ADAR1 and PACT contribute to efficient translation of transcripts containing HIV-1 trans-activating response (TAR) element.

Human immunodeficiency virus type 1 (HIV-1) has evolved various measures to counter the host cell's innate antiviral response during the course of infection. Interferon (IFN)-stimulated gene products are produced following HIV-1 infection to limit viral replication, but viral proteins and RNAs counteract their effect. One such mechanism is specifically directed against the IFN-induced Protein Kinase PKR, which is centrally important to the cellular antiviral response. In the presence of viral RNAs, PKR is activated and phosphorylates the translation initiation factor eIF2α. This shuts down the synthesis of both host and viral proteins, allowing the cell to mount an effective antiviral response. PACT (protein activator of PKR) is a cellular protein activator of PKR, primarily functioning to activate PKR in response to cellular stress. Recent studies have indicated that during HIV-1 infection, PACT's normal cellular function is compromised and that PACT is unable to activate PKR. Using various reporter systems and in vitro kinase assays, we establish in this report that interactions between PACT, ADAR1 and HIV-1-encoded Tat protein diminish the activation of PKR in response to HIV-1 infection. Our results highlight an important pathway by which HIV-1 transcripts subvert the host cell's antiviral activities to enhance their translation.

Endocytosis-mediated HIV-1 entry and its significance in the elusive behavior of the virus in astrocytes.

Astrocytes protect neurons but also evoke a proinflammatory response to injury and viral infections including HIV. We investigated the mechanism of HIV-1 infection in primary astrocytes, which showed minimal but productive viral infection independent of CXCR4. As with ectopic-CD4-expressing astrocytes, lysosomotropic agents led to increased HIV-1 infection in wild-type but not Rabs 5, 7, and 11-ablated astrocytes. Instead, HIV-1 infection was decreased in Rab-depleted astrocytes, corroborating viral entry by endocytosis. HIV-1 produced persistent infection in astrocytes (160 days); no evidence of latent infection was seen. Notably, one caveat is that endosomal modifiers enhanced wild-type HIV-1 infection (M- and T-tropic) in astrocytes, suggesting endocytic entry of the virus. Impeding endocytosis by inhibition of Rab 5, 7 or 11 will inhibit HIV infection in astrocytes. Although the contribution of such low-level infection in astrocytes to neurological complications is unclear, it may serve as an elusive viral reservoir in the central nervous system.

Relationship between heat shock protein 70 expression and life span in Daphnia.

The longevity of an organism is directly related to its ability to effectively cope with cellular stress. Heat shock response (HSR) protects the cells against accumulation of damaged proteins after exposure to elevated temperatures and also in aging cells. To understand the role of Hsp70 in regulating life span of Daphnia, we examined the expression of Hsp70 in two ecotypes that exhibit strikingly different life spans. Daphnia pulicaria, the long lived ecotype, showed a robust Hsp70 induction as compared to the shorter lived Daphnia pulex. Interestingly, the short-lived D. pulex isolates showed no induction of Hsp70 at the mid point in their life span. In contrast to this, the long-lived D. pulicaria continued to induce Hsp70 expression at an equivalent age. We further show that the Hsp70 expression was induced at transcriptional level in response to heat shock. The transcription factor responsible for Hsp70 induction, heat shock factor-1 (HSF-1), although present in aged organisms did not exhibit DNA-binding capability. Thus, the decline of Hsp70 induction in old organisms could be attributed to a decline in HSF-1's DNA-binding activity. These results for the first time, present a molecular analysis of the relationship between HSR and life span in Daphnia.

STAT1 requirement for PKR-induced cell cycle arrest in vascular smooth muscle cells in response to heparin.

Interferons (IFNs) are a family of cytokines that exhibit antiviral, antiproliferative, and immunomodulatory properties. PKR (protein kinase, RNA activated) is of central importance in mediating the antiproliferative actions of IFNs. Our research has established that PKR inhibits vascular smooth muscle cell (VSMC) proliferation by regulating G1 to S transition. Many cardiovascular diseases result from complications of atherosclerosis, a chronic and progressive inflammatory condition often characterized by excessive proliferation of VSMC. Thus, an effective method for inhibiting VSMC proliferation is likely to arrest atherosclerosis and restenosis at early stages. Our research establishes that PKR activation in VSMC leads to a G1 arrest brought about by an inhibition of cyclin-dependent kinase 2 (Cdk2) activity by p27(kip1). In quiescent VSMC, p27(kip1) levels are high and when stimulated by serum/growth factors, p27(kip1) levels drop by destabilization of the protein. Under conditions that lead to activation of PKR, there is a marked inhibition of p27(kip1) down-regulation due to increased stability of p27(kip1) protein. In order to understand the mechanism of heparin-induced stabilization of p27(kip1) in VSMC, we examined the involvement of the Signal Transducer and Activator of Transcription-1 (STAT1), which is an important player in mediating antiproliferative effects of IFNs. Our results demonstrate that PKR overexpression in VSMC leads to an increase in p27(kip1) protein levels and this increase requires the catalytic activity of PKR. PKR activation induced by antiproliferative agent heparin leads to phosphorylation of STAT1 on serine 727, which is essential for the cell cycle block. STAT1 null VSMCs are largely defective in heparin-induced cell cycle arrest and in PKR null cells the STAT1 phosphorylation in response to heparin was absent. These results establish that heparin causes STAT1 phosphorylation on serine 727 via activation of PKR and that this event is required for the G1 arrest in VSMC.

Bryostatin modulates latent HIV-1 infection via PKC and AMPK signaling but inhibits acute infection in a receptor independent manner.

HIV's ability to establish long-lived latent infection is mainly due to transcriptional silencing in resting memory T lymphocytes and other non dividing cells including monocytes. Despite an undetectable viral load in patients treated with potent antiretrovirals, current therapy is unable to purge the virus from these latent reservoirs. In order to broaden the inhibitory range and effectiveness of current antiretrovirals, the potential of bryostatin was investigated as an HIV inhibitor and latent activator. Bryostatin revealed antiviral activity against R5- and X4-tropic viruses in receptor independent and partly via transient decrease in CD4/CXCR4 expression. Further, bryostatin at low nanomolar concentrations robustly reactivated latent viral infection in monocytic and lymphocytic cells via activation of Protein Kinase C (PKC) -alpha and -delta, because PKC inhibitors rottlerin and GF109203X abrogated the bryostatin effect. Bryostatin specifically modulated novel PKC (nPKC) involving stress induced AMP Kinase (AMPK) inasmuch as an inhibitor of AMPK, compound C partially ablated the viral reactivation effect. Above all, bryostatin was non-toxic in vitro and was unable to provoke T-cell activation. The dual role of bryostatin on HIV life cycle may be a beneficial adjunct to the treatment of HIV especially by purging latent virus from different cellular reservoirs such as brain and lymphoid organs.

Essential role of PACT-mediated PKR activation in tunicamycin-induced apoptosis.

Cellular stresses such as disruption of calcium homeostasis, inhibition of protein glycosylation, and reduction of disulfide bonds result in accumulation of misfolded proteins in the endoplasmic reticulum (ER) and lead to cell death by apoptosis. Tunicamycin, which is an inhibitor of protein glycosylation, induces ER stress and apoptosis. In this study, we examined the involvement of double-stranded RNA (dsRNA)-activated protein kinase (PKR) and its protein activator PACT in tunicamycin-induced apoptosis. We demonstrate for the first time that PACT is phosphorylated in response to tunicamycin and is responsible for PKR activation by direct interaction. Furthermore, PACT-induced PKR activation is essential for tunicamycin-induced apoptosis, since PACT as well as PKR null cells are markedly resistant to tunicamycin and show defective eIF2alpha phosphorylation and C/EBP homologous protein (CHOP, also known as GADD153) induction especially at low concentrations of tunicamycin. Reconstitution of PKR and PACT expression in the null cells renders them sensitive to tunicamycin, thus demonstrating that PACT-induced PKR activation plays an essential function in induction of apoptosis.

Interaction of human tRNA-dihydrouridine synthase-2 with interferon-induced protein kinase PKR.

PKR is an interferon (IFN)-induced protein kinase, which is involved in regulation of antiviral innate immunity, stress signaling, cell proliferation and programmed cell death. Although a low amount of PKR is expressed ubiquitously in all cell types in the absence of IFNs, PKR expression is induced at transcriptional level by IFN. PKR's enzymatic activity is activated by its binding to one of its activators. Double-stranded (ds) RNA, protein activator PACT and heparin are the three known activators of PKR. Activation of PKR in cells leads to a general block in protein synthesis due to phosphorylation of eIF2alpha on serine 51 by PKR. PKR activation is regulated very tightly in mammalian cells and a prolonged activation of PKR leads to apoptosis. Thus, positive and negative regulation of PKR activation is important for cell viability and function. The studies presented here describe human dihydrouridine synthase-2 (hDUS2) as a novel regulator of PKR. We originally identified hDUS2 as a protein interacting with PACT in a yeast two-hybrid screen. Further characterization revealed that hDUS2 also interacts with PKR through its dsRNA binding/dimerization domain and inhibits its kinase activity. Our results suggest that hDUS2 may act as a novel inhibitor of PKR in cells.

Identification of the heparin-binding domains of the interferon-induced protein kinase, PKR.

PKR is an interferon-induced serine-threonine protein kinase that plays an important role in the mediation of the antiviral and antiproliferative actions of interferons. PKR is present at low basal levels in cells and its expression is induced at the transcriptional level by interferons. PKR's kinase activity stays latent until it binds to its activator. In the case of virally infected cells, double-stranded (ds) RNA serves as PKR's activator. The dsRNA binds to PKR via two copies of an evolutionarily conserved motif, thus inducing a conformational change, unmasking the ATP-binding site and leading to autophosphorylation of PKR. Activated PKR then phosphorylates the alpha-subunit of the protein synthesis initiation factor 2 (eIF2alpha) thereby inducing a general block in the initiation of protein synthesis. In addition to dsRNA, polyanionic agents such as heparin can also activate PKR. In contrast to dsRNA-induced activation of PKR, heparin-dependent PKR activation has so far remained uncharacterized. In order to understand the mechanism of heparin-induced PKR activation, we have mapped the heparin-binding domains of PKR. Our results indicate that PKR has two heparin-binding domains that are nonoverlapping with its dsRNA-binding domains. Although both these domains can function independently of each other, they function cooperatively when present together. Point mutations created within these domains rendered PKR defective in heparin-binding, thereby confirming their essential role. In addition, these mutants were defective in kinase activity as determined by both in vitro and in vivo assays.

Regulation of vascular smooth muscle proliferation by heparin: inhibition of cyclin-dependent kinase 2 activity by p27(kip1).

Uncontrolled proliferation of vascular smooth muscle cells (VSMCs) contribute to intimal hyperplasia during atherosclerosis and restenosis. Heparin is an antiproliferative agent for VSMCs and has been shown to block VSMC proliferation both in tissue culture systems and in animals. Despite the well documented antiproliferative actions of heparin, its cellular targets largely remain unknown. In an effort to characterize the mechanism of the antiproliferative property of heparin, we have analyzed the effect of heparin on cell cycle in VSMC. Our results indicate that the heparin-induced block in G(1) to S phase transition is imposed by p27(kip1)-mediated inhibition of cyclin-dependent kinase 2 activity. Further analysis of p27(kip1) mRNA levels showed that the increase in p27(kip1) protein levels in heparin-treated VSMC occurs at posttranscriptional levels. We present evidence that heparin causes stabilization of p27(kip1) protein during G(1) phase and thereby prevents activation of cyclin-dependent kinase 2.

Contribution of double-stranded RNA-activated protein kinase toward antiproliferative actions of heparin on vascular smooth muscle cells.

OBJECTIVE: The proliferation of vascular smooth muscle cells (VSMCs) in blood vessels after endothelial injury contributes to the onset of atherosclerosis. Heparin is a potent antiproliferative agent for VSMCs in vivo and in vitro. Although heparin has shown promise in suppressing VSMC proliferation after invasive procedures in laboratory animals, the mechanism of its antiproliferative actions is largely unknown. Here, we present evidence for the first time that the antiproliferative action of heparin is in part mediated by its ability to activate double-stranded RNA-activated protein kinase (PKR), an interferon-induced protein kinase. METHODS AND RESULTS: We have analyzed the VSMC proliferation by cell-cycle analysis and correlated it to the kinase activity of PKR in the presence of heparin. Heparin treatment of VSMCs results in activation of PKR by direct binding and results in a block in G1- to S-phase transition. PKR-null cells are largely insensitive to the antiproliferative actions of heparin, and inhibition of PKR in VSMCs results in a partial abrogation of the antiproliferative effects of heparin. CONCLUSIONS: These results invoke the involvement of novel PKR-dependent regulatory pathways in mediating the antiproliferative actions of heparin.