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Jovan Pavlovic - One of the best experts on this subject based on the ideXlab platform.
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human mxa protein confers resistance to semliki forest virus and inhibits the amplification of a semliki forest virus based replicon in the absence of Viral Structural Proteins
Journal of Virology, 1998Co-Authors: Heinrich Landis, Angela Simonjodicke, Andreas Kloti, Claudio Di Paolo, J J Schnorr, Sibylle Schneiderschaulies, Hans Peter Hefti, Jovan PavlovicAbstract:Mx Proteins form a small family of interferon (IFN)-induced GTPases with potent antiViral activity against various negative-strand RNA viruses. To examine the antiViral spectrum of human MxA in homologous cells, we stably transfected HEp-2 cells with a plasmid directing the expression of MxA cDNA. HEp-2 cells are permissive for many viruses and are unable to express endogenous MxA in response to IFN. Experimental infection with various RNA and DNA viruses revealed that MxA-expressing HEp-2 cells were protected not only against influenza virus and vesicular stomatitis virus (VSV) but also against Semliki Forest virus (SFV), a togavirus with a single-stranded RNA genome of positive polarity. In MxA-transfected cells, Viral yields were reduced up to 1,700-fold, and the degree of inhibition correlated well with the expression level of MxA. Furthermore, expression of MxA prevented the accumulation of 49S RNA and 26S RNA, indicating that SFV was inhibited early in its replication cycle. Very similar results were obtained with MxA-transfected cells of the human monocytic cell line U937. The results demonstrate that the antiViral spectrum of MxA is not restricted to negative-strand RNA viruses but also includes SFV, which contains an RNA genome of positive polarity. To test whether MxA protein exerts its inhibitory activity against SFV in the absence of Viral Structural Proteins, we took advantage of a recombinant vector based on the SFV replicon. The vector contains only the coding sequence for the Viral nonStructural Proteins and the bacterial LacZ gene, which was cloned in place of the Viral Structural genes. Upon transfection of vector-derived recombinant RNA, expression of the beta-galactosidase reporter gene was strongly reduced in the presence of MxA. This finding indicates that Viral components other than the Structural Proteins are the target of MxA action.
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human mxa protein confers resistance to semliki forest virus and inhibits the amplification of a semliki forest virus based replicon in the absence of Viral Structural Proteins
Journal of Virology, 1998Co-Authors: Heinrich Landis, Angela Simonjodicke, Andreas Kloti, Claudio Di Paolo, J J Schnorr, Sibylle Schneiderschaulies, Hans Peter Hefti, Jovan PavlovicAbstract:SFV is a member of the family Togaviridae (genus Alphavirus), a family of mosquito-borne, positive-strand RNA viruses which has a large host range and whose most common complication is encephalitis (17). The virus enters the cells via receptor-mediated endocytosis (22). The uncoating of the nucleocapsids depends on ribosomes which release the capsid Proteins from the nucleocapsid and sequester them (35). In contrast to negative-strand RNA viruses, which transport their RNA transcriptase within the virion into the cells, the liberated genomic 49S RNA of Semliki Forest virus (SFV) serves directly as mRNA for the synthesis of the RNA polymerase. For replication, which occurs in the cytoplasm, the parental 49S positive-strand RNA is transcribed into a 49S negative-strand RNA, which in turn serves as a template for either the synthesis of progeny 49S positive-strand genomic RNA or subgenomic 26S mRNA directing the synthesis of Structural Proteins (15). SFV infection is strongly impaired in mice following treatment with type I interferon (IFN) (7). IFN also mediates a very potent activity against SFV replication in cell cultures, and the virus is widely used as challenging agent in virus yield reduction assays (20). IFN-α treatment leads to reduced Viral protein levels and hinders virus-mediated host shutoff (24). However, little is known about the molecular mechanisms of this antiViral action. The antiViral effect of IFNs is mediated by several IFN-induced Proteins which inhibit the multiplication of viruses by distinct mechanisms (for reviews see references 31 and 37). Some members of the Mx protein family were shown to contribute to this antiViral state by inhibiting the multiplication of different negative-strand RNA viruses (5, 6, 8, 16, 23, 27, 33, 39, 43, 44, 46). The Mx Proteins form a small group of GTPases (9, 25, 29) and are synthesized under the stringent control of IFN type I (1, 38). The molecular mechanism of Mx action still remains unclear, but the GTPase activity appears to be essential for antiViral function (28). The antiViral properties of Mx Proteins differ and are influenced by the intracellular localization of a particular Mx protein (14, 45, 47). Murine Mx1, which accumulates in the nucleus (4, 11), appears to be specific for Orthomyxoviridae (8, 27, 39, 43). The protein interferes with influenza virus replication at the level of primary transcription (18, 19, 26), suggesting an interaction with the Viral polymerase complex. Indeed, overexpression of PB2, a subunit of the influenza virus polymerase complex, leads to a partial neutralization of the antiViral effect of Mx1 (12, 41). The human MxA protein, which accumulates in the cytoplasm, has a broader activity, inhibiting the multiplication of influenza virus, Thogoto virus (Orthomyxoviridae), vesicular stomatitus virus (VSV) (Rhabdoviridae), measles virus (MV), human parainfluenza virus type 3 (Paramyxoviridae), and several members of the family Bunyaviridae (5, 6, 16, 27, 32, 33, 44). In contrast to Mx1, MxA appears to block the multiplication of influenza virus at a poorly defined cytoplasmic step following primary transcription (26). In the case of VSV, MxA inhibits primary transcription (40). For MV, the situation is clearly different. First, the protective effect of MxA against MV was detected only in the human monocytic cell line U937 and in the glioblastoma cell line U87. Furthermore, MxA inhibited the multiplication of MV at the level of either Viral RNA synthesis or synthesis of Viral glycoProteins, depending on the cell line used (32, 33). We report here that the antiViral specificity of MxA is extended to SFV, a positive-strand RNA virus. The activity of MxA against SFV appears to be either cell type or species specific, since no inhibitory effect was found in MxA-transfected mouse 3T3 fibroblasts (27). The fact that the accumulation of Viral RNAs and Proteins was inhibited points to a block occurring early in the replicative cycle. In order to define potential Viral targets of MxA, we took advantage of an SFV replicon-based vector coding only for the Viral replicase. The Viral Structural genes are replaced by the bacterial LacZ reporter gene (21). Upon transfection into cells, vector-derived recombinant RNA is amplified by virtue of its self-encoded replicase, and as a consequence large quantities of β-galactosidase (β-Gal) are produced. In MxA-transfected HEp-2 cells but not in mouse 3T3 cells, expression of β-Gal was dramatically reduced. These results demonstrate that the SFV Structural Proteins are not the target of MxA action and further suggest the involvement of species-specific cellular factors.
Eric Jan - One of the best experts on this subject based on the ideXlab platform.
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molecular analysis of the factorless internal ribosome entry site in cricket paralysis virus infection
Scientific Reports, 2016Co-Authors: Craig H Kerr, Sunnie R Thompson, Zi Wang, Christopher J Jang, Eric JanAbstract:The dicistrovirus Cricket Paralysis virus contains a unique dicistronic RNA genome arrangement, encoding two main open reading frames that are driven by distinct internal ribosome entry sites (IRES). The intergenic region (IGR) IRES adopts an unusual structure that directly recruits the ribosome and drives translation of Viral Structural Proteins in a factor-independent manner. While Structural, biochemical, and biophysical approaches have provided mechanistic details into IGR IRES translation, these studies have been limited to in vitro systems and little is known about the behavior of these IRESs during infection. Here, we examined the role of previously characterized IGR IRES mutations on Viral yield and translation in CrPV-infected Drosophila S2 cells. Using a recently generated infectious CrPV clone, introduction of a subset of mutations that are known to disrupt IRES activity failed to produce virus, demonstrating the physiological relevance of specific Structural elements within the IRES for virus infection. However, a subset of mutations still led to virus production, thus revealing the key IRES-ribosome interactions for IGR IRES translation in infected cells, which highlights the importance of examining IRES activity in its physiological context. This is the first study to examine IGR IRES translation in its native context during virus infection.
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Switch from Cap- to Factorless IRES-Dependent 0 and +1 Frame Translation during Cellular Stress and Dicistrovirus Infection
2014Co-Authors: Qing S Wang, Eric JanAbstract:Internal ribosome entry sites (IRES) are utilized by a subset of cellular and Viral mRNAs to initiate translation during cellular stress and virus infection when canonical cap-dependent translation is compromised. The intergenic region (IGR) IRES of the Dicistroviridae uses a streamlined mechanism in which it can directly recruit the ribosome in the absence of initiation factors and initiates translation using a non-AUG codon. A subset of IGR IRESs including that from the honey bee viruses can also direct translation of an overlapping +1 frame gene. In this study, we systematically examined cellular conditions that lead to IGR IRES-mediated 0 and +1 frame translation in Drosophila S2 cells. Towards this, a novel bicistronic reporter that exploits the 2A “stop-go” peptide was developed to allow the detection of IRES-mediated translation in vivo. Both 0 and +1 frame translation by the IGR IRES are stimulated under a number of cellular stresses and in S2 cells infected by cricket paralysis virus, demonstrating a switch from cap-dependent to IRES-dependent translation. The regulation of the IGR IRES mechanism ensures that both 0 frame Viral Structural Proteins and +1 frame ORFx protein are optimally expressed during virus infection.
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Switch from Cap- to Factorless IRES-Dependent 0 and +1 Frame Translation during Cellular Stress and Dicistrovirus Infection
2014Co-Authors: Qing S Wang, Eric JanAbstract:Internal ribosome entry sites (IRES) are utilized by a subset of cellular and Viral mRNAs to initiate translation during cellular stress and virus infection when canonical cap-dependent translation is compromised. The intergenic region (IGR) IRES of the Dicistroviridae uses a streamlined mechanism in which it can directly recruit the ribosome in the absence of initiation factors and initiates translation using a non-AUG codon. A subset of IGR IRESs including that from the honey bee viruses can also direct translation of an overlapping +1 frame gene. In this study, we systematically examined cellular conditions that lead to IGR IRES-mediated 0 and +1 frame translation in Drosophila S2 cells. Towards this, a novel bicistronic reporter that exploits the 2A ‘‘stop-go’ ’ peptide was developed to allow the detection of IRES-mediated translation in vivo. Both 0 and +1 frame translation by the IGR IRES are stimulated under a number of cellular stresses and in S2 cells infected by cricket paralysis virus, demonstrating a switch from cap-dependent to IRES-dependent translation. The regulation of the IGR IRES mechanism ensures that both 0 frame Viral Structural Proteins and +1 frame ORFx protein are optimally expressed durin
Pei Yong Shi - One of the best experts on this subject based on the ideXlab platform.
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a high throughput assay using dengue 1 virus like particles for drug discovery
Antiviral Research, 2010Co-Authors: Min Qing, Wei Liu, Zhiming Yuan, Pei Yong ShiAbstract:Abstract Dengue virus (DENV) is a mosquito-borne flavivirus responsible for 50–100 million human infections each year. The development of DENV chemotherapy requires high-throughput screening (HTS) assays. A dengue virus-like particle (VLP) has been constructed using Viral Structural Proteins to package a Renilla luciferase reporter replicon. VLP could be produced by either the sequential electroporation of the replicon RNAs and the Structural gene RNAs or by electroporating replicon RNA into a stable cell line expressing the Structural Proteins. In both approaches, the key to produce high titer VLP (3 × 10 6 foci-forming unit/ml) is to use low temperature (30 °C) in the packaging step. In addition, exogenous expression of host protease furin increased VLP infectivity. The infection could be blocked by antibodies against Viral envelope protein and by an inhibitor of Viral NS5 polymerase, but not by an inhibitor of host alpha-glucosidase (castanospermine). The VLP infection assay was optimized for HTS in a 384-well format with consistent and robust signal, providing a simple and rapid cell-based assay for screening inhibitors against DENV entry, translation, and replication in an HTS format.
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a high throughput assay using dengue 1 virus like particles for drug discovery
Antiviral Research, 2010Co-Authors: Min Qing, Wei Liu, Zhiming Yuan, Pei Yong ShiAbstract:Dengue virus (DENV) is a mosquito-borne flavivirus responsible for 50-100 million human infections each year. The development of DENV chemotherapy requires high-throughput screening (HTS) assays. A dengue virus-like particle (VLP) has been constructed using Viral Structural Proteins to package a Renilla luciferase reporter replicon. VLP could be produced by either the sequential electroporation of the replicon RNAs and the Structural gene RNAs or by electroporating replicon RNA into a stable cell line expressing the Structural Proteins. In both approaches, the key to produce high titer VLP (3x10(6)foci-forming unit/ml) is to use low temperature (30 degrees C) in the packaging step. In addition, exogenous expression of host protease furin increased VLP infectivity. The infection could be blocked by antibodies against Viral envelope protein and by an inhibitor of Viral NS5 polymerase, but not by an inhibitor of host alpha-glucosidase (castanospermine). The VLP infection assay was optimized for HTS in a 384-well format with consistent and robust signal, providing a simple and rapid cell-based assay for screening inhibitors against DENV entry, translation, and replication in an HTS format.
Min Qing - One of the best experts on this subject based on the ideXlab platform.
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a high throughput assay using dengue 1 virus like particles for drug discovery
Antiviral Research, 2010Co-Authors: Min Qing, Wei Liu, Zhiming Yuan, Pei Yong ShiAbstract:Abstract Dengue virus (DENV) is a mosquito-borne flavivirus responsible for 50–100 million human infections each year. The development of DENV chemotherapy requires high-throughput screening (HTS) assays. A dengue virus-like particle (VLP) has been constructed using Viral Structural Proteins to package a Renilla luciferase reporter replicon. VLP could be produced by either the sequential electroporation of the replicon RNAs and the Structural gene RNAs or by electroporating replicon RNA into a stable cell line expressing the Structural Proteins. In both approaches, the key to produce high titer VLP (3 × 10 6 foci-forming unit/ml) is to use low temperature (30 °C) in the packaging step. In addition, exogenous expression of host protease furin increased VLP infectivity. The infection could be blocked by antibodies against Viral envelope protein and by an inhibitor of Viral NS5 polymerase, but not by an inhibitor of host alpha-glucosidase (castanospermine). The VLP infection assay was optimized for HTS in a 384-well format with consistent and robust signal, providing a simple and rapid cell-based assay for screening inhibitors against DENV entry, translation, and replication in an HTS format.
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a high throughput assay using dengue 1 virus like particles for drug discovery
Antiviral Research, 2010Co-Authors: Min Qing, Wei Liu, Zhiming Yuan, Pei Yong ShiAbstract:Dengue virus (DENV) is a mosquito-borne flavivirus responsible for 50-100 million human infections each year. The development of DENV chemotherapy requires high-throughput screening (HTS) assays. A dengue virus-like particle (VLP) has been constructed using Viral Structural Proteins to package a Renilla luciferase reporter replicon. VLP could be produced by either the sequential electroporation of the replicon RNAs and the Structural gene RNAs or by electroporating replicon RNA into a stable cell line expressing the Structural Proteins. In both approaches, the key to produce high titer VLP (3x10(6)foci-forming unit/ml) is to use low temperature (30 degrees C) in the packaging step. In addition, exogenous expression of host protease furin increased VLP infectivity. The infection could be blocked by antibodies against Viral envelope protein and by an inhibitor of Viral NS5 polymerase, but not by an inhibitor of host alpha-glucosidase (castanospermine). The VLP infection assay was optimized for HTS in a 384-well format with consistent and robust signal, providing a simple and rapid cell-based assay for screening inhibitors against DENV entry, translation, and replication in an HTS format.
Scott C Weaver - One of the best experts on this subject based on the ideXlab platform.
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ires driven expression of the capsid protein of the venezuelan equine encephalitis virus tc 83 vaccine strain increases its attenuation and safety
PLOS Neglected Tropical Diseases, 2013Co-Authors: Mathilde Guerbois, Eugenia Volkova, Naomi L Forrester, Shannan L Rossi, Ilya Frolov, Scott C WeaverAbstract:The live-attenuated TC-83 strain is the only licensed veterinary vaccine available to protect equids against Venezuelan equine encephalitis virus (VEEV) and to protect humans indirectly by preventing equine amplification. However, TC-83 is reactogenic due to its reliance on only two attenuating point mutations and has infected mosquitoes following equine vaccination. To increase its stability and safety, a recombinant TC-83 was previously engineered by placing the expression of the Viral Structural Proteins under the control of the Internal Ribosome Entry Site (IRES) of encephalomyocarditis virus (EMCV), which drives translation inefficiently in insect cells. However, this vaccine candidate was poorly immunogenic. Here we describe a second generation of the recombinant TC-83 in which the subgenomic promoter is maintained and only the capsid protein gene is translated from the IRES. This VEEV/IRES/C vaccine candidate did not infect mosquitoes, was stable in its attenuation phenotype after serial murine passages, and was more attenuated in newborn mice but still as protective as TC-83 against VEEV challenge. Thus, by using the IRES to modulate TC-83 capsid protein expression, we generated a vaccine candidate that combines efficient immunogenicity and efficacy with lower virulence and a reduced potential for spread in nature.
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ires dependent replication of venezuelan equine encephalitis virus makes it highly attenuated and incapable of replicating in mosquito cells
Virology, 2008Co-Authors: Eugenia Volkova, Naomi L Forrester, Scott C Weaver, Elena I Frolova, Justin R Darwin, Ilya FrolovAbstract:The development of infectious cDNA for different alphaviruses opened an opportunity to explore their attenuation by extensively modifying the Viral genomes, an approach that might minimize or exclude the reversion to the wild-type, pathogenic phenotype. Moreover, the genomes of such alphaviruses can be engineered to contain RNA elements that would be functional only in cells of vertebrate, but not insect, origin. In the present study, we developed a recombinant VEEV that is more attenuated than TC-83 and capable of replicating only in vertebrate cells. This phenotype was achieved by rendering the translation of the Viral Structural Proteins, and ultimately Viral replication, dependent on the internal ribosome entry site of encephalomyocarditis virus (EMCV IRES). This recombinant virus was viable, but required additional, adaptive mutations in nsP2 that strongly increased its replication rates. In spite of efficient replication in cultured vertebrate cells, the genetically modified VEEV demonstrated a highly attenuated phenotype in newborn mice, and yet induced protective immunity against VEEV infection.
