition of protein synthesis. After heat shock of interferon-treated HeLa cells, the double-stranded RNA-dependent autophosphorylation of p68 kinase in cytoplasmic extracts is greatly reduced whereas the phosphorylation of other cellular proteins is not affected. In vivo, autophosphorylation of p68 kinase is also reduced in heat-shocked cells whereas there is no apparent effect on the phosphorylation state of other proteins. In such cells, the interferon-mediated antiviral response becomes modified according to the virus challenge, i.e. these cells remain resistant to vesicular stomatitis virus but become partially sensitive to encephalomyocarditis virus (EMCV) infection. The reduction in the activity of p68 kinase is due to its reduced nonionic detergent solubility occurring during the heat shock period. The resultant reduced detergent extractibility of p68 kinase is dependent on the intensity of the thermal stress. In contrast to the effect after a heat shock, arsenite treatment of interferon-treated HeLa cells induces heat shock proteins, but neither modifies the antiviral response nor affects the extractibility of p68 kinase. These results indicate that the degradation of the anti-EMCV response and reduced p68 kinase activity occur in response to heat treatment independently of the induction of heat shock proteins. The role of p68 kinase in the mechanism of the antiviral response against EMCV and vesicular stomatitis virus is discussed.

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. The 69 and 100 kDa 2-5A synthetases were induced in different types of human cells, such as Daudi, BJAB, HeLa and differentiated HL-60 cells. These enzymes were not detectable nor induced in MRC5 and undifferentiated HL-60 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. Dephosphorylation of the Mr = 68,000 peptide by the phosphatase is directly associated with a loss in kinase activity which can be restored by incubation with double-stranded RNA in the presence of ATP. The results demonstrate that the eIF-2 alpha kinase can undergo cyclic activation-inactivation that appears to be directly related to the phosphorylation state of the Mr = 68,000 peptide. They strongly support the previous conclusion that double-stranded RNA is required only for activation of the kinase and phosphorylation of the Mr = 68,000 peptide.

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).poly(C). Our study suggest that p68 kinase has high- and low-affinity binding sites: the high-affinity binding site is responsible for the activation and the low-affinity binding site for the inhibition of kinase activity. This is in accord with the fact that autophosphorylation of p68 kinase occurs at low concentrations of dsRNA whereas high concentrations of dsRNA inhibit its autophosphorylation. We have also investigated the binding of adenoviral VAI RNA to the purified p68 kinase and have found that the affinity of this binding is lower than that of poly(I).poly(C). We show that VAI RNA can activate or inhibit autophosphorylation of p68 kinase in a dose-dependent manner, i.e. activation at less than or equal to 1 microgram/ml or inhibition at greater than 1 microgram/ml of VAI RNA. In spite of its lower affinity of binding, VAI RNA cannot be displaced by poly(I).poly(C) or reovirus dsRNA. These data confirm our previous results to illustrate that VAI RNA can bind p68 kinase and cause its inactivation irreversably.

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. In interferon-treated cells, they restore virus sensitivity 4-6 h after the establishment of antiviral protection. In the present study we investigate the effect of sarcolectins on the steady state levels of two double-stranded RNA dependent enzymes, 2-5A (p chi (A2'p)nA) synthetase and protein kinase. Several authors have previously emphasized the role of these enzymes in the mechanism of interferon's antiviral action. Interferon promotes a 4-8 fold increase in protein kinase and 2-5A synthetase in cells. Addition of sarcolectin 5 h after interferon results in a dramatic reduction in the steady state levels of both these enzymes, as shown by their decreased activity and yield observed in Western blot assays. The degradation of the antiviral response in sarcolectin-treated cells might therefore be at least partially attributed to a reduced synthesis of protein kinase and 2-5A synthetase. Since there are no direct interactions between sarcolectins and interferon or its receptors, it can be postulated that sarcolectins exert their effect through these interferon-dependent proteins. We postulate that the opposing biological effects of interferon and sarcolectins strike a balance which may, however, be modified in one direction or the other, depending on their respective concentrations.

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. The basal activity level of this enzyme could be reduced in normal mice by treatment with anti-mouse IFN (alpha + beta) antibody. This treatment also reduced the levels of 2-5A synthetase and 1-8 mRNAs. Thus, physiological levels of circulating IFN maintain elevated levels of IFN-induced mRNAs in mice. Furthermore, changes in 2-5A synthetase enzyme activity reflect the changes in gene expression in vivo.

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. When activated by dsRNA in the presence of Mn2+ and ATP, p68 kinase is autophosphorylated and is then capable of catalyzing phosphorylation of histone in the absence of dsRNA. Whereas binding of 8-azido-[alpha-32P] ATP (8-N3ATP) to p68 kinase is dependent on both dsRNA and Mn2+, phosphorylated p68 kinase binds 8-N3ATP independent of dsRNA. This is consistent with a dsRNA requirement for the autophosphorylation of p68 kinase, but not for the phosphorylation of exogenous substrates. p68 kinase is mainly associated with the ribosomal pellet. It could be recovered efficiently by a buffer containing both high salt and a nonionic detergent. Synthesis of p68 kinase is induced several-fold by interferon in different types of human cells. Partial proteolysis of [35S]methionine and an 8-N3ATP-labeled p68 kinase preparation by Staphylococcus aureus V8 protease indicated the presence of a major Mr 48,000 polypeptide (p48) with a specific ATP-binding site. p48 probably contains the catalytic unit of p68 kinase and is analogous to a similar protein which we have previously described as a distinct protein present in a complexed form with p68 kinase. We now believe that the presence of p48 in previously purified kinase preparations was due to partial degradation of p68 kinase.

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. The second phosphorylation step can take place in the absence of dsRNA. Here we show that, besides dsRNA other polyanions, especially heparin, can also activate the p68 kinase for the autophosphorylation reaction. Heparin activation of the p68 kinase is reversible since it can be prevented by addition of antithrombin III, heparin-binding protein. However, when antithrombin III is added after autophosphorylation of the p68 kinase then phosphorylation of histone is not affected. The p68 kinase binds to heparin-Sepharose. Further evidence that the p68 kinase can be activated by heparin was provided by photoaffinity labeling with 8-azido-[alpha-32P]ATP. This ATP analog can bind to the p68 kinase only in the presence of heparin or dsRNA. Thus suggesting that the activation of the p68 kinase triggers a conformational modification allowing the binding of ATP. Basic proteins, histone and protamine, prevent the activation process induced by heparin. This is probably due to binding of these basic proteins to heparin and thus sequestering the activator of the protein kinase.

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. However, in spite of this activation, the level of p68 kinase is rapidly decreased in virus-infected cells. The half-life of p68 kinase in uninfected cells is 6 to 7 hr, whereas in EMCV-infected cells it is 2 to 3 hr. This decrease in the level of p68 kinase is dependent on the multiplicity of virus infection and it seems to be specific since other cellular proteins as well as the activity of 2'-5'-oligoadenylate synthetase are not modified. Decreased levels of p68 kinase are also observed in cells infected with VSV and vaccinia virus. In the absence of virus infection, decreased levels of p68 kinase occur in cells following incubation with poly(I).poly(C).

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. The monoclonal antibodies immunoprecipitate either a 69- or a 100-kd 2-5A synthetase. These purified 2-5A synthetases in immune complex preparations are active, i.e. addition of poly(I).poly(C) and ATP results in the synthesis of 2-5A. Both 2-5A synthetases are composed of several subspecies with similar isoelectric points in the range of 7-8 but have different subcellular localizations: 100-kd synthetase is recovered from the microsomal pellet whereas 69-kd synthetase is found to be associated with cell membranes as well as with the microsomal pellet. Different types of interferon-treated human cells express both or either forms of these enzymes. The 69- and 100-kd 2-5A synthetases were also identified by electrophoretic transfer immunoblot analysis using rabbit polyclonal antibodies against a synthetic peptide common on both 46- and 40-kd 2-5A synthetases. These results indicate that small and large size isozymes share a common peptide sequence.