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Stephan Becker - One of the best experts on this subject based on the ideXlab platform.
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The Ebola Virus Nucleoprotein Recruits the Host PP2A-B56 Phosphatase to Activate Transcriptional Support Activity of VP30.
Molecular Cell, 2017Co-Authors: Thomas Kruse, Nadine Biedenkopf, Emil Peter Thrane Hertz, Erik Dietzel, Gertrud Stalmann, Blanca López-méndez, Norman E. Davey, Jakob Nilsson, Stephan BeckerAbstract:Summary Transcription of the Ebola virus genome depends on the Viral Transcription factor VP30 in its unphosphorylated form, but the underlying molecular mechanism of VP30 dephosphorylation is unknown. Here we show that the Ebola virus nucleoprotein (NP) recruits the host PP2A-B56 protein phosphatase through a B56-binding LxxIxE motif and that this motif is essential for VP30 dephosphorylation and Viral Transcription. The LxxIxE motif and the binding site of VP30 in NP are in close proximity, and both binding sites are required for the dephosphorylation of VP30. We generate a specific inhibitor of PP2A-B56 and show that it suppresses Ebola virus Transcription and infection. This work dissects the molecular mechanism of VP30 dephosphorylation by PP2A-B56, and it pinpoints this phosphatase as a potential target for therapeutic intervention.
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rna binding of ebola virus vp30 is essential for activating Viral Transcription
Journal of Virology, 2016Co-Authors: Nadine Biedenkopf, Stephan Becker, Julia Schlereth, Arnold Grunweller, Roland K HartmannAbstract:ABSTRACT The template for Ebola virus (EBOV) Transcription and replication is the helical Viral nucleocapsid composed of the Viral negative-sense (−) RNA genome, which is complexed by the nucleoprotein (NP), VP35, polymerase L, VP24, and VP30. While Viral replication is exerted by polymerase L and its cofactor VP35, EBOV mRNA synthesis is regulated by the Viral nucleocapsid protein VP30, an essential EBOV-specific Transcription factor. VP30 is a homohexameric phosphoprotein containing a nonconventional zinc finger. The Transcriptional support activity of VP30 is strongly influenced by its phosphorylation state. We studied here how RNA binding contributed to VP309s function in Transcriptional activation. Using a novel mobility shift assay and the 3′-terminal 154 nucleotides of the EBOV genome as a standard RNA substrate, we detected that RNA binding of VP30 was severely impaired by VP30 mutations that (i) destroy the protein9s capability to form homohexamers, (ii) disrupt the integrity of its zinc finger domain, (iii) mimic its fully phosphorylated state, or (iv) alter the putative RNA binding region. RNA binding of the mutant VP30 proteins correlated strongly with their Transcriptional support activity. Furthermore, we showed that the interaction between VP30 and the polymerase cofactor VP35 is RNA dependent, while formation of VP30 homohexamers and VP35 homotetramers is not. Our data indicate that RNA binding of VP30 is essential for its Transcriptional support activity and stabilizes complexes of VP35/L polymerase with the (−) RNA template to favor productive Transcriptional initiation in the presence of termination-active RNA secondary structures. IMPORTANCE Ebola virus causes severe fevers with unusually high case fatality rates. The recent outbreak of Ebola virus in West Africa claimed more than 11,000 lives and threatened to destabilize a whole region because of its dramatic effects on the public health systems. It is currently not completely understood how Ebola virus manages to balance Viral Transcription and replication in the infected cells. This study shows that Transcriptional support activity of the Ebola virus Transcription factor VP30 is highly correlated with its ability to bind Viral RNA. The interaction between VP30 and VP35, the Ebola virus polymerase cofactor, is dependent on the presence of RNA as well. Our data contribute to the understanding of the dynamic interplay between nucleocapsid proteins and the Viral RNA template in order to promote Viral RNA synthesis.
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Dynamic Phosphorylation of VP30 is essential for Ebola virus life cycle
Journal of Virology, 2016Co-Authors: Nadine Biedenkopf, Clemens Lier, Stephan BeckerAbstract:ABSTRACT Ebola virus is the causative agent of a severe fever with high fatality rates in humans and nonhuman primates. The regulation of Ebola virus Transcription and replication currently is not well understood. An important factor regulating Viral Transcription is VP30, an Ebola virus-specific Transcription factor associated with the Viral nucleocapsid. Previous studies revealed that the phosphorylation status of VP30 impacts Viral Transcription. Together with NP, L, and the polymerase cofactor VP35, nonphosphorylated VP30 supports Viral Transcription. Upon VP30 phosphorylation, Viral Transcription ceases. Phosphorylation weakens the interaction between VP30 and the polymerase cofactor VP35 and/or the Viral RNA. VP30 thereby is excluded from the Viral Transcription complex, simultaneously leading to increased Viral replication which is supported by NP, L, and VP35 alone. Here, we use an infectious virus-like particle assay and recombinant viruses to show that the dynamic phosphorylation of VP30 is critical for the cotransport of VP30 with nucleocapsids to the sites of Viral RNA synthesis, where VP30 is required to initiate primary Viral Transcription. We further demonstrate that a single serine residue at amino acid position 29 was sufficient to render VP30 active in primary Transcription and to generate a recombinant virus with characteristics comparable to those of wild-type virus. In contrast, the rescue of a recombinant virus with a single serine at position 30 in VP30 was unsuccessful. Our results indicate critical roles for phosphorylated and dephosphorylated VP30 during the Viral life cycle. IMPORTANCE The current Ebola virus outbreak in West Africa has caused more than 28,000 cases and 11,000 fatalities. Very little is known regarding the molecular mechanisms of how the Ebola virus transcribes and replicates its genome. Previous investigations showed that the Transcriptional support activity of VP30 is activated upon VP30 dephosphorylation. The current study reveals that the situation is more complex and that primary Transcription as well as the rescue of recombinant Ebola virus also requires the transient phosphorylation of VP30. VP30 encodes six N-proximal serine residues that serve as phosphorylation acceptor sites. The present study shows that the dynamic phosphorylation of serine at position 29 alone is sufficient to activate primary Viral Transcription. Our results indicate a series of phosphorylation/dephosphorylation events that trigger binding to and release from the nucleocapsid and Transcription complex to be essential for the full activity of VP30.
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Phosphorylation of ebola virus VP30 influences the composition of the Viral nucleocapsid complex: Impact on Viral Transcription and replication
Journal of Biological Chemistry, 2013Co-Authors: Nadine Biedenkopf, Bettina Hartlieb, Thomas Hoenen, Stephan BeckerAbstract:Ebola virus is a non-segmented negative-sense RNA virus causing severe hemorrhagic fever with high fatality rates in humans and nonhuman primates. For Transcription of the Viral genome four Viral proteins are essential: the nucleoprotein NP, the polymerase L, the polymerase cofactor VP35, and VP30. VP30 represents an essential Ebola virus-specific Transcription factor whose activity is regulated via its phosphorylation state. In contrast to Viral Transcription, VP30 is not required for Viral replication. Using a minigenome assay, we show that phosphorylation of VP30 inhibits Viral Transcription while Viral replication is increased. Concurrently, phosphorylation of VP30 reciprocally regulates a newly described interaction of VP30 with VP35, and strengthens the interaction with NP. Our results indicate a critical role of VP30 phosphorylation for Viral Transcription and replication, suggesting a mechanism by which VP30 phosphorylation modulates the composition of the Viral polymerase complex presumably forming a transcriptase in the presence of non-phosphorylated VP30 or a replicase in the presence of phosphorylated VP30.
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Oligomerization of Ebola virus VP30 is essential for Viral Transcription and can be inhibited by a synthetic peptide.
Journal of Biological Chemistry, 2003Co-Authors: Bettina Hartlieb, Jens Modrof, Elke Mühlberger, Hans-dieter Klenk, Stephan BeckerAbstract:Abstract Transcription of Ebola virus (EBOV)-specific mRNA is driven by the nucleocapsid proteins NP, VP35, and L. This process is further dependent on VP30, an essential EBOV-specific Transcription factor. The present study addresses the self-assembly of VP30 and the functional significance of this process for Viral Transcription and propagation. Essential for oligomerization of VP30 is a region spanning amino acids 94–112. Within this region a cluster of four leucine residues is of critical importance. Mutation of only one of these leucine residues resulted in oligomerization-deficient VP30 molecules that were no longer able to support EBOV-specific Transcription. The essential role of homo-oligomerization for the function of VP30 was further corroborated by the finding that mixed VP30 oligomers consisting of VP30 and Transcriptionally inactive VP30 mutants were impaired in their ability to support EBOV Transcription. The dominant negative effect of these VP30 mutants was dependent on their ability to bind to VP30. The oligomerization of VP30 could be dose dependently inhibited by a 25-mer peptide (E30pep-wt) derived from the presumed oligomerization domain (IC50,1 μm). A control peptide (E30pep-3LA), in which three leucines were changed to alanine, had no inhibitory effect. Thus, E30pep-wt seemed to bind efficiently to VP30 and consequently blocked the oligomerization of the protein. When E30pep-wt was transfected into EBOV-infected cells, the peptide inhibited Viral replication suggesting that inhibition of VP30 oligomerization represents a target for EBOV antiViral drugs.
Nadine Biedenkopf - One of the best experts on this subject based on the ideXlab platform.
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The Ebola Virus Nucleoprotein Recruits the Host PP2A-B56 Phosphatase to Activate Transcriptional Support Activity of VP30.
Molecular Cell, 2017Co-Authors: Thomas Kruse, Nadine Biedenkopf, Emil Peter Thrane Hertz, Erik Dietzel, Gertrud Stalmann, Blanca López-méndez, Norman E. Davey, Jakob Nilsson, Stephan BeckerAbstract:Summary Transcription of the Ebola virus genome depends on the Viral Transcription factor VP30 in its unphosphorylated form, but the underlying molecular mechanism of VP30 dephosphorylation is unknown. Here we show that the Ebola virus nucleoprotein (NP) recruits the host PP2A-B56 protein phosphatase through a B56-binding LxxIxE motif and that this motif is essential for VP30 dephosphorylation and Viral Transcription. The LxxIxE motif and the binding site of VP30 in NP are in close proximity, and both binding sites are required for the dephosphorylation of VP30. We generate a specific inhibitor of PP2A-B56 and show that it suppresses Ebola virus Transcription and infection. This work dissects the molecular mechanism of VP30 dephosphorylation by PP2A-B56, and it pinpoints this phosphatase as a potential target for therapeutic intervention.
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rna binding of ebola virus vp30 is essential for activating Viral Transcription
Journal of Virology, 2016Co-Authors: Nadine Biedenkopf, Stephan Becker, Julia Schlereth, Arnold Grunweller, Roland K HartmannAbstract:ABSTRACT The template for Ebola virus (EBOV) Transcription and replication is the helical Viral nucleocapsid composed of the Viral negative-sense (−) RNA genome, which is complexed by the nucleoprotein (NP), VP35, polymerase L, VP24, and VP30. While Viral replication is exerted by polymerase L and its cofactor VP35, EBOV mRNA synthesis is regulated by the Viral nucleocapsid protein VP30, an essential EBOV-specific Transcription factor. VP30 is a homohexameric phosphoprotein containing a nonconventional zinc finger. The Transcriptional support activity of VP30 is strongly influenced by its phosphorylation state. We studied here how RNA binding contributed to VP309s function in Transcriptional activation. Using a novel mobility shift assay and the 3′-terminal 154 nucleotides of the EBOV genome as a standard RNA substrate, we detected that RNA binding of VP30 was severely impaired by VP30 mutations that (i) destroy the protein9s capability to form homohexamers, (ii) disrupt the integrity of its zinc finger domain, (iii) mimic its fully phosphorylated state, or (iv) alter the putative RNA binding region. RNA binding of the mutant VP30 proteins correlated strongly with their Transcriptional support activity. Furthermore, we showed that the interaction between VP30 and the polymerase cofactor VP35 is RNA dependent, while formation of VP30 homohexamers and VP35 homotetramers is not. Our data indicate that RNA binding of VP30 is essential for its Transcriptional support activity and stabilizes complexes of VP35/L polymerase with the (−) RNA template to favor productive Transcriptional initiation in the presence of termination-active RNA secondary structures. IMPORTANCE Ebola virus causes severe fevers with unusually high case fatality rates. The recent outbreak of Ebola virus in West Africa claimed more than 11,000 lives and threatened to destabilize a whole region because of its dramatic effects on the public health systems. It is currently not completely understood how Ebola virus manages to balance Viral Transcription and replication in the infected cells. This study shows that Transcriptional support activity of the Ebola virus Transcription factor VP30 is highly correlated with its ability to bind Viral RNA. The interaction between VP30 and VP35, the Ebola virus polymerase cofactor, is dependent on the presence of RNA as well. Our data contribute to the understanding of the dynamic interplay between nucleocapsid proteins and the Viral RNA template in order to promote Viral RNA synthesis.
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Dynamic Phosphorylation of VP30 is essential for Ebola virus life cycle
Journal of Virology, 2016Co-Authors: Nadine Biedenkopf, Clemens Lier, Stephan BeckerAbstract:ABSTRACT Ebola virus is the causative agent of a severe fever with high fatality rates in humans and nonhuman primates. The regulation of Ebola virus Transcription and replication currently is not well understood. An important factor regulating Viral Transcription is VP30, an Ebola virus-specific Transcription factor associated with the Viral nucleocapsid. Previous studies revealed that the phosphorylation status of VP30 impacts Viral Transcription. Together with NP, L, and the polymerase cofactor VP35, nonphosphorylated VP30 supports Viral Transcription. Upon VP30 phosphorylation, Viral Transcription ceases. Phosphorylation weakens the interaction between VP30 and the polymerase cofactor VP35 and/or the Viral RNA. VP30 thereby is excluded from the Viral Transcription complex, simultaneously leading to increased Viral replication which is supported by NP, L, and VP35 alone. Here, we use an infectious virus-like particle assay and recombinant viruses to show that the dynamic phosphorylation of VP30 is critical for the cotransport of VP30 with nucleocapsids to the sites of Viral RNA synthesis, where VP30 is required to initiate primary Viral Transcription. We further demonstrate that a single serine residue at amino acid position 29 was sufficient to render VP30 active in primary Transcription and to generate a recombinant virus with characteristics comparable to those of wild-type virus. In contrast, the rescue of a recombinant virus with a single serine at position 30 in VP30 was unsuccessful. Our results indicate critical roles for phosphorylated and dephosphorylated VP30 during the Viral life cycle. IMPORTANCE The current Ebola virus outbreak in West Africa has caused more than 28,000 cases and 11,000 fatalities. Very little is known regarding the molecular mechanisms of how the Ebola virus transcribes and replicates its genome. Previous investigations showed that the Transcriptional support activity of VP30 is activated upon VP30 dephosphorylation. The current study reveals that the situation is more complex and that primary Transcription as well as the rescue of recombinant Ebola virus also requires the transient phosphorylation of VP30. VP30 encodes six N-proximal serine residues that serve as phosphorylation acceptor sites. The present study shows that the dynamic phosphorylation of serine at position 29 alone is sufficient to activate primary Viral Transcription. Our results indicate a series of phosphorylation/dephosphorylation events that trigger binding to and release from the nucleocapsid and Transcription complex to be essential for the full activity of VP30.
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Phosphorylation of ebola virus VP30 influences the composition of the Viral nucleocapsid complex: Impact on Viral Transcription and replication
Journal of Biological Chemistry, 2013Co-Authors: Nadine Biedenkopf, Bettina Hartlieb, Thomas Hoenen, Stephan BeckerAbstract:Ebola virus is a non-segmented negative-sense RNA virus causing severe hemorrhagic fever with high fatality rates in humans and nonhuman primates. For Transcription of the Viral genome four Viral proteins are essential: the nucleoprotein NP, the polymerase L, the polymerase cofactor VP35, and VP30. VP30 represents an essential Ebola virus-specific Transcription factor whose activity is regulated via its phosphorylation state. In contrast to Viral Transcription, VP30 is not required for Viral replication. Using a minigenome assay, we show that phosphorylation of VP30 inhibits Viral Transcription while Viral replication is increased. Concurrently, phosphorylation of VP30 reciprocally regulates a newly described interaction of VP30 with VP35, and strengthens the interaction with NP. Our results indicate a critical role of VP30 phosphorylation for Viral Transcription and replication, suggesting a mechanism by which VP30 phosphorylation modulates the composition of the Viral polymerase complex presumably forming a transcriptase in the presence of non-phosphorylated VP30 or a replicase in the presence of phosphorylated VP30.
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oligomerization of ebola virus vp40 is essential for particle morphogenesis and regulation of Viral Transcription
Journal of Virology, 2010Co-Authors: Nadine Biedenkopf, Thomas Hoenen, Florian Zielecki, S Jung, Allison Groseth, Heinz FeldmannAbstract:The morphogenesis and budding of virus particles represent an important stage in the life cycle of viruses. For Ebola virus, this process is driven by its major matrix protein, VP40. Like the matrix proteins of many other nonsegmented, negative-strand RNA viruses, VP40 has been demonstrated to oligomerize and to occur in at least two distinct oligomeric states: hexamers and octamers, which are composed of antiparallel dimers. While it has been shown that VP40 oligomers are essential for the Viral life cycle, their function is completely unknown. Here we have identified two amino acids essential for oligomerization of VP40, the mutation of which blocked virus-like particle production. Consistent with this observation, oligomerization-deficient VP40 also showed impaired intracellular transport to budding sites and reduced binding to cellular membranes. However, other biological functions, such as the interaction of VP40 with the nucleoprotein, NP, remained undisturbed. Furthermore, both wild-type VP40 and oligomerization-deficient VP40 were found to negatively regulate Viral genome replication, a novel function of VP40, which we have recently reported. Interestingly, while wild-type VP40 was also able to negatively regulate Viral genome Transcription, oligomerization-deficient VP40 was no longer able to fulfill this function, indicating that regulation of Viral replication and Transcription by VP40 are mechanistically distinct processes. These data indicate that VP40 oligomerization not only is a prerequisite for intracellular transport of VP40 and efficient membrane binding, and as a consequence virion morphogenesis, but also plays a critical role in the regulation of Viral Transcription by VP40.
Thomas Hoenen - One of the best experts on this subject based on the ideXlab platform.
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Phosphorylation of ebola virus VP30 influences the composition of the Viral nucleocapsid complex: Impact on Viral Transcription and replication
Journal of Biological Chemistry, 2013Co-Authors: Nadine Biedenkopf, Bettina Hartlieb, Thomas Hoenen, Stephan BeckerAbstract:Ebola virus is a non-segmented negative-sense RNA virus causing severe hemorrhagic fever with high fatality rates in humans and nonhuman primates. For Transcription of the Viral genome four Viral proteins are essential: the nucleoprotein NP, the polymerase L, the polymerase cofactor VP35, and VP30. VP30 represents an essential Ebola virus-specific Transcription factor whose activity is regulated via its phosphorylation state. In contrast to Viral Transcription, VP30 is not required for Viral replication. Using a minigenome assay, we show that phosphorylation of VP30 inhibits Viral Transcription while Viral replication is increased. Concurrently, phosphorylation of VP30 reciprocally regulates a newly described interaction of VP30 with VP35, and strengthens the interaction with NP. Our results indicate a critical role of VP30 phosphorylation for Viral Transcription and replication, suggesting a mechanism by which VP30 phosphorylation modulates the composition of the Viral polymerase complex presumably forming a transcriptase in the presence of non-phosphorylated VP30 or a replicase in the presence of phosphorylated VP30.
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oligomerization of ebola virus vp40 is essential for particle morphogenesis and regulation of Viral Transcription
Journal of Virology, 2010Co-Authors: Nadine Biedenkopf, Thomas Hoenen, Florian Zielecki, S Jung, Allison Groseth, Heinz FeldmannAbstract:The morphogenesis and budding of virus particles represent an important stage in the life cycle of viruses. For Ebola virus, this process is driven by its major matrix protein, VP40. Like the matrix proteins of many other nonsegmented, negative-strand RNA viruses, VP40 has been demonstrated to oligomerize and to occur in at least two distinct oligomeric states: hexamers and octamers, which are composed of antiparallel dimers. While it has been shown that VP40 oligomers are essential for the Viral life cycle, their function is completely unknown. Here we have identified two amino acids essential for oligomerization of VP40, the mutation of which blocked virus-like particle production. Consistent with this observation, oligomerization-deficient VP40 also showed impaired intracellular transport to budding sites and reduced binding to cellular membranes. However, other biological functions, such as the interaction of VP40 with the nucleoprotein, NP, remained undisturbed. Furthermore, both wild-type VP40 and oligomerization-deficient VP40 were found to negatively regulate Viral genome replication, a novel function of VP40, which we have recently reported. Interestingly, while wild-type VP40 was also able to negatively regulate Viral genome Transcription, oligomerization-deficient VP40 was no longer able to fulfill this function, indicating that regulation of Viral replication and Transcription by VP40 are mechanistically distinct processes. These data indicate that VP40 oligomerization not only is a prerequisite for intracellular transport of VP40 and efficient membrane binding, and as a consequence virion morphogenesis, but also plays a critical role in the regulation of Viral Transcription by VP40.
Danielle Blondel - One of the best experts on this subject based on the ideXlab platform.
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hsp70 protein positively regulates rabies virus infection
Journal of Virology, 2012Co-Authors: Xavier Lahaye, Aurore Vidy, Baptiste Fouquet, Danielle BlondelAbstract:The Hsp70 chaperone plays a central role in multiple processes within cells, including protein translation, folding, intracellular trafficking, and degradation. This protein is implicated in the replication of numerous viruses. We have shown that rabies virus infection induced the cellular expression of Hsp70, which accumulated in Negri body-like structures, where Viral Transcription and replication take place. In addition, Hsp70 is present in both nucleocapsids purified from infected cells and in purified virions. Hsp70 has been shown to interact with the nucleoprotein N. The downregulation of Hsp70, using specific chaperone inhibitors, such as quercetin or RNA interference, resulted in a significant decrease of the amount of Viral mRNAs, Viral proteins, and virus particles. These results indicate that Hsp70 has a proViral function during rabies virus infection and suggest that Hsp70 is involved in at least one stage(s) of the Viral life cycle, such as Viral Transcription, translation, and/or production. The mechanism by which Hsp70 controls Viral infection will be discussed.
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rabies virus Transcription and replication
Advances in Virus Research, 2011Co-Authors: Aurelie Albertini, Rob W H Ruigrok, Danielle BlondelAbstract:Abstract Rabies virus (RABV) is a negative-stranded RNA virus. Its genome is tightly encapsidated by the Viral nucleoprotein (N) and this RNA–N complex is the template for Transcription and replication by the Viral RNA-dependent RNA polymerase (L) and its cofactor, the phosphoprotein (P). We present molecular, structural, and cellular aspects of RABV Transcription and replication. We first summarize the characteristics and molecular biology of both RNA synthesis processes. We then discuss biochemical and structural data on the Viral proteins (N, P, and L) and their interactions with regard to their role in Viral Transcription and replication. Finally, we review evidence that rabies Viral Transcription and replication take place in cytoplasmic inclusion bodies formed in RABV-infected cells and discuss the role of this cellular compartmentalization.
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functional characterization of negri bodies nbs in rabies virus infected cells evidence that nbs are sites of Viral Transcription and replication
Journal of Virology, 2009Co-Authors: Xavier Lahaye, Aurore Vidy, Carole Pomier, Linda Obiang, Francis Harper, Yves Gaudin, Danielle BlondelAbstract:Rabies virus infection induces the formation of cytoplasmic inclusion bodies that resemble Negri bodies found in the cytoplasm of some infected nerve cells. We have studied the morphogenesis and the role of these Negri body-like structures (NBLs) during Viral infection. The results indicate that these spherical structures (one or two per cell in the initial stage of infection), composed of the Viral N and P proteins, grow during the virus cycle before appearing as smaller structures at late stages of infection. We have shown that the microtubule network is not necessary for the formation of these inclusion bodies but is involved in their dynamics. In contrast, the actin network does not play any detectable role in these processes. These inclusion bodies contain Hsp70 and ubiquitinylated proteins, but they are not misfolded protein aggregates. NBLs, in fact, appear to be functional structures involved in the Viral life cycle. Specifically, using in situ fluorescent hybridization techniques, we show that all Viral RNAs (genome, antigenome, and every mRNA) are located inside the inclusion bodies. Significantly, short-term RNA labeling in the presence of BrUTP strongly suggests that the NBLs are the sites where Viral Transcription and replication take place.
Roland K Hartmann - One of the best experts on this subject based on the ideXlab platform.
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rna binding of ebola virus vp30 is essential for activating Viral Transcription
Journal of Virology, 2016Co-Authors: Nadine Biedenkopf, Stephan Becker, Julia Schlereth, Arnold Grunweller, Roland K HartmannAbstract:ABSTRACT The template for Ebola virus (EBOV) Transcription and replication is the helical Viral nucleocapsid composed of the Viral negative-sense (−) RNA genome, which is complexed by the nucleoprotein (NP), VP35, polymerase L, VP24, and VP30. While Viral replication is exerted by polymerase L and its cofactor VP35, EBOV mRNA synthesis is regulated by the Viral nucleocapsid protein VP30, an essential EBOV-specific Transcription factor. VP30 is a homohexameric phosphoprotein containing a nonconventional zinc finger. The Transcriptional support activity of VP30 is strongly influenced by its phosphorylation state. We studied here how RNA binding contributed to VP309s function in Transcriptional activation. Using a novel mobility shift assay and the 3′-terminal 154 nucleotides of the EBOV genome as a standard RNA substrate, we detected that RNA binding of VP30 was severely impaired by VP30 mutations that (i) destroy the protein9s capability to form homohexamers, (ii) disrupt the integrity of its zinc finger domain, (iii) mimic its fully phosphorylated state, or (iv) alter the putative RNA binding region. RNA binding of the mutant VP30 proteins correlated strongly with their Transcriptional support activity. Furthermore, we showed that the interaction between VP30 and the polymerase cofactor VP35 is RNA dependent, while formation of VP30 homohexamers and VP35 homotetramers is not. Our data indicate that RNA binding of VP30 is essential for its Transcriptional support activity and stabilizes complexes of VP35/L polymerase with the (−) RNA template to favor productive Transcriptional initiation in the presence of termination-active RNA secondary structures. IMPORTANCE Ebola virus causes severe fevers with unusually high case fatality rates. The recent outbreak of Ebola virus in West Africa claimed more than 11,000 lives and threatened to destabilize a whole region because of its dramatic effects on the public health systems. It is currently not completely understood how Ebola virus manages to balance Viral Transcription and replication in the infected cells. This study shows that Transcriptional support activity of the Ebola virus Transcription factor VP30 is highly correlated with its ability to bind Viral RNA. The interaction between VP30 and VP35, the Ebola virus polymerase cofactor, is dependent on the presence of RNA as well. Our data contribute to the understanding of the dynamic interplay between nucleocapsid proteins and the Viral RNA template in order to promote Viral RNA synthesis.
