Inhibition of interferon-mediated antiviral responses by influenza A viruses and other negative-strand RNA viruses.
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Animals↗
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A viral dsRNA element of the chestnut blight fungus with a distinct genetic organization.
We have sequenced overlapping complementary DNA clones representing the viral double-stranded (ds) RNA from hypovirulent strain NB58 of the chestnut blight fungus Cryphonectria parasitica. Cryphonectria hypovirus 2-NB58 (CHV2-NB58) dsRNA contains 12,507 base pairs, excluding the poly(A) tail at the 3' end of the plus strand, and is organizationally similar to the largest dsRNA from the virus of strain EP713 (CHV1-713; identical to HAV; Shapira et al., (1991), EMBO J. 10, 731-739). CHV2-NB58 and CHV1-713 dsRNAs share approximately 60% nucleotide sequence identity. On the poly(A)-containing strand of CHV2-NB58, a 487-residue nontranslated region precedes two open reading frames, designated ORF A (438 codons) and ORF B (3291 codons). The connecting pentanucleotide sequence UAAUG (1802-1806) terminates ORF A and initiates ORF B. In contrast to the 69-kDa ORF A product of CHV1-713, the 50-kDa CHV2-NB58 ORF A product did not undergo autoproteolysis under the conditions tested, nor were motifs associated with cysteine proteases present in the CHV2-NB58 ORF A sequence. CHV2-NB58 ORF B products appear to be homologous with CHV1-713 ORF B products, and the motifs involved in autoproteolysis of the N-terminal 48 kDa of CHV1-713 ORF B were identified in the CHV2-NB58 ORF B product. Motifs associated with RNA polymerase and helicase activities were highly conserved between CHV2-NB58 and CHV1-713 and were found at similar genomic positions in the C-terminal half of ORF B.
Amino Acid Sequence↗
Negative regulation of interferon-regulatory factor 3-dependent innate antiviral response by the prolyl isomerase Pin1.
Recognition of double-stranded RNA activates interferon-regulatory factor 3 (IRF3)-dependent expression of antiviral factors. Although the molecular mechanisms underlying the activation of IRF3 have been studied, the mechanisms by which IRF3 activity is reduced have not. Here we report that activation of IRF3 is negatively regulated by the peptidyl-prolyl isomerase Pin1. After stimulation by double-stranded RNA, induced phosphorylation of the Ser339-Pro340 motif of IRF3 led to its interaction with Pin1 and finally polyubiquitination and then proteasome-dependent degradation of IRF3. Suppression of Pin1 by RNA interference or genetic deletion resulted in enhanced IRF-3-dependent production of interferon-beta, with consequent reduction of virus replication. These results elucidate a previously unknown mechanism for controlling innate antiviral responses by negatively regulating IRF3 activity via Pin1.
Animals↗
MAK10, a glucose-repressible gene necessary for replication of a dsRNA virus of Saccharomyces cerevisiae, has T cell receptor alpha-subunit motifs.
The MAK10 gene is necessary for the propagation of the L-A dsRNA virus of the yeast Saccharomyces cerevisiae. We have isolated MAK10 from selected phage lambda genomic DNA clones that map near MAK10. This gene encodes a 733-amino acid protein with several regions of similarity to T cell receptor alpha-subunit V (variable) regions. We show that MAK10 is essential for optimal growth on nonfermentable carbon sources independent of its effect on L-A. Although loss of L-A by mak10-1 mutants is partially suppressed by loss of the mitochondrial genome, no such suppression of a mak10::URA3 mutation was observed. Using MAK10-lacZ fusions we show that MAK10 is expressed at a very low level and that it is glucose repressed. The highest levels of expression were seen in tup1 and cyc8 mutants, known to be defective in glucose repression. These results suggest that the mitochondrial genome and L-A dsRNA compete for the MAK10 protein.
Acetyltransferases↗
Yeast 20 S RNA replicon. Replication intermediates and encoded putative RNA polymerase.
The 20 S RNA genome is a circular single-stranded replicon, present in most laboratory yeast strains, whose copy number is induced 10,000-fold by transfer of cells to acetate medium without a carbon source. We have sequenced most of the 20 S RNA genome, and the (+) strand has a long open reading frame with the potential to encode a protein with homology to viral RNA-dependent RNA polymerases. The presence of a typical cAMP-dependent phosphorylation site in the putative RNA polymerase suggests that the acetate amplification of the 20 S RNA genome might be mediated by cAMP, a signal known to transmit the same nutritional status information to the sporulation-control system. Our inability to clone across the gap in the sequence suggests either autocatalytic cleavage of the RNA in the reverse transcriptase reaction, an unusual linkage of 5' and 3' ends of a fundamentally linear molecule, or a structure unusually resistant to reverse transcription. The identity of our sequence with that of the accompanying paper (Rodriguez-Cousino, N., Esteban, L.M., and Esteban, R. (1991) J. Biol. Chem. 266, 12772-12778) for W double-stranded RNA (dsRNA) suggests that W is the replicative form of 20 S RNA. The presence of single-stranded (+) and (-) strands and greater than unit length molecules suggests a rolling circle mode of replication as has been suggested for viroids.
Amino Acid Sequence↗
Mismatched double-stranded RNA: polyI:polyC12U.
Ampligen [polyI:polyC12U] is a mismatched double-stranded RNA that acts by inducing interferon production (immunomodulator) and by activating an intracellular enzyme (RNase-L) against viral RNA transcripts (antiviral). Ampligen, currently under development by Hemispherx Biopharma in the US, acts on the immunological system through T-lymphocyte stimulation and is indicated for the treatment of chronic fatigue syndrome and acquired immunodeficiency deficiency syndrome (AIDS), as part of the combined therapy. Ampligen is available for licensing worldwide. In February 2004, Fujisawa Deutschland GmbH, a subsidiary of Fujisawa Pharmaceutical Co., entered into an option agreement with Hemispherx Biopharma with the intent of becoming a distributor for Ampligen for the potential treatment of chronic fatigue syndrome in Germany, Switzerland and Austria. An option fee of 400,000 euros was paid pursuant to the terms of the option agreement and upon execution of the Distribution Agreement, Fujisawa will pay Hemispherx fees and milestone payments with a potential worth of several millions of dollars. In September 2003, Hemispherx Biopharma Inc. entered into an agreement with Guangdong Medicine Group Corporation to organise clinical trials, marketing, sales and distribution for both of its lead compounds, Ampligen and Alferon N in the People's Republic of China. The agreement stipulates that the Guangdong Medicine Group Corporation (GMC) will conduct clinical trials with Ampligen for the treatment of HIV. All costs related to the trials are to be covered by GMC. Additionally, GMC has to develop and implement marketing and promotional programmes. In May 2003, Hemispherx Biopharma and the Center for Cell and Gene Therapy entered into a research project agreement that will see Ampligen implemented in a protocol used in patients with relapsed EBV-positive Hodgkin's Lymphoma. In March 2002, Esteve and Hemispherx Biopharma entered into a collaborative agreement under which Esteve will be the sole distributor of Ampligen in Spain, Portugal and Andorra for the treatment of chronic fatigue syndrome. Under this agreement, in addition to other terms, Esteve will also collaborate in the drug product development by conducting clinical studies in Spain in patients coinfected with HIV/HCV. In July 2001 Hemispherx Biopharma announced that it had formed a strategic alliance with Empire Health Resources for clinical trials of Ampligen in the treatment of HIV and hepatitis C virus infections. Empire Health Resources, a healthcare management firm, will be responsible for accrual and retention of patients for HIV trials, and protocols for trials in patients with hepatitis C or both HIV and hepatitis C infections. Hemispherx has entered into a collaboration with RED Laboratories, and RED Laboratories NV expects that this will facilitate the continued development of Ampligen. Hemispherx has also entered into an agreement with Schering Plough to use a Schering facility as its principal manufacturing platform in the US. This agreement may be expanded to include other territories. Hemispherx and AOP Orphan Pharmaceuticals have signed a marketing agreement for Ampligen for the treatment of chronic fatigue syndrome for Austria, the Czech Republic, Poland and Hungary. In an arrangement between Hemispherx and Bioclones, Bioclones has certain marketing rights for Ampligen in the Southern Hemisphere, UK and Ireland. In the US, Ampligen has been granted orphan drug status for the treatment of AIDS, renal cell carcinoma (phase II, completed), chronic fatigue syndrome (phase III) and invasive/metastatic malignant melanoma (phase II). In August 2004, Hemispherx announced that it intends to use the proceeds from the private placement of company stock to complete the clinical work for its immunotherapeutics/ antivirals Ampligen and Oragens. Previously, Hemispherx submitted an application to the EMEA for the approval of Ampligen for the treatment of chronic fatigue syndrome; the first stage of th;) for the treatment of chronic fatigue syndrome; the first stage of the regulatory review has been cleared. In 2000, Hemispherx Europe (Hemispherx) obtained orphan drug status for Ampligen for the treatment of chronic fatigue syndrome in the EU, providing Hemispherx with 10 years of marketing exclusivity following the launch of the drug, as well as potential financial research benefits for the agent. In February 2000, Crystaal Corporation (now Biovail Pharmaceuticals Canada) acquired exclusive marketing rights to Ampligen in Canada, where it submitted an NDA for the agent for the treatment of chronic fatigue syndrome. In the meantime, Ampligen has been available since May 1996 under the Canadian Emergency Drug Release Programme for the treatment of chronic fatigue syndrome and immune dysfunction syndrome by Rivex Pharma (Helix BioPharma). Bioclones has initiated clinical studies with Ampligen for the treatment of chronic fatigue syndrome in Australia. The active substance for Ampligen is manufactured by F.H. Faulding Ltd. Clinical treatment programmes for chronic fatigue syndrome in other Pacific Rim countries are planned. Ampligen is available for severe chronic fatigue syndrome on a named patient, cost-recovery basis in South Africa. Hemispherx has developed a 'ready-to-use' liquid formulation of the drug and has begun treating patients with chronic fatigue syndrome in ongoing clinical trials. Hemispherx has also developed an oral version of the drug (Oragen), which is undergoing preclinical evaluation. In February 2001, Hemispherx Biopharma announced that it was initiating phase II/III trials of Ampligen in the treatment of late-stage, multidrug-resistant strains of HIV in the European Union. Patients treated in these studies will have exhausted all other treatment options. In July 2001, Hemispherx stated that Ampligen was being evaluated in a phase IIb trial in patients with HIV in the US. The trial, comprising two studies, REARMI and REARMII (Research/Evaluation of Ampligen for Retroviral Mutations I and II), will evaluate the ability of Ampligen to prevent the emergence of mutated, drug-resistant strains of the virus. 'Several hundred' patients currently on antiretroviral therapy and at risk of viral relapse will be enrolled at centres in Connecticut, New York, Florida and California. A second phase IIb study evaluating the effect of Ampligen on structured treatment interruptions (STI) is also underway. Final results from this study were reported in December 2002. NIH sponsored studies of potential therapies for SARS have identified Ampligen as having unusually high and consistent antiviral activity against human coronavirus, the pathogen implicated as the causative agent of the disease. Ampligen demonstrated very high potency at very low concentrations (0.4 microg/mL) and had a favourable safety profile. In October 2003, Hemispherx announced that, based on these promising new results, the company will stockpile injectible and/or oral formats of Ampligen and Alferon N. Independent researchers have demonstrated the antiviral activity of Ampligen against flaviviruses (West Nile virus, Equine Encephalitis virus, Dengue fever virus and Japanese Encephalitis virus) as well as virus classes associated with bioterrorism. In an animal study, Ampligen was shown to prevent destruction of nerve cells, reduce virus concentrations in the brain and blood stream and increase survival rates. Researchers at the Rega Institute in Belgium have published results from an animal study demonstrating that Ampligen was superior at protecting mice against coxsackie B3 virus-induced myocarditis compared with pegylated interferon. In May 2004 Hemispherx announced that it had filed an expanded US patent application covering the use of Ampligen for the potential treatment and prevention of severe acute respiratory syndrome (SARS) and dreaded emerging viruses.
Animals↗
Analysis of the dsRNAs of apple chlorotic leaf spot virus.
Double-stranded RNAs were isolated from plants infected with five different isolates of apple chlorotic leaf spot virus (ACLSV). Analysis by PAGE and by Northern blot hybridization showed that six major species of viral dsRNA of approximately 7.5, 6.4, 5.4, 2.2, 1.1 and 1.0 kbp can be detected in infected plants, irrespective of the ACLSV isolate used. In addition to the dsRNA of 7.5 kbp corresponding to the full-length genome, the size and position on the genome of the 2.2 and 1.1 kbp species indicate that these are very probably double-stranded forms of subgenomic RNAs allowing the expression of the internal open reading frames coding respectively for the ACLSV 50K and coat proteins. The subgenomic messenger for the coat protein was indeed detected in total RNA preparations from infected plants. Surprisingly, the two most abundant dsRNA species, of 6.4 and 5.4 kbp, were found to be 5'-coterminal with the genomic RNA. A model for the expression of the genome of ACLSV and for the production of the molecules 5'-coterminal with the genomic RNA is presented.
Base Sequence↗
"Superkiller" mutations suppress chromosomal mutations affecting double-stranded RNA killer plasmid replication in saccharomyces cerevisiae.
Saccharomyces cerevisiae strains carrying a 1.5 x 10(6)-dalton double-stranded RNA genome in virus-like particles (killer plasmid) secrete a protein toxin that kills strains not carrying this plasmid. At least 28 chromosomal genes (mak genes) are required to maintain or replicate this plasmid. Recessive mutations in any of four other chromosomal genes (ski for superkiller) result in enhanced toxin production. We report that many ski- mak- double mutants are able to maintain the killer plasmid, indicating that the SKI products have an effect on plasmid replication. The ski1-1 mutation suppresses (bypasses) all mak mutations tested except mak16-1. A variant killer plasmid is described that confers the superkiller phenotype and, like chromosomal ski mutations, makes several mak genes dispensable for plasmid replication.
Genes, Lethal↗
Transcription and in vitro translation of the dsRNA virus isolated from Rhizoctonia solani.
A segmented double-stranded dsRNA virus has been isolated from virulent strains of Rhizoctonia solani. The dsRNA genome has mol. wts. of 1.45 and 1.32 X 10(6). Two full-size transcripts with mol. wts. of 0.74 and 0.66 X 10(6) (2.2 kb and 2 kb, respectively) were synthesized by the virus-associated RNA-dependent RNA polymerase and resolved by denaturing polyacrylamide gel electrophoresis. The transcripts cross-hybridized to the viral dsRNA isolated from a number of strains. The transcripts did not hybridize with the genomic DNA. An unencapsidated species of dsRNA with mol. wt. of 1.6 X 10(6) did not hybridize with the viral transcripts. No cross-hybridization between the two viral dsRNA segments was obtained. The viral-encoded proteins were studied by in vitro translation using the rabbit reticulocyte lysate system. The transcripts served as mRNA for the synthesis of the major capsid protein of 55 kD, and a number of other products. The viral coat protein was immunoprecipitated with antibodies against purified virus particles. Partial proteolysis of the major in vitro product and the authentic capsid protein using Staphylococcus aureus V8 protease produced similar peptide patterns. Denatured viral dsRNA also directed the synthesis of proteins identical to those translated from the transcripts in vitro.
Animals↗
Characterization of the Palyam serogroup viruses (Reoviridae: Orbivirus).
31 Palyam serogroup viruses were examined by complement-fixation and plaque-reduction neutralization tests and by PAGE of the segmented, double-stranded (ds) RNA genome. Although the viruses were indistinguishable by complement-fixation tests, 10 distinct virus serotypes were identified by plaque-reduction neutralization methods. Palyam group viruses which were distinct by the neutralization test had unique dsRNA profiles, whereas those agents which were indistinct by the neutralization test had identical dsRNA profiles. 20 isolations of 3 Palyam serotypes were made from bovines and Culicoides midges in Australia over a 9-year period. When the genome of these isolates was examined electrophoretically, the dsRNA profiles of virus isolates within a given serotype were identical.
Animals↗
Characterisation of several heterogeneous species of defective RNAs derived from RNA 3 of cucumber mosaic virus.
Preparations of double-stranded RNAs (dsRNAs) extracted from Nicotiana tabacum cv Xanthi plants infected with a subgroup IB isolate of Cucumber mosaic virus (CMV) were found to contain a heterogeneous population of defective RNAs (D-RNAs) derived from RNA 3. Characterised D-RNAs ranged in size from 1.5 to 1.9 kb and were derived either by a single in-frame deletion within the 3a or 3b genes or by means of double in-frame deletions within both genes. Also, northern blot hybridisation showed two other types of RNA derived from RNA 3: (a) RNA species of ca. 0.7 kb containing the 3'-terminus but lacking the 5'-terminus, which could be 3'-coterminal subgenomic of D-RNAs derived from the 3b gene and (b) RNA species of unknown origin of ca. 0.8 kb containing the 5'-terminus but lacking the 3'-terminus.
Base Sequence↗
Short interfering RNA accumulation correlates with host recovery in DNA virus-infected hosts, and gene silencing targets specific viral sequences.
Viruses are both inducers and targets of posttranscriptional gene silencing (PTGS), a natural defense mechanism in plants. Here we report molecular evidence of the ability of single-stranded DNA (ssDNA) viruses to induce PTGS in infected plants irrespective of the severity of or recovery from the symptoms. Our results reveal that five distinct species of cassava-infecting geminiviruses were capable of triggering PTGS by producing two classes of virus-specific short interfering RNAs (siRNAs) of 21 to 26 nucleotides in two plant hosts, tobacco (Nicotiana benthamiana) and cassava (Manihot esculenta, Crantz). However, the efficacy of virus-induced PTGS varied depending on the intrinsic features of the virus and its interaction with the plant host. We found that symptom recovery over time in plants infected with the isolates of African cassava mosaic virus (ACMV-[CM]) or Sri Lankan cassava mosaic virus was associated with a much higher level of virus-derived siRNA accumulation compared to plants infected with viruses that do not show symptom recovery. Furthermore, we determined that the C terminus of AC1 that overlaps with the N terminus of AC2 early viral genes involved in virus replication were the primary targets for ACMV-[CM]-induced PTGS, whereas the C terminus of BC1 was targeted for the East African cassava mosaic Cameroon virus. In addition, our results reveal the possibility for double-stranded RNA formation during transcription in ssDNA viruses, which explains in part how these viruses can trigger PTGS in plants.
Base Sequence↗
IFN-alpha enhances TLR3-mediated antiviral cytokine expression in human endothelial and epithelial cells by up-regulating TLR3 expression.
TLRs play a critical role in early innate immune response to virus infection. TLR3 together with TLR7 and TLR8 constitute a powerful system to detect genetic material of RNA viruses. TLR3 has been shown to bind viral dsRNA whereas TLR7 and TLR8 are receptors for viral single-stranded RNA. In this report we show that TLR7 or TLR8 are not expressed in human epithelial A549 cells or in HUVECs. Accordingly, A549 cells and HUVECs were unresponsive to TLR7/8 ligand R848. TLR3 was expressed at a higher level in HUVECs than in A549 cells. The TLR3 ligand poly(I:C) up-regulated IFN-beta, IL-28, IL-29, STAT1, and TLR3 expression in HUVECs but not in A549 cells. An enhanced TLR3 expression by transfection or by IFN-alpha stimulation conferred poly(I:C) responsiveness in A549 cells. Similarly, IFN-alpha pretreatment strongly enhanced poly(I:C)-induced activation of IFN-beta, IL-28, and IL-29 genes also in HUVECs. In conclusion, our results suggest that IFN-alpha-induced up-regulation of TLR3 expression is involved in dsRNA activated antiviral response in human epithelial and endothelial cells.
Cell Line, Tumor↗
Regulation of the antiviral and anticellular activities of interferon by exogenous double-stranded RNA.
The effects of double-stranded RNA (dsRNA) on interferon (IFN)-induced antiviral and anticellular activities was investigated by introducing poly(I)-poly(C) into mouse L-cells. Coprecipitation of dsRNA with calcium phosphate enabled its efficient penetration into cells in culture. Rate of cellular protein synthesis was inhibited by dsRNA only in cultures pretreated with IFN. Moreover, the anticellular effect of IFN, as measured by the inhibition of cell DNA synthesis, was also enhanced by dsRNA. The kinetics of dsRNA-mediated inhibition of protein synthesis were relatively slow as compared with the inhibitory effect of 2'-5' oligoadenylic acid (2'5'A), which was also introduced into cells by the calcium phosphate coprecipitation technique. To analyze the effects of dsRNA on the antiviral state induced by IFN, vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMC), replications were followed by measuring viral-specific RNA synthesis in the cell. Introduction of dsRNA after the infection had no effect on VSV and EMC replication in control cells, and it enhanced, to a small extent, the antiviral state of cells pretreated with IFN. In contrast, introduction of 2'5'A into virus-infected cells inhibited VSV and EMC replications regardless of IFN pretreatment. This work demonstrated that the role of dsRNA in regulating the antiviral and anticellular activities of IFN could be studied by introducing exogenous dsRNA into cells in culture by the calcium phosphate coprecipitation technique.
2',5'-Oligoadenylate Synthetase↗
Single- and double-stranded RNAs associated with an isolate of beet soil-borne virus.
Ethidium bromide staining of electrophoretically separated ssRNAs and dsRNAs as well as northern blot analyses with cDNA clones suggested that the genome of the Ahlum serotype of beet soil-borne virus (BSBV) consists of two major ssRNA species of approximately 3.6 and 3.2 kb, respectively, and possibly a minor ssRNA of approximately 6.0 kb. A few of our clones hybridized with both the 3.6-kb and the 3.2-kb RNAs, the majority of the clones, however, hybridized only with the 3.2-kb RNA. The 3.2-kb RNA is, therefore, apparently not a degradation product or a partially deleted form of the 3.6-kb RNA. None of our clones hybridized with the faint band(s) of the 6.0-kb ssRNA(s) which was produced by RNA extracts of some of our virus preparations. A fourth ssRNA of approximately 3.0 kb, which hybridized with the same clones as the 3.2-kb RNA, was found at relatively high concentrations in RNA extracts from purified virus, but not in total RNA extracts from leaves. Its origin is unknown. It is apparently not derived from the 3.2-kb RNA by the loss of a VPg or a poly(A) tail. Hybridization tests with 32P-labelled poly(dT) suggested that none of the RNAs of BSBV is polyadenylated. With respect to size and the lack of a poly(A) tail the RNAs of BSBV are more similar to those of definitive furoviruses than to those of beet necrotic yellow vein virus which is only a possible member of the furovirus group and has RNAs which readily hybridized with poly(dT).
Blotting, Northern↗
Biophysical characterization of the complex between double-stranded RNA and the N-terminal domain of the NS1 protein from influenza A virus: evidence for a novel RNA-binding mode.
The influenza virus nonstructural protein 1 encoded by influenza A virus (NS1A protein) is a multifunctional protein involved in both protein-protein and protein-RNA interactions. NS1A binds nonspecifically to double-stranded RNA (dsRNA) and to specific protein targets, and regulates several post-transcriptional processes. The N-terminal structural domain corresponding to the first 73 amino acids of the NS1 protein from influenza A/Udorn/72 virus [NS1A(1-73)] possesses all of the dsRNA binding activities of the full-length protein. Both NMR and X-ray crystallography of this domain have demonstrated that it is a symmetric homodimer which forms a six-helix chain fold, a unique structure that differs from that of the predominant class of dsRNA-binding domains, termed dsRBDs, that are found in a large number of eukaryotic and prokaryotic proteins. Here we describe biophysical experiments on complexes containing NS1A(1-73) and a short 16 bp synthetic dsRNA duplex. From sedimentation equilibrium measurements, we determined that the dimeric NS1A(1-73) binds to the dsRNA duplex with a 1:1 stoichiometry, yielding a complex with an apparent dissociation constant (K(d)) of approximately 1 microM. Circular dichroism and nuclear magnetic resonance (NMR) data demonstrate that the conformations of both NS1A(1-73) and dsRNA in the complex are similar to their free forms, indicating little or no structural change in the protein or RNA upon complex formation. NMR chemical shift perturbation experiments show that the dsRNA-binding epitope of NS1A(1-73) is associated with helices 2 and 2'. Analytical gel filtration and gel shift studies of the interaction between NS1A(1-73) and different double-stranded nucleic acids indicate that NS1A(1-73) recognizes canonical A-form dsRNA, but does not bind to dsDNA or dsRNA-DNA hybrids, which feature B-type or A/B-type intermediate conformations, respectively. On the basis of these results, we propose a three-dimensional model of the complex in which NS1A(1-73) sits astride the minor groove of A-form RNA with a few amino acids in the helix 2-helix 2' face forming an electrostatically stabilized interaction with the phosphodiester backbone. This mode of dsRNA binding differs from that observed for any other dsRNA-binding protein.
Carbon Isotopes↗
Tentative identification of RNA-dependent RNA polymerases of dsRNA viruses and their relationship to positive strand RNA viral polymerases.
Amino acid sequence stretches similar to the four most conserved segments of positive strand RNA viral RNA-dependent RNA polymerases have been identified in proteins of four dsRNA viruses belonging to three families, i.e. P2 protein of bacteriophage phi 6 (Cystoviridae), RNA 2 product of infectious bursa disease virus (Birnaviridae), lambda 3 protein of reovirus, and VP1 of bluetongue virus (Reoviridae). High statistical significance of the observed similarity was demonstrated, allowing identification of these proteins as likely candidates for RNA-dependent RNA polymerases. Based on these observations, and on the previously reported sequence similarity between the RNA polymerases of a yeast dsRNA virus and those of positive strand RNA viruses, a possible evolutionary relationship between the two virus classes is discussed.
Amino Acid Sequence↗
Role for bovine viral diarrhea virus Erns glycoprotein in the control of activation of beta interferon by double-stranded RNA.
Production of alpha/beta interferon in response to viral double-stranded RNA (dsRNA) produced during viral replication is a first line of defense against viral infections. Here we demonstrate that the Erns glycoprotein of the pestivirus bovine viral diarrhea virus can act as an inhibitor of dsRNA-induced responses of cells. This effect is seen whether Erns is constitutively expressed in cells or exogenously added to the culture medium. The Erns effect is specific to dsRNA since activation of NF-kappaB in cells infected with Semliki Forest virus or treated with tumor necrosis factor alpha was not affected. We also show that Erns contains a dsRNA-binding activity, and its RNase is active against dsRNA at a low pH. Both the dsRNA binding and RNase activities are required for the inhibition of dsRNA signaling, and we discuss here a model to account for these observations.
Animals↗