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PubMed2002

Structural comparisons of empty and full cytoplasmic polyhedrosis virus. Protein-RNA interactions and implications for endogenous RNA transcription mechanism.

Viruses in the family Reoviridae are capable of transcription within the intact capsids. As the only single-shelled and thus the simplest member of the Reoviridae, cytoplasmic polyhedrosis virus (CPV) provides an attractive system for studying endogenous transcription. We report the structures of the full and empty CPV determined at 13-A resolution by electron cryomicroscopy. The structure of the empty CPV reveals a density attributed to the transcription enzyme complex, which is attached to the internal surface of the capsid shell below each of the 12 turrets. The full capsid has an identical capsid shell but contains additional internal densities contributed by the genomic double-stranded (ds) RNA. The RNA densities proximal to the capsid shell are organized into layers with a dodecahedral appearance, suggesting a genome organization of dsRNA segments each having a cone shape spooling around a transcription enzyme complex. Our structures also suggest that the capsid shell serves as a scaffold for appropriate positioning of the RNA genome, whereas nascent mRNA release takes place through the constricted central channel of the turret. Based on these observations, a detailed moving template transcription mechanism is proposed that may provide insight into the well coordinated and highly efficient endogenous RNA transcription of dsRNA viruses.

Capsid↗
PubMed2004

Serine 18 phosphorylation of RAX, the PKR activator, is required for PKR activation and consequent translation inhibition.

It is now apparent that the double-stranded (ds)RNA-dependent protein kinase, PKR, is a regulator of diverse cellular responses to stress. Recently, the murine dsRNA-binding protein RAX and its human ortholog PACT were identified as cellular activators of PKR. Previous reports demonstrate that following stress, RAX/PACT associates with and activates PKR resulting in eIF2alpha phosphorylation, consequent translation inhibition, and cell death via apoptosis. Although RAX/PACT is phosphorylated during stress, any regulatory role for this post-translational modification has been uncertain. Now we have discovered that RAX is phosphorylated on serine 18 in both human and mouse cells. The non-phosphorylatable form of RAX, RAX(S18A), although still able to bind dsRNA and associate with PKR, fails to activate PKR following stress. Furthermore, stable expression of RAX(S18A) results in a dominant-negative effect characterized by deficiency of eukaryotic initiation factor 2 alpha subunit phosphorylation, delay of translation inhibition, and failure to undergo rapid apoptosis following removal of interleukin-3. We propose that the ability of RAX to activate PKR is regulated by a sequential mechanism featuring RAX association with PKR, RAX phosphorylation at serine 18, and activation of PKR.

Amino Acid Sequence↗
PubMed2004

Double-stranded RNAs from the helminth parasite Schistosoma activate TLR3 in dendritic cells.

Stimulation of dendritic cells (DCs) by the egg stage of the helminth parasite Schistosoma mansoni activates a signaling pathway resulting in type I interferon (IFN) and IFN-stimulated gene (ISG) expression. Here, we demonstrate that S. mansoni eggs disjointedly activate myeloid differentiation factor 88 (MyD88)-dependent and MyD88-independent pathways in DCs. Inflammatory cytokine expression and NF-kappa B activation in DCs from MyD88-deficient mice were impaired, whereas signaling transducer activator of transcription (STAT) 1(Tyr701) phosphorylation and ISG expression were intact in MyD88 or Toll-like receptor (TLR)4-deficient counterparts. Accordingly, we analyzed distinct TLR members for their ability to respond to schistosome eggs and established that TLR3 resulted in the activation of NF-kappa B and the positive regulatory domain III-I site from IFN-beta promoter. Unexpectedly, egg-derived RNA possessed RNase A-resistant and RNase III-sensitive structures capable of triggering TLR3 activation, suggesting the involvement of double-stranded (ds) structures. Moreover, DCs from TLR3-deficient mice displayed a complete loss of signaling transducer activator of transcription 1 phosphorylation and ISG expression in response to egg-derived dsRNA. Finally, TLR3-deficient DCs showed a reduced response to schistosome eggs relative to wild-type cells. Collectively, our data suggest for the first time that dsRNA from a non-viral pathogen may act as an inducer of the innate immune system through TLR3.

Animals↗
PubMed2006

Double-stranded RNA-dependent protein kinase phosphorylation of the alpha-subunit of eukaryotic translation initiation factor 2 mediates apoptosis.

As the molecular processes of complex cell stress signaling pathways are defined, the subsequent challenge is to elucidate how each individual event influences the final biological outcome. Phosphorylation of the translation initiation factor 2 (eIF2alpha)atSer(51) is a molecular signal that inhibits translation in response to activation of any of four diverse eIF2alpha stress kinases. We used gene targeting to replace the wild-type Ser(51) allele with an Ala in the eIF2alpha gene to test the hypothesis that translational control through eIF2alpha phosphorylation is a central death stimulus in eukaryotic cells. Homozygous eIF2alpha mutant mouse embryo fibroblasts were resistant to the apoptotic effects of dsRNA, tumor necrosis factor-alpha, and serum deprivation. TNFalpha treatment induced eIF2alpha phosphorylation and activation of caspase 3 primarily through the dsRNA-activated eIF2alpha kinase PKR. In addition, expression of a phospho-mimetic Ser(51) to Asp mutant eIF2alpha-activated caspase 3, indicating that eIF2alpha phosphorylation is sufficient to induce apoptosis. The proapoptotic effects of PKR-mediated eIF2alpha phosphorylation contrast with the anti-apoptotic response upon activation of the PKR-related endoplasmic reticulum eIF2alpha kinase, PERK. Therefore, divergent fates of death and survival can be mediated through phosphorylation at the same site within eIF2alpha. We propose that eIF2alpha phosphorylation is fundamentally a death signal, yet it may promote either death or survival, depending upon coincident signaling events.

Animals↗
PubMed2005

Double-stranded RNA stimulates the expression of monocyte chemoattractant protein-1 in BEAS-2B bronchial epithelial cells.

BEAS-2B bronchial epithelial cells were treated with polyinosinic-polycytidylic acid (poly IC), a synthetic double-stranded RNA (dsRNA) analog, and the expressions of monocyte chemoattractant protein-1 (MCP-1) mRNA and protein were analyzed by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay. Poly IC enhanced the expression of MCP-1 and release of mononuclear cell chemotactic activity, which were inhibited by dexamethasone pre-treatment. The poly IC-induced up-regulation of MCP-1 was blocked by 2-aminopurine, a specific inhibitor of dsRNA-dependent protein kinase, but not by nuclear factor (NF)-kappaB inhibitor SN50.

Base Sequence↗
PubMed1990

Flexibility difference between double-stranded RNA and DNA as revealed by gel electrophoresis.

A systematic study of agarose gel electrophoresis of double-stranded RNA in the kilobase range of sizes was performed. The dsRNA to dsDNA relative mobility was found to depend on gel concentration: in low density gels RNA moves slower and in high density gels - faster than DNA of the same molecular size. The electrophoretic differences were interpreted within the reptation theory to be mainly due to the molecular stiffness differences. The dsRNA persistence length was roughly estimated to be about twice as great as that of DNA.

Bacteriophages↗
PubMed1992

Atomic force microscopy imaging of double stranded DNA and RNA.

A procedure for imaging long DNA and double stranded RNA (dsRNA) molecules using Atomic Force Microscopy (AFM) is described. Stable binding of double stranded DNA molecules to the flat mica surface is achieved by chemical modification of freshly cleaved mica under mild conditions with 3-aminopropyltriethoxy silane. We have obtained striking images of intact lambda DNA, Hind III restriction fragments of lambda DNA and dsRNA from reovirus. These images are stable under repeated scanning and measured contour lengths are accurate to within a few percent. This procedure leads to strong DNA attachment, allowing imaging under water. The widths of the DNA images lie in the range of 20 to 80nm for data obtained in air with commercially available probes. The work demonstrates that AFM is now a routine tool for simple measurements such as a length distribution. Improvement of substrate and sample preparation methods are needed to achieve yet higher resolution.

Aluminum Silicates↗
PubMedDate not supplied

Influenza virus induction of apoptosis by intrinsic and extrinsic mechanisms.

It is now firmly established that apoptosis is an important mechanism of influenza virus-induced cell death both in vivo and in vitro. Data are predominantly from experiments with influenza A virus and in vitro experimental systems. Multiple influenza virus factors have been identified that can activate intrinsic or extrinsic apoptotic induction pathways. Currently there is no evidence for influenza virus directly accessing the apoptosis execution factors. The best-studied influenza virus inducers of apoptosis are dsRNA, NS1, NA, and a newly described gene product PB1-F2. PB1-F2 is the only influenza virus factor to date identified to act intrinsically by localization and interaction with the mitochondrial-dependent apoptotic pathway. Both dsRNA and NA have been shown to act via an extrinsic mechanism involving proapoptotic host-defense molecules: PKR by induction of Fas-Fas ligand and NA by activation of TGF-beta. PKR is capable of controlling several important cell-signaling pathways and therefore may have multiple effects; a predominant one is increased interferon (IFN) production and activity. NS1 has been shown to be both proapoptotic and antiapoptotic. Use of influenza virus NS1 deletion mutants has provided evidence for NS1 interference with apoptosis, IFN induction, and related cell-signaling pathways. Influenza virus also has important exocrine paracrine effects, which are likely mediated via TNF family ligands and oxygen, free radicals capable of inducing apoptosis. Little is known about activation of inhibitors of apoptosis such as inhibitory apoptotic proteins. Whether all these factors always have a role in influenza virus-induced apoptosis is unknown. The kinetics of synthesis of influenza virus factors affecting apoptosis during the replication cycle may be an important aspect of apoptosis induction.

Apoptosis↗
PubMedDate not supplied

Double-stranded RNA induces the synthesis of retinoic acid-inducible gene-I in vascular endothelial cells.

Viral infection induces various responses in vascular endothelial cells. Polyinosinic-polycytidylic acid (poly IC) is a synthetic double-stranded RNA (dsRNA), and treatment of cells with poly IC mimics the viral infection to the cells. Retinoic acid-inducible gene-I (RIG-I) is a protein belonging to the DExH-box family and designated as a putative RNA helicase. RIG-I is considered to play a role in antiviral responses through the regulation of gene expressions. In the present study, the authors treated human umbilical vein endothelial cells (HUVECs) with poly IC and found that poly IC induced the expression of RIG-I. The poly IC-induced RIG-I expression was inhibited by the preincubation of the cells with 2-aminopurine, an inhibitor of dsRNA-dependent protein kinase (PKR). Immunohistochemical examination revealed high levels of RIG-I immunoreactivity in vascular endothelial cells in the thalamus from rats inoculated with hantavirus. Induction of RIG-I by poly IC may be involved in the antiviral responses in endothelial cells.

Animals↗
PubMed2004

Double-stranded RNA mediates selective gene silencing of protein phosphatase type 1 delta isoform in HEK-293 cells.

The reversible phosphorylation of proteins mediates cellular signals in eukaryotic cells. RNA interference inhibits the expression of genes and proteins in a sequence-specific manner and provides a tool to study the functions of target molecules. The effect of RNA interference on protein phosphatase isoforms in HEK-293 cells was examined. Protein phosphatase 1 delta (PP1delta) sequence-specific double-stranded RNA (dsRNA) inhibited mRNA and protein expression of the PP1delta. This RNA interference did not affect the expression of lphaand gamma1 isoforms of PP1. Transfection of antisense RNA specific for PP1delta also suppressed the expression of PP1delta. It was further demonstrated by an in vitro RNA cleavage assay that extracts of HEK-293 cells catalyzed the processing of dsRNA. This cell line had much stronger mRNA expression of Dicer, an RNase III-like enzyme, than did human osteoblastic MG63 cells. The present results show that RNA interference is a useful tool to distinguish between PP1 isoforms.

Cell Line↗
PubMed2006

Silencing of HIV-1 gene expression by siRNAs in transduced cells.

The RNA interference (RNAi) phenomenon is a recently observed process in which the introduction of a double-stranded RNA (dsRNA) into cells causes the specific degradation of an mRNA containing the same sequence. To study dsRNA-mediated gene interference targeted to the env gene (NL4-3: 7490-7508) in HIV-1 infected cells, we constructed tandem-type and hairpin-type siRNA expression vectors, which were under the control of two U6 promoters. We also constructed lentiviral-based siRNA expression vectors for further assessment of their antiviral activity in transduced cells. At both the transient plasmid and lentiviral-mediated RNA expression levels, the siRNA encoding the env fragment exhibited sequence-specific suppression of target gene expression and strongly inhibited (> or = 90%) HIV-1 infection in the cells, as compared to the antisense RNA expression vector. Targeting the HIV-1 env gene with siRNAs encoding the env gene fragment (7490-7508) might be an effective strategy for gene therapy applications in HIV-1/AIDS treatment and management.

Animals↗
PubMed2006

Inhibition of hcv replication in HCV replicon by shRNAs.

We show that the vector-derived long dsRNA specifically inhibits the replication of HCV RNA in HCV replicon. We designed a long dsRNA targeted to the full-length HCV IRES/core elements (1-to 377-nt). Our results revealed that the replication of HCV RNA was reduced to near background levels in a sequence-specific manner by the long dsRNAs in the HCV replicon. We also designed four shRNAs against several regions (120- to 139-nt, 260- to 279-nt, 330- to 349-nt, and 340- to 359-nt) of the HCV IRES/Core elements. The two HCV IRES/core-specific shRNAs, 330- to 349-nt and 340- to 359-nt, containing the AUG initiation codon sequence showed stronger HCV inhibitory effects than the other two shRNAs, 120- to 139-nt and 260- to 279-nt.

Hepacivirus↗
PubMed1980

Molecular basis of reovirus virulence. Role of the M2 gene.

The mammalian reoviruses (serotype 1, strain Lang and serotype 3, strain Dearing) differ in their sensitivity to digestion by chymotrypsin. We have found that the M2 double-stranded RNA (dsRNA) genome segment (encoding the micro1C outer capsid polypeptide) is responsible for this property. In addition to determining response to protease treatement in vitro, we have found that the M2 genome segment also determines the ability of these two viruses successfully to initiate local and systemic infection in newborn mice after peroral inoculation. Thus the M2 dsRNA segment defines a new virulence gene of the mammalian reoviruses.

Animals↗
PubMed2002

Nuclear factor 90 mediates activation of the cellular antiviral expression cascade.

Viral infection triggers a cascade of interferon response genes, but the mechanisms that prime such innate antiviral defenses are poorly understood. Among candidate cellular mediators of the antiviral response are the double-stranded RNA (dsRNA)-binding proteins. Here we show that a C-terminal variant of the ubiquitous dsRNA-binding protein, nuclear factor 90 (NF90ctv), can activate the interferon response genes in the absence of viral infection. NF90ctv-expressing cells were infected with the syncytium-inducing HIV-1 strain NL4-3 and were shown to inhibit viral replication. To gain insight into this mechanism of protection, we analyzed the expression profiles of NF90ctv-positive cells as compared with parental cells transduced with the empty vector. Of the 5600 genes represented on the expression arrays, 90 displayed significant (4-fold or more) changes in mRNA levels in NF90-expressing cells. About 50% are known interferon alpha/beta-stimulated genes. The microarray expression data were confirmed by quantitative reverse transcriptase-polymerase chain reaction analysis of six representative interferon-inducible genes. Electrophoretic mobility shift assays showed that the biological response is mediated by the activation of transcription factors in NF90ctv-expressing cells. Functional significance of the activated transcription complex was evaluated by transfection assays with luciferase reporter constructs driven by the interferon-inducible promoter from the 2'-5'-oligoadenylate synthetase (p69) gene. Resistance to HIV-1, caused by the expression of NF90ctv in the cell culture system, appears to be mediated in part by the induction of interferon response genes. This leads to a hypothesis as to the mechanism of action of NF90 in mediating endogenous antiviral responses.

Amino Acid Sequence↗
PubMed2000

Cloning and promoter analysis of the chicken interferon regulatory factor-3 gene.

Interferon regulatory factors (IRFs) are a family of DNA-binding proteins involved in mediating the cellular response to interferons (IFNs) and viral infection. Although extensively studied in mammals, IRFs of other vertebrates have been less well characterized. Previously, we cloned chicken interferon regulatory factor-3 (chIRF-3) mRNA, which is rapidly and transiently induced by double-stranded (ds)RNA. The chIRF-3 mRNA encodes a protein distinct from any known mammalian IRF. Here, we show that chIRF-3 is activated additively by type I and type II IFNs. To delineate the sequence elements required to regulate chIRF-3 expression, we cloned chlRF-3 and 0.48 kb of 5' flanking sequence. Computer analysis of the proximal promoter revealed three putative binding sites for nuclear factor (NF)-kappaB, two overlapping interferon-stimulated response elements (ISREs), and an interferon gamma activating sequence (GAS). The presence of both GAS and ISRE consensus sequences in the chIRF-3 promoter is unique among IRF family members. Both type I and II IFNs, as well as dsRNA and IRF-1, trans-activate the promoter in short-term transfection experiments. Mutational analysis of the promoter demonstrated that the putative NF-kappaB binding sites are needed for stimulation by dsRNA but not by either type I or type II IFN and that both the overlapping ISREs and GAS are required for full induction by type I or type II IFN.

Animals↗
PubMed2000

Transient resistance of influenza virus to interferon action attributed to random multiple packaging and activity of NS genes.

Interferon (IFN) action survival curves for an avian influenza virus (AIV) in chicken or quail cells showed that 40-60% of the virions in a stock of virus were highly sensitive to the inhibitory effects of chicken IFN-alpha (ChIFN-alpha), whereas the rest were up to 100 times less sensitive. This greater resistance to IFN was transient, that is, was not a stable characteristic, in that virus stocks grown from plaques that formed in the presence of 50-800 U/ml IFN gave rise to virus populations that contained both sensitive and resistant virions. If AIV was serially passaged several times in the presence of IFN, the proportion of transiently IFN-resistant virus was greater. We propose a model to account for this transient resistance of AIV to IFN action based on the reported inactivation of the dsRNA-dependent protein kinase (PKR) and its activator dsRNA by the NS1 protein of influenza virus and also on the increase in the survival of AIV in IFN-treated cells exposed to 2-aminopurine, a known inhibitor of PKR. We suggest that IFN-resistant AIV is generated from a random packaging event that results in virions that contain two or more copies of RNA segment 8, the gene segment that encodes the NS1 protein of AIV, and that these virions will produce correspondingly elevated levels of NS1. The experimental data fit well to theoretical curves based on this model and constructed from the fraction of virus in the population expected by chance to contain one, two, or three copies of the NS gene when packaging an average of 12 influenza gene segments that include the 8 segments essential for infectivity.

2-Aminopurine↗
PubMed2002

Poly I:CLC induction of the interferon system in mice: an initial study of four detection methods.

Induction of a large number of the components of the interferon (IFN) system (IFN genes, their mRNAs, IFN proteins, IFN receptors, IFN signaling molecules, the IFN response genes, and their effector proteins) has been studied. Less well studied is the comparative induction of these components in vivo. Induction of IFN by double-stranded RNA (dsRNA) treatment mimics certain aspects of viral infection and induces the components of the IFN system. To determine the comparative sensitivity of detection of induction in mice, we initially studied the limiting concentrations of polyribinosinic-polyribocytidylic acid, polylysine complex (poly I:CLC, a synthetic dsRNA preparation), for induction of four representative components of the IFN system: (1) IFN in serum, (2) the IFN response gene mRNA ISG54 in spleen and liver, (3) the IFN-beta mRNA in spleen, and (4) resistance of mice to Banzi viral infection. The results of this initial study showed that resistance to infection was 7-fold more sensitive for detection of the IFN response than was ISG54 mRNA and 70-fold more sensitive than either IFN-beta mRNA or IFN production in serum. In comparison, mouse cells in vitro treated with poly I:CLC were 3-10-fold less sensitive to its antiviral action than is the mouse. The results demonstrate that in the four tests in mice, the most sensitive indicator of poly I:CLC induction of the IFN system was protection against Banzi viral infection, followed by ISG54 mRNA levels, IFN-beta mRNA, and IFN protein levels. It is hypothesized that the highest sensitivity of mouse protection may be due to priming by the initial poly I:CLC-induced IFN of the subsequent Banzi virus-induced IFN, resulting in rapid and high concentrations of IFN at the local site of viral replication. Future studies are needed to study other molecular components of the IFN system to identify those that cause the unanticipated high sensitivity of mice to protection against Banzi virus.

Animals↗
PubMed2003

Function of ubiquitin-like domain of chicken 2'-5'-oligoadenylate synthetase in conformational stability.

2'-5'-Oligoadenylate synthetase (OAS), an interferon (IFN) induced enzyme, synthesizes 2'-5'-oligoadenylate (2-5A) from ATP when activated by dsRNA. Chicken OAS (ChOAS) has a ubiquitin-like (UbL) domain of two consecutive sequences (UbL1 and UbL2) at its carboxyl-terminus. The OAS gene has at least two alleles, OAS*A and OAS*B. OAS-A is the wild-type (wt) and OAS-B is a mutant deleted of a highly hydrophobic region of UbL1. To study the function of the UbL domain, enzymatic and physiologic properties were compared between OAS-A and OAS-B. OAS-B was more susceptible to trypsin than OAS-A and was converted very quickly into p38, deleting a greater part of the UbL domain. The p38 has the enzymatic activity to synthesize 2-5A. Thermal inactivation of OAS-B occurred at a lower temperature than that of OAS-A and p38, with loss of the ability to bind dsRNA. In contrast to OAS-A, the content of OAS-B in erythrocytes decreased during growth to a very low level. However, red blood cells (RBC) from anemic B/B chickens synthesized OAS-B at a high level comparable to A/A, although OAS-B levels decreased sharply again during maturation to erythrocytes. Thus, OAS-B carrying the mutated UbL domain is unstable compared with OAS-A in vitro and in vivo, and the wt UbL domain may contribute to the stability of the protein structure of ChOAS.

2',5'-Oligoadenylate Synthetase↗