The Experts below are selected from a list of 1431 Experts worldwide ranked by ideXlab platform
Alexander A Khromykh - One of the best experts on this subject based on the ideXlab platform.
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a noncoding rna produced by arthropod borne flaviViruses inhibits the cellular exoribonuclease xrn1 and alters host mrna stability
RNA, 2012Co-Authors: Stephanie L Moon, Alexander A Khromykh, Carol J Wilusz, John R. Anderson, Yutaro Kumagai, Jeffrey WiluszAbstract:All arthropod-borne flaviViruses generate a short noncoding RNA (sfRNA) from the viral 3′ untranslated region during infection due to stalling of the cellular 5′-to-3′ exonuclease XRN1. We show here that formation of sfRNA also inhibits XRN1 activity. Cells infected with Dengue or Kunjin Viruses accumulate uncapped mRNAs, decay intermediates normally targeted by XRN1. XRN1 repression also resulted in the increased overall stability of cellular mRNAs in flaviVirus-infected cells. Importantly, a mutant Kunjin Virus that cannot form sfRNA but replicates to normal levels failed to affect host mRNA stability or XRN1 activity. Expression of sfRNA in the absence of viral infection demonstrated that sfRNA formation was directly responsible for the stabilization of cellular mRNAs. Finally, numerous cellular mRNAs were differentially expressed in an sfRNA-dependent fashion in a Kunjin Virus infection. We conclude that flaviViruses incapacitate XRN1 during infection and dysregulate host mRNA stability as a result of sfRNA formation.
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regulated cleavages at the west nile Virus ns4a 2k ns4b junctions play a major role in rearranging cytoplasmic membranes and golgi trafficking of the ns4a protein
Journal of Virology, 2006Co-Authors: Jojanneke Roosendaal, Edwin G. Westaway, Alexander A Khromykh, Jason M. MackenzieAbstract:A common feature associated with the replication of most RNA Viruses is the formation of a unique membrane environment encapsulating the viral replication complex. For their part, flaviViruses are no exception, whereupon infection causes a dramatic rearrangement and induction of unique membrane structures within the cytoplasm of infected cells. These Virus-induced membranes, termed paracrystalline arrays, convoluted membranes, and vesicle packets, all appear to have specific functions during replication and are derived from different organelles within the host cell. The aim of this study was to identify which protein(s) specified by the Australian strain of West Nile Virus, Kunjin Virus (KUNV), are responsible for the dramatic membrane alterations observed during infection. Thus, we have shown using immunolabeling of ultrathin cryosections of transfected cells that expression of the KUNV polyprotein intermediates NS4A-4B and NS2B-3-4A, as well as that of individual NS4A proteins with and without the C-terminal transmembrane domain 2K, resulted in different degrees of rearrangement of cytoplasmic membranes. The formation of the membrane structures characteristic for Virus infection required coexpression of an NS4A-NS4B cassette with the viral protease NS2B-3pro which was shown to be essential for the release of the individual NS4A and NS4B proteins. Individual expression of NS4A protein retaining the C-terminal transmembrane domain 2K resulted in the induction of membrane rearrangements most resembling Virus-induced structures, while removal of the 2K domain led to a less profound membrane rearrangement but resulted in the redistribution of the NS4A protein to the Golgi apparatus. The results show that cleavage of the KUNV polyprotein NS4A-4B by the viral protease is the key initiation event in the induction of membrane rearrangement and that the NS4A protein intermediate containing the uncleaved C-terminal transmembrane domain plays an essential role in these membrane rearrangements.
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Kunjin Virus replicons an rna based non cytopathic viral vector system for protein production vaccine and gene therapy applications
Expert Opinion on Biological Therapy, 2006Co-Authors: Gorben P Pijlman, Andreas Suhrbier, Alexander A KhromykhAbstract:The application of viral vectors for gene expression and delivery is rapidly evolving, with several entering clinical trials. However, a number of issues, including safety, gene expression levels, cell selectivity and antivector immunity, are driving the search for new vector systems. A number of replicon-based vectors derived from positive-strand RNA Viruses have recently been developed, and this paper reviews the current knowledge on the first flaviVirus replicon system, which is based on the Australian flaviVirus Kunjin (KUN). Like most replicon systems, KUN replicons can be delivered as DNA, RNA or Virus-like particles, they replicate their RNA in the cytoplasm and direct prolonged high-level gene expression. However, unlike most alphaVirus replicon systems, KUN replicons are non-cytopathic, with transfected cells able to divide, allowing the establishment of cell lines stably expressing replicon RNA and heterologous genes. As vaccine vectors KUN replicons can induce potent, long-lived, protective, immunogen-specific CD8+ T cell immunity, a feature potentially related to extended production of antigen and double-stranded RNA-induced 'danger signals'. The identification of KUN replicon mutants that induce increased levels of IFN-alpha/beta has also spawned investigation of KUN replicons for use in cancer gene therapy. The unique characteristics of KUN replicons may thus make them suitable for specific protein production, vaccine and gene therapy applications.
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Kunjin Virus replicons an rna based non cytopathic viral vector system for protein production vaccine and gene therapy applications
Expert Opinion on Biological Therapy, 2006Co-Authors: Gorben P Pijlman, Andreas Suhrbier, Alexander A KhromykhAbstract:The application of viral vectors for gene expression and delivery is rapidly evolving, with several entering clinical trials. However, a number of issues, including safety, gene expression levels, cell selectivity and antivector immunity, are driving the search for new vector systems. A number of replicon-based vectors derived from positive-strand RNA Viruses have recently been developed, and this paper reviews the current knowledge on the first flaviVirus replicon system, which is based on the Australian flaviVirus Kunjin (KUN). Like most replicon systems, KUN replicons can be delivered as DNA, RNA or Virus-like particles, they replicate their RNA in the cytoplasm and direct prolonged high-level gene expression. However, unlike most alphaVirus replicon systems, KUN replicons are non-cytopathic, with transfected cells able to divide, allowing the establishment of cell lines stably expressing replicon RNA and heterologous genes. As vaccine vectors KUN replicons can induce potent, long-lived, protective, im...
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inhibition of interferon signaling by the new york 99 strain and Kunjin subtype of west nile Virus involves blockage of stat1 and stat2 activation by nonstructural proteins
Journal of Virology, 2005Co-Authors: Alexander A Khromykh, Wen Jun Liu, Xiang Ju Wang, Vladislav V Mokhonov, Pei Yong Shi, R E RandallAbstract:The interferon (IFN) response is the first line of defense against viral infections, and the majority of Viruses have developed different strategies to counteract IFN responses in order to ensure their survival in an infected host. In this study, the abilities to inhibit IFN signaling of two closely related West Nile Viruses, the New York 99 strain (NY99) and Kunjin Virus (KUN), strain MRM61C, were analyzed using reporter plasmid assays, as well as immunofluorescence and Western blot analyses. We have demonstrated that infections with both NY99 and KUN, as well as transient or stable transfections with their replicon RNAs, inhibited the signaling of both alpha/beta IFN (IFN-α/β) and gamma IFN (IFN-γ) by blocking the phosphorylation of STAT1 and its translocation to the nucleus. In addition, the phosphorylation of STAT2 and its translocation to the nucleus were also blocked by KUN, NY99, and their replicons in response to treatment with IFN-α. IFN-α signaling and STAT2 translocation to the nucleus was inhibited when the KUN nonstructural proteins NS2A, NS2B, NS3, NS4A, and NS4B, but not NS1 and NS5, were expressed individually from the pcDNA3 vector. The results clearly demonstrate that both NY99 and KUN inhibit IFN signaling by preventing STAT1 and STAT2 phosphorylation and identify nonstructural proteins responsible for this inhibition.
Jason M. Mackenzie - One of the best experts on this subject based on the ideXlab platform.
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west nile Virus differentially modulates the unfolded protein response to facilitate replication and immune evasion
Journal of Virology, 2011Co-Authors: Rebecca L Ambrose, Jason M. MackenzieAbstract:For intracellular survival it is imperative that Viruses have the capacity to manipulate various cellular responses, including metabolic and biosynthetic pathways. The unfolded protein response (UPR) is induced by various external and internal stimuli, including the accumulation of misfolded proteins in the endoplasmic reticulum (ER). Our previous studies have indicated that the replication and assembly of the flaviVirus West Nile Virus strain Kunjin Virus (WNV(KUN)) is intimately associated with the ER. Thus, we sought to determine whether the UPR was induced during WNV(KUN) infection. WNV(KUN) induces UPR signaling during replication, which is coordinated with peak replication. Interestingly, signaling is biased toward the ATF6/IRE-1 arm of the response, with high levels of Xbp-1 activation but negligible eukaryotic translation initiation factor 2α phosphorylation and downstream transcription. We show that the PERK-mediated response may partially regulate replication, since external UPR stimulation had a limiting effect on early replication events and cells deficient for PERK demonstrated increased replication and Virus release. Significantly, we show that the WNV(KUN) hydrophobic nonstructural proteins NS4A and NS4B are potent inducers of the UPR, which displayed a high correlation in inhibiting Jak-STAT signaling in response to alpha interferon (IFN-α). Sequential removal of the transmembrane domains of NS4A showed that reducing hydrophobicity decreased UPR signaling and restored IFN-α-mediated activation. Overall, these results suggest that WNV(KUN) can stimulate the UPR to facilitate replication and that the induction of a general ER stress response, regulated by hydrophobic WNV(KUN) proteins, can potentiate the inhibition of the antiviral signaling pathway.
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the endoplasmic reticulum provides the membrane platform for biogenesis of the flaviVirus replication complex
Journal of Virology, 2010Co-Authors: Leah Gillespie, Antje Hoenen, Gary Morgan, Jason M. MackenzieAbstract:The cytoplasmic replication of positive-sense RNA Viruses is associated with a dramatic rearrangement of host cellular membranes. These Virus-induced changes result in the induction of vesicular structures that envelop the Virus replication complex (RC). In this study, we have extended our previous observations on the intracellular location of West Nile Virus strain Kunjin Virus (WNV(KUN)) to show that the Virus-induced recruitment of host proteins and membrane appears to occur at a pre-Golgi step. To visualize the WNV(KUN) replication complex, we performed three-dimensional (3D) modeling on tomograms from WNV(KUN) replicon-transfected cells. These analyses have provided a 3D representation of the replication complex, revealing the open access of the replication complex with the cytoplasm and the fluidity of the complex to the rough endoplasmic reticulum. In addition, we provide data that indicate that a majority of the viral RNA species housed within the RC is in a double-stranded RNA (dsRNA) form.
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west nile Virus strain Kunjin ns5 polymerase is a phosphoprotein localized at the cytoplasmic site of viral rna synthesis
Journal of General Virology, 2007Co-Authors: Jason M. Mackenzie, Mark T Kenney, Edwin G. WestawayAbstract:Using West Nile Virus strain Kunjin Virus (WNVKUN) as a model system for flaviVirus replication, we showed that the Virus replication complex (RC) is associated with the dsRNA template located in induced membranes only in the cytoplasm. In this report we established for the first time that the RNA-dependent RNA polymerase NS5 is located in flaviVirus-induced membranes, including the site of viral RNA replication. We found no evidence for nuclear localization of the essential RC components NS5 and its dsRNA template for WNVKUN or the closely related WNV strain Sarafend, by immuno-electron microscopy or by immunofluorescence. Metabolic radiolabelling with [32P]orthophosphate revealed that WNVKUN NS5 was phosphorylated and this was confirmed by Western blotting with antibodies specific for phosphorylated serine and threonine only. These observations of a cytoplasmic location for the WNV polymerase and its phosphorylation state correspond to the characteristics of the hepatitis C Virus RNA polymerase NS5B.
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regulated cleavages at the west nile Virus ns4a 2k ns4b junctions play a major role in rearranging cytoplasmic membranes and golgi trafficking of the ns4a protein
Journal of Virology, 2006Co-Authors: Jojanneke Roosendaal, Edwin G. Westaway, Alexander A Khromykh, Jason M. MackenzieAbstract:A common feature associated with the replication of most RNA Viruses is the formation of a unique membrane environment encapsulating the viral replication complex. For their part, flaviViruses are no exception, whereupon infection causes a dramatic rearrangement and induction of unique membrane structures within the cytoplasm of infected cells. These Virus-induced membranes, termed paracrystalline arrays, convoluted membranes, and vesicle packets, all appear to have specific functions during replication and are derived from different organelles within the host cell. The aim of this study was to identify which protein(s) specified by the Australian strain of West Nile Virus, Kunjin Virus (KUNV), are responsible for the dramatic membrane alterations observed during infection. Thus, we have shown using immunolabeling of ultrathin cryosections of transfected cells that expression of the KUNV polyprotein intermediates NS4A-4B and NS2B-3-4A, as well as that of individual NS4A proteins with and without the C-terminal transmembrane domain 2K, resulted in different degrees of rearrangement of cytoplasmic membranes. The formation of the membrane structures characteristic for Virus infection required coexpression of an NS4A-NS4B cassette with the viral protease NS2B-3pro which was shown to be essential for the release of the individual NS4A and NS4B proteins. Individual expression of NS4A protein retaining the C-terminal transmembrane domain 2K resulted in the induction of membrane rearrangements most resembling Virus-induced structures, while removal of the 2K domain led to a less profound membrane rearrangement but resulted in the redistribution of the NS4A protein to the Golgi apparatus. The results show that cleavage of the KUNV polyprotein NS4A-4B by the viral protease is the key initiation event in the induction of membrane rearrangement and that the NS4A protein intermediate containing the uncleaved C-terminal transmembrane domain plays an essential role in these membrane rearrangements.
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assembly and maturation of the flaviVirus Kunjin Virus appear to occur in the rough endoplasmic reticulum and along the secretory pathway respectively
Journal of Virology, 2001Co-Authors: Jason M. Mackenzie, Edwin G. WestawayAbstract:The intracellular assembly site for flaviViruses in currently not known but is presumed to be located within the lumen of the rough endoplasmic reticulum (RER). Building on previous studies involving immunofluorescence (IF) and cryoimmunoelectron microscopy of Kunjin Virus (KUN)-infected cells, we sought to identify the steps involved in the assembly and maturation of KUN. Thus, using antibodies directed against envelope protein E in IF analysis, we found the accumulation of E within regions coincident with the RER and endosomal compartments. Immunogold labeling of cryosections of infected cells indicated that E and minor envelope protein prM were localized to reticulum membranes continuous with KUN-induced convoluted membranes (CM) or paracrystalline arrays (PC) and that sometimes the RER contained immunogold-labeled Virus particles. Both proteins were also observed to be labeled in membranes at the periphery of the induced CM or PC structures, but the latter were very seldom labeled internally. Utilizing drugs that inhibit protein and/or membrane traffic throughout the cell, we found that the secretion of KUN particles late in infection was significantly affected in the presence of brefeldin A and that the infectivity of secreted particles was severely affected in the presence of monensin and N-nonyl-deoxynojirimycin. Nocodazole did not appear to affect maturation, suggesting that microtubules play no role in assembly or maturation processes. Subsequently, we showed that the exit of intact virions from the RER involves the transport of individual virions within individual vesicles en route to the Golgi apparatus. The results suggest that the assembly of virions occurs within the lumen of the RER and that subsequent maturation occurs via the secretory pathway.
Edwin G. Westaway - One of the best experts on this subject based on the ideXlab platform.
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west nile Virus strain Kunjin ns5 polymerase is a phosphoprotein localized at the cytoplasmic site of viral rna synthesis
Journal of General Virology, 2007Co-Authors: Jason M. Mackenzie, Mark T Kenney, Edwin G. WestawayAbstract:Using West Nile Virus strain Kunjin Virus (WNVKUN) as a model system for flaviVirus replication, we showed that the Virus replication complex (RC) is associated with the dsRNA template located in induced membranes only in the cytoplasm. In this report we established for the first time that the RNA-dependent RNA polymerase NS5 is located in flaviVirus-induced membranes, including the site of viral RNA replication. We found no evidence for nuclear localization of the essential RC components NS5 and its dsRNA template for WNVKUN or the closely related WNV strain Sarafend, by immuno-electron microscopy or by immunofluorescence. Metabolic radiolabelling with [32P]orthophosphate revealed that WNVKUN NS5 was phosphorylated and this was confirmed by Western blotting with antibodies specific for phosphorylated serine and threonine only. These observations of a cytoplasmic location for the WNV polymerase and its phosphorylation state correspond to the characteristics of the hepatitis C Virus RNA polymerase NS5B.
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regulated cleavages at the west nile Virus ns4a 2k ns4b junctions play a major role in rearranging cytoplasmic membranes and golgi trafficking of the ns4a protein
Journal of Virology, 2006Co-Authors: Jojanneke Roosendaal, Edwin G. Westaway, Alexander A Khromykh, Jason M. MackenzieAbstract:A common feature associated with the replication of most RNA Viruses is the formation of a unique membrane environment encapsulating the viral replication complex. For their part, flaviViruses are no exception, whereupon infection causes a dramatic rearrangement and induction of unique membrane structures within the cytoplasm of infected cells. These Virus-induced membranes, termed paracrystalline arrays, convoluted membranes, and vesicle packets, all appear to have specific functions during replication and are derived from different organelles within the host cell. The aim of this study was to identify which protein(s) specified by the Australian strain of West Nile Virus, Kunjin Virus (KUNV), are responsible for the dramatic membrane alterations observed during infection. Thus, we have shown using immunolabeling of ultrathin cryosections of transfected cells that expression of the KUNV polyprotein intermediates NS4A-4B and NS2B-3-4A, as well as that of individual NS4A proteins with and without the C-terminal transmembrane domain 2K, resulted in different degrees of rearrangement of cytoplasmic membranes. The formation of the membrane structures characteristic for Virus infection required coexpression of an NS4A-NS4B cassette with the viral protease NS2B-3pro which was shown to be essential for the release of the individual NS4A and NS4B proteins. Individual expression of NS4A protein retaining the C-terminal transmembrane domain 2K resulted in the induction of membrane rearrangements most resembling Virus-induced structures, while removal of the 2K domain led to a less profound membrane rearrangement but resulted in the redistribution of the NS4A protein to the Golgi apparatus. The results show that cleavage of the KUNV polyprotein NS4A-4B by the viral protease is the key initiation event in the induction of membrane rearrangement and that the NS4A protein intermediate containing the uncleaved C-terminal transmembrane domain plays an essential role in these membrane rearrangements.
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NOTES Significance in Replication of the Terminal Nucleotides of
2003Co-Authors: The Flavivirus Genome, Natasha Kondratieva, Er A Khromykh, Jean-yves Sgro, Ann Palmenberg, Edwin G. WestawayAbstract:Point mutations that resulted in a substitution of the conserved 3-penultimate cytidine in genomic RNA or the RNA negative strand of the self-amplifying replicon of the FlaviVirus Kunjin Virus completely blocked in vivo replication. Similarly, substitutions of the conserved 3-terminal uridine in the RNA negative or positive strand completely blocked replication or caused much-reduced replication, respectively. The same preference for cytidine in the 3-terminal dinucleotide was noted in reports of the in vitro activity of the RNA-dependent RNA polymerase (RdRp) for the other genera of Flaviviridae that also employ a double-stranded RNA (dsRNA) template to initiate asymmetric semiconservative RNA positive-strand synthesis. The Kunjin Virus replicon results were interpreted in the context of a proposed model for initiation of RNA synthesis based on the solved crystal structure of the RdRp of 6 bacteriophage, which also replicates efficiently using a dsRNA template with conserved 3-penultimate cytidines and a 3-terminal pyrimidine. A previously untested substitution of the conserved pentanucleotide at the top of the 3-terminal stem-loop of all FlaviVirus species also blocked detect-able in vivo replication of the Kunjin Virus replicon RNA. The Flaviviridae comprise three genera of positive-strand RNA Viruses (FlaviVirus, PestiVirus, and HepaciVirus). Their in-fectious genomes have a single open reading frame and n
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assembly and maturation of the flaviVirus Kunjin Virus appear to occur in the rough endoplasmic reticulum and along the secretory pathway respectively
Journal of Virology, 2001Co-Authors: Jason M. Mackenzie, Edwin G. WestawayAbstract:The intracellular assembly site for flaviViruses in currently not known but is presumed to be located within the lumen of the rough endoplasmic reticulum (RER). Building on previous studies involving immunofluorescence (IF) and cryoimmunoelectron microscopy of Kunjin Virus (KUN)-infected cells, we sought to identify the steps involved in the assembly and maturation of KUN. Thus, using antibodies directed against envelope protein E in IF analysis, we found the accumulation of E within regions coincident with the RER and endosomal compartments. Immunogold labeling of cryosections of infected cells indicated that E and minor envelope protein prM were localized to reticulum membranes continuous with KUN-induced convoluted membranes (CM) or paracrystalline arrays (PC) and that sometimes the RER contained immunogold-labeled Virus particles. Both proteins were also observed to be labeled in membranes at the periphery of the induced CM or PC structures, but the latter were very seldom labeled internally. Utilizing drugs that inhibit protein and/or membrane traffic throughout the cell, we found that the secretion of KUN particles late in infection was significantly affected in the presence of brefeldin A and that the infectivity of secreted particles was severely affected in the presence of monensin and N-nonyl-deoxynojirimycin. Nocodazole did not appear to affect maturation, suggesting that microtubules play no role in assembly or maturation processes. Subsequently, we showed that the exit of intact virions from the RER involves the transport of individual virions within individual vesicles en route to the Golgi apparatus. The results suggest that the assembly of virions occurs within the lumen of the RER and that subsequent maturation occurs via the secretory pathway.
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Essential Role of Cyclization Sequences in FlaviVirus RNA Replication
Journal of Virology, 2001Co-Authors: Alexander A Khromykh, Hedije Meka, Kimberley J. Guyatt, Edwin G. WestawayAbstract:A possible role in RNA replication for interactions between conserved complementary (cyclization) sequences in the 5'- and 3'-terminal regions of FlaviVirus RNA was previously suggested but never tested in vivo. Using the M-fold program for RNA secondary-structure predictions, we examined for the first time the base-pairing interactions between the covalently linked 5' genomic region (first similar to 160 nucleotides) and the 3' untranslated region (last similar to 115 nucleotides) for a range of mosquito borne FlaviVirus species. Base-pairing occurred as predicted for the previously proposed conserved cyclization sequences. In order to obtain experimental evidence of the predicted interactions, the putative cyclization sequences (5' or 3') in the replicon RNA of the mosquito-borne Kunjin Virus,were mutated either separately, to destroy base-pairing, or simultaneously, to restore the complementarity. None of the RNAs with separate mutations in only the 5' or only the 3' cyclization sequences was able to replicate after transfection into BHK cells, while replicon RNA with simultaneous compensatory mutations in both cyclization sequences was replication competent. This was detected by immunofluorescence for expression of the major nonstructural protein NS3 and by Northern blot analysis for amplification and accumulation of replicon RNA. We then used the M-fold program to analyze RNA secondary structure of the covalently linked 5'- and 3'-terminal regions of three tick borne Virus species and identified a previously undescribed additional pair of conserved complementary sequences in locations similar to those of the mosquito borne species. They base-paired with DeltaG values of approximately -20 kcal, equivalent or greater in stability than those calculated for the originally proposed cyclization sequences. The results show that the base-pairing between 5' and 3' complementary sequences, rather than the nucleotide sequence per se, is essential for the replication of mosquito-borne Kunjin Virus RNA and that more than one pair of cyclization sequences might be involved in the replication of the tick-borne FlaviVirus species.
Roy A Hall - One of the best experts on this subject based on the ideXlab platform.
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in situ reactions of monoclonal antibodies with a viable mutant of murray valley encephalitis Virus reveal an absence of dimeric ns1 protein
Journal of General Virology, 2007Co-Authors: David C Clark, Mario Lobigs, M J Howard, Eva Lee, Kerri Clark, B J Blitvich, Roy A HallAbstract:Studies on the NS1 protein of flaviViruses have concluded that formation of a stable homodimer is required for Virus replication. However, previous work has reported that substitution of a conserved proline by leucine at residue 250 in NS1 of Kunjin Virus (KUNV) eliminated dimerization, but allowed Virus replication to continue. To assess whether this substitution has similar effects on other flaviViruses, it was introduced into an infectious clone of Murray Valley encephalitis Virus (MVEV). Consistent with studies of KUNV, the mutant Virus (MVEVNS1-250Leu) produced high levels of monomeric NS1 and the NS1 homodimer could not be detected. In contrast, wild-type MVEV cultures contained predominantly dimeric NS1. Retarded Virus growth in Vero cells and loss of neuroinvasiveness for weanling mice revealed further similarities between MVEVNS1-250Leu and the corresponding KUNV mutant. To confirm that the lack of detection of dimeric NS1 in mutant Virus samples was not due to denaturation of unstable dimers during Western blotting, a mAb (2E3) specific for the MVEV NS1 homodimer was produced. When NS1 protein was fixed in situ in mammalian and arthropod cells infected with wild-type or mutant Virus, 2E3 reacted strongly with the former, but not the latter. These results confirmed that Pro250 in NS1 is important for dimerization and that substitution of this residue by leucine represents a conserved marker of attenuation for Viruses of the Japanese encephalitis Virus serocomplex. The inability to detect dimeric NS1 in supernatant or cell monolayers of cultures productively infected with mutant Virus also suggests that dimerization of the protein may not be essential for Virus replication.
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dna vaccine coding for the full length infectious Kunjin Virus rna protects mice against the new york strain of west nile Virus
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Roy A Hall, D J Nisbet, Kim Pham, Alyssa T Pyke, Greg A Smith, Alexander A KhromykhAbstract:A plasmid DNA directing transcription of the infectious full-length RNA genome of Kunjin (KUN) Virus in vivo from a mammalian expression promoter was used to vaccinate mice intramuscularly. The KUN viral cDNA encoded in the plasmid contained the mutation in the NS1 protein (Pro-250 to Leu) previously shown to attenuate KUN Virus in weanling mice. KUN Virus was isolated from the blood of immunized mice 3-4 days after DNA inoculation, demonstrating that infectious RNA was being transcribed in vivo; however, no symptoms of Virus-induced disease were observed. By 19 days postimmunization, neutralizing antibody was detected in the serum of immunized animals. On challenge with lethal doses of the virulent New York strain of West Nile (WN) or wild-type KUN Virus intracerebrally or intraperitoneally, mice immunized with as little as 0.1-1 μg of KUN plasmid DNA were solidly protected against disease. This finding correlated with neutralization data in vitro showing that serum from KUN DNA-immunized mice neutralized KUN and WN Viruses with similar efficiencies. The results demonstrate that delivery of an attenuated but replicating KUN Virus via a plasmid DNA vector may provide an effective vaccination strategy against virulent strains of WN Virus.
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polymerase chain reaction tests for the identification of ross river Kunjin and murray valley encephalitis Virus infections in horses
Australian Veterinary Journal, 2003Co-Authors: M J Studdert, Roy A Hall, J K Azuolas, J R Vasey, Nino Ficorilli, Jinan HuangAbstract:Objective To develop and validate specific, sensitive and rapid diagnostic tests using RT-PCR for the detection of Ross River Virus (RRV), Kunjin Virus (KV) and Murray Valley encephalitis Virus (MVEV) infections in horses. Methods Primer sets based on nucleotide sequence encoding the envelope glycoprotein E2 of RRV and on the nonstructural protein 5 (NS5) of KV and MVEV were designed and used in single round PCRs to test for the respective Viruses in infected cell cultures and, in the case of RRV, in samples of horse blood and synovial fluid. Results The primer pairs designed for each of the three Viruses amplified a product of expected size from prototype Viruses that were grown in cell culture. The identity of each of the products was confirmed by nucleotide sequencing indicating that in the context used the RT-PCRs were specific. RRV was detected in serums from 8 horses for which there were clinical signs consistent with RRV infection such that an acute-phase serum sample was taken and submitted for RRV serology testing. The RRV RT-PCR was analytically sensitive in that it was estimated to detect as little as 50 TCID50 of RRV per mL of serum and was specific in that the primer pairs did not amplify other products from the 8 serum samples. The RRV primers also detected Virus in three independent mosquito pools known to contain RRV by Virus isolation in cell culture. Samples from horses suspected to be infected with KV and MVEV were not available. Conclusion Despite much anecdotal and serological evidence for infection of horses with RRV actual infection and associated clinical disease are infrequently confirmed. The availability of a specific and analytically sensitive RT-PCR for the detection of RRV provides additional opportunities to confirm the presence of this Virus in clinical samples. The RTPCR primers for the diagnosis of KV and MVEV infections were shown to be specific for cell culture grown Viruses but the further validation of these tests requires the availability of appropriate clinical samples from infected horses.
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loss of dimerisation of the nonstructural protein ns1 of Kunjin Virus delays viral replication and reduces virulence in mice but still allows secretion of ns1
Virology, 1999Co-Authors: Roy A Hall, Alexander A Khromykh, Jason M. Mackenzie, J Scherret, Tatiana Khromykh, J S MackenzieAbstract:Abstract The flaviVirus nonstructural protein NS1 has been implicated in viral RNA replication, although its precise role has not been identified. In its native state NS1 exists as a heat labile homodimer that is thought to be required for NS1 function and secretion. However, we have recently identified a cDNA clone of KUN Virus (FLSD) that replicates efficiently in cell culture but produces and secretes NS1 in monomeric form. Sequence analysis of the NS1 gene in FLSD revealed a single amino acid substitution (proline250 to leucine) when compared with the parental KUN Virus. When site-directed mutagenesis was used to substitute leucine250 with proline in FLSD to produce the clone 250pro, dimerisation was fully restored. Furthermore, time course experiments revealed that 250pro replicated in Vero cells significantly faster than FLSD and produced 100-fold more infectious Virus early (12–24 h) in infection. This correlated with our observations that FLSD required approximately 10-fold more infectious Virus than 250pro to produce disease in weanling mice after intraperitoneal inoculation. Taken together our results indicate that mutation from proline to leucine at residue 250 in KUN NS1 ablates dimer formation, slows Virus replication, and reduces virulence in mice.
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glycosylation and antigenic variation among Kunjin Virus isolates
Virology, 1995Co-Authors: S C Adams, A K Broom, Leanne M Sammels, A Hartnett, M J Howard, R J Coelen, J S Mackenzie, Roy A HallAbstract:Previous studies have found Kunjin (KUN) Virus isolates from within Australia to be genetically homogenous and that the envelope protein of the type strain (MRM61C) was unglycosylated and lacked a potential glycosylation site. We investigated the extent of antigenic variation between KUN Virus isolates from Australia and Sarawak using an immunoperoxidase assay and a panel of six monoclonal antibodies. The glycosylation status of the E protein of each Virus was also determined by N glycosidase F (PNGase F) digestion and limited sequence analysis. The results showed that KUN Viruses isolated within Australia oscillated between three antigenic types defined by two epitopes whose expression was influenced by passage history and host cell type. In contrast an isolate from Sarawak formed a stable antigenic type that was not influenced by passage history and was distinct from all Australian isolates. PNGase F digestions of KUN isolates indicated that 19 of the 33 Viruses possessed a glycosylated E protein. Nucleotide sequence of the 5' third of the E gene of selected KUN isolates revealed that a single base change in PNGase F sensitive strains changed the tripeptide N-Y-F (amino acids 154-156 of the published sequence) to the potential glycosylation site N-Y-S. Further analysis revealed that passage history also had a significant influence on glycosylation.
Dmitri I Svergun - One of the best experts on this subject based on the ideXlab platform.
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crystal structure and activity of Kunjin Virus ns3 helicase protease and helicase domain assembly in the full length ns3 protein
Journal of Molecular Biology, 2007Co-Authors: E. Mastrangelo, M. Milani, P. V. Konarev, Michela Bollati, Barbara Selisko, Frederic Peyrane, V Pandini, Graziella Sorrentino, Bruno Canard, Dmitri I SvergunAbstract:Flaviviral NS3 is a multifunctional protein displaying N-terminal protease activity in addition to C-terminal helicase, nucleoside 5'-triphosphatase (NTPase), and 5'-terminal RNA triphosphatase (RTPase) activities. NS3 is held to support the separation of RNA daughter and template strands during viral replication. In addition, NS3 assists the initiation of replication by unwinding the RNA secondary structure in the 3' non-translated region (NTR). We report here the three-dimensional structure (at 3.1 angstrom resolution) of the NS3 helicase domain (residues 186-619; NS3:186-619) from Kunjin Virus, an Australian variant of the West Nile Virus. As for homologous helicases, NS3:186-619 is composed of three domains, two of which are structurally related and held to host the NTPase and RTPase active sites. The third domain (C-terminal) is involved in RNA binding/ recognition. The NS3:186-619 construct occurs as a dimer in solution and in the crystals. We show that NS3:186-619 displays both ATPase and RTPase activities, that it can unwind a double-stranded RNA substrate, being however inactive on a double-stranded DNA substrate. Analysis of different constructs shows that full length NS3 displays increased helicase activity, suggesting that the protease domain plays an assisting role in the RNA unwinding process. The structural interaction between the helicase and protease domain has been assessed using small angle X-ray scattering on full length NS3, disclosing that the protease and helicase domains build a rather elongated molecular assembly differing from that observed in the NS3 protein from hepatitis C Virus. (C) 2007 Elsevier Ltd. All rights reserved.
