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Richard K. Plemper - One of the best experts on this subject based on the ideXlab platform.
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the unstructured paramyxoVirus nucleocapsid protein tail domain modulates viral pathogenesis through regulation of transcriptase activity
Journal of Virology, 2018Co-Authors: Vidhi D Thakkar, Bevan Sawatsky, Renata Da Fontoura Budaszewski, Julien Sourimant, Katrin Wabbel, Veronika Von Messling, Negar Makhsous, Alexander L. Greninger, Richard K. PlemperAbstract:The paramyxoVirus replication machinery comprises the viral large (L) protein and phosphoprotein (P-protein) in addition to the nucleocapsid (N) protein, which encapsidates the single-stranded RNA genome. Common to paramyxoVirus N proteins is a C-terminal tail (Ntail). The mechanistic role and relevance for Virus replication of the structurally disordered central Ntail section are unknown. Focusing initially on members of the MorbilliVirus genus, a series of measles Virus (MeV) and canine distemper Virus (CDV) N proteins were generated with internal deletions in the unstructured tail section. N proteins with large tail truncations remained bioactive in mono- and polycistronic minireplicon assays and supported efficient replication of recombinant Viruses. Bioactivity of Ntail mutants extended to N proteins derived from highly pathogenic Nipah Virus. To probe an effect of Ntail truncations on viral pathogenesis, recombinant CDVs were analyzed in a lethal CDV/ferret model of morbilliVirus disease. The recombinant Viruses displayed different stages of Attenuation ranging from ameliorated clinical symptoms to complete survival of infected animals, depending on the molecular nature of the Ntail truncation. Reinfection of surviving animals with pathogenic CDV revealed robust protection against a lethal challenge. The highly attenuated Virus was genetically stable after ex vivo passaging and recovery from infected animals. Mechanistically, gradual viral Attenuation coincided with stepwise altered viral transcriptase activity in infected cells. These results identify the central Ntail section as a determinant for viral pathogenesis and establish a novel platform to engineer gradual Virus Attenuation for next-generation paramyxoVirus vaccine design. IMPORTANCE Investigating the role of the paramyxoVirus N protein tail domain (Ntail) in Virus replication, we demonstrated in this study that the structurally disordered central Ntail region is a determinant for viral pathogenesis. We show that internal deletions in this Ntail region of up to 55 amino acids in length are compatible with efficient replication of recombinant Viruses in cell culture but result in gradual viral Attenuation in a lethal canine distemper Virus (CDV)/ferret model. Mechanistically, we demonstrate a role of the intact Ntail region in the regulation of viral transcriptase activity. Recombinant Viruses with Ntail truncations induce protective immunity against lethal challenge of ferrets with pathogenic CDV. This identification of the unstructured central Ntail domain as a nonessential paramyxoVirus pathogenesis factor establishes a foundation for harnessing Ntail truncations for vaccine engineering against emerging and reemerging members of the paramyxoVirus family.
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the unstructured paramyxoVirus nucleocapsid protein tail domain modulates viral pathogenesis through regulation of transcriptase activity
Journal of Virology, 2018Co-Authors: Vidhi D Thakkar, Bevan Sawatsky, Renata Da Fontoura Budaszewski, Julien Sourimant, Katrin Wabbel, Veronika Von Messling, Negar Makhsous, Alexander L. Greninger, Richard K. PlemperAbstract:The paramyxoVirus replication machinery comprises the viral large (L) protein and phosphoprotein (P-protein) in addition to the nucleocapsid (N) protein, which encapsidates the single-stranded RNA genome. Common to paramyxoVirus N proteins is a C-terminal tail (Ntail). The mechanistic role and relevance for Virus replication of the structurally disordered central Ntail section are unknown. Focusing initially on members of the MorbilliVirus genus, a series of measles Virus (MeV) and canine distemper Virus (CDV) N proteins were generated with internal deletions in the unstructured tail section. N proteins with large tail truncations remained bioactive in mono- and polycistronic minireplicon assays and supported efficient replication of recombinant Viruses. Bioactivity of Ntail mutants extended to N proteins derived from highly pathogenic Nipah Virus. To probe an effect of Ntail truncations on viral pathogenesis, recombinant CDVs were analyzed in a lethal CDV/ferret model of morbilliVirus disease. The recombinant Viruses displayed different stages of Attenuation ranging from ameliorated clinical symptoms to complete survival of infected animals, depending on the molecular nature of the Ntail truncation. Reinfection of surviving animals with pathogenic CDV revealed robust protection against a lethal challenge. The highly attenuated Virus was genetically stable after ex vivo passaging and recovery from infected animals. Mechanistically, gradual viral Attenuation coincided with stepwise altered viral transcriptase activity in infected cells. These results identify the central Ntail section as a determinant for viral pathogenesis and establish a novel platform to engineer gradual Virus Attenuation for next-generation paramyxoVirus vaccine design. IMPORTANCE Investigating the role of the paramyxoVirus N protein tail domain (Ntail) in Virus replication, we demonstrated in this study that the structurally disordered central Ntail region is a determinant for viral pathogenesis. We show that internal deletions in this Ntail region of up to 55 amino acids in length are compatible with efficient replication of recombinant Viruses in cell culture but result in gradual viral Attenuation in a lethal canine distemper Virus (CDV)/ferret model. Mechanistically, we demonstrate a role of the intact Ntail region in the regulation of viral transcriptase activity. Recombinant Viruses with Ntail truncations induce protective immunity against lethal challenge of ferrets with pathogenic CDV. This identification of the unstructured central Ntail domain as a nonessential paramyxoVirus pathogenesis factor establishes a foundation for harnessing Ntail truncations for vaccine engineering against emerging and reemerging members of the paramyxoVirus family.
Vidhi D Thakkar - One of the best experts on this subject based on the ideXlab platform.
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the unstructured paramyxoVirus nucleocapsid protein tail domain modulates viral pathogenesis through regulation of transcriptase activity
Journal of Virology, 2018Co-Authors: Vidhi D Thakkar, Bevan Sawatsky, Renata Da Fontoura Budaszewski, Julien Sourimant, Katrin Wabbel, Veronika Von Messling, Negar Makhsous, Alexander L. Greninger, Richard K. PlemperAbstract:The paramyxoVirus replication machinery comprises the viral large (L) protein and phosphoprotein (P-protein) in addition to the nucleocapsid (N) protein, which encapsidates the single-stranded RNA genome. Common to paramyxoVirus N proteins is a C-terminal tail (Ntail). The mechanistic role and relevance for Virus replication of the structurally disordered central Ntail section are unknown. Focusing initially on members of the MorbilliVirus genus, a series of measles Virus (MeV) and canine distemper Virus (CDV) N proteins were generated with internal deletions in the unstructured tail section. N proteins with large tail truncations remained bioactive in mono- and polycistronic minireplicon assays and supported efficient replication of recombinant Viruses. Bioactivity of Ntail mutants extended to N proteins derived from highly pathogenic Nipah Virus. To probe an effect of Ntail truncations on viral pathogenesis, recombinant CDVs were analyzed in a lethal CDV/ferret model of morbilliVirus disease. The recombinant Viruses displayed different stages of Attenuation ranging from ameliorated clinical symptoms to complete survival of infected animals, depending on the molecular nature of the Ntail truncation. Reinfection of surviving animals with pathogenic CDV revealed robust protection against a lethal challenge. The highly attenuated Virus was genetically stable after ex vivo passaging and recovery from infected animals. Mechanistically, gradual viral Attenuation coincided with stepwise altered viral transcriptase activity in infected cells. These results identify the central Ntail section as a determinant for viral pathogenesis and establish a novel platform to engineer gradual Virus Attenuation for next-generation paramyxoVirus vaccine design. IMPORTANCE Investigating the role of the paramyxoVirus N protein tail domain (Ntail) in Virus replication, we demonstrated in this study that the structurally disordered central Ntail region is a determinant for viral pathogenesis. We show that internal deletions in this Ntail region of up to 55 amino acids in length are compatible with efficient replication of recombinant Viruses in cell culture but result in gradual viral Attenuation in a lethal canine distemper Virus (CDV)/ferret model. Mechanistically, we demonstrate a role of the intact Ntail region in the regulation of viral transcriptase activity. Recombinant Viruses with Ntail truncations induce protective immunity against lethal challenge of ferrets with pathogenic CDV. This identification of the unstructured central Ntail domain as a nonessential paramyxoVirus pathogenesis factor establishes a foundation for harnessing Ntail truncations for vaccine engineering against emerging and reemerging members of the paramyxoVirus family.
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the unstructured paramyxoVirus nucleocapsid protein tail domain modulates viral pathogenesis through regulation of transcriptase activity
Journal of Virology, 2018Co-Authors: Vidhi D Thakkar, Bevan Sawatsky, Renata Da Fontoura Budaszewski, Julien Sourimant, Katrin Wabbel, Veronika Von Messling, Negar Makhsous, Alexander L. Greninger, Richard K. PlemperAbstract:The paramyxoVirus replication machinery comprises the viral large (L) protein and phosphoprotein (P-protein) in addition to the nucleocapsid (N) protein, which encapsidates the single-stranded RNA genome. Common to paramyxoVirus N proteins is a C-terminal tail (Ntail). The mechanistic role and relevance for Virus replication of the structurally disordered central Ntail section are unknown. Focusing initially on members of the MorbilliVirus genus, a series of measles Virus (MeV) and canine distemper Virus (CDV) N proteins were generated with internal deletions in the unstructured tail section. N proteins with large tail truncations remained bioactive in mono- and polycistronic minireplicon assays and supported efficient replication of recombinant Viruses. Bioactivity of Ntail mutants extended to N proteins derived from highly pathogenic Nipah Virus. To probe an effect of Ntail truncations on viral pathogenesis, recombinant CDVs were analyzed in a lethal CDV/ferret model of morbilliVirus disease. The recombinant Viruses displayed different stages of Attenuation ranging from ameliorated clinical symptoms to complete survival of infected animals, depending on the molecular nature of the Ntail truncation. Reinfection of surviving animals with pathogenic CDV revealed robust protection against a lethal challenge. The highly attenuated Virus was genetically stable after ex vivo passaging and recovery from infected animals. Mechanistically, gradual viral Attenuation coincided with stepwise altered viral transcriptase activity in infected cells. These results identify the central Ntail section as a determinant for viral pathogenesis and establish a novel platform to engineer gradual Virus Attenuation for next-generation paramyxoVirus vaccine design. IMPORTANCE Investigating the role of the paramyxoVirus N protein tail domain (Ntail) in Virus replication, we demonstrated in this study that the structurally disordered central Ntail region is a determinant for viral pathogenesis. We show that internal deletions in this Ntail region of up to 55 amino acids in length are compatible with efficient replication of recombinant Viruses in cell culture but result in gradual viral Attenuation in a lethal canine distemper Virus (CDV)/ferret model. Mechanistically, we demonstrate a role of the intact Ntail region in the regulation of viral transcriptase activity. Recombinant Viruses with Ntail truncations induce protective immunity against lethal challenge of ferrets with pathogenic CDV. This identification of the unstructured central Ntail domain as a nonessential paramyxoVirus pathogenesis factor establishes a foundation for harnessing Ntail truncations for vaccine engineering against emerging and reemerging members of the paramyxoVirus family.
Stanley Perlman - One of the best experts on this subject based on the ideXlab platform.
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recombinant receptor binding domains of multiple middle east respiratory syndrome coronaViruses mers covs induce cross neutralizing antibodies against divergent human and camel mers covs and antibody escape mutants
Journal of Virology, 2017Co-Authors: Wanbo Tai, Stanley Perlman, Craig Fett, Yufei Wang, Guangyu Zhao, Shibo Jiang, Yusen ZhouAbstract:Middle East respiratory syndrome coronaVirus (MERS-CoV) binds to cellular receptor dipeptidyl peptidase 4 (DPP4) via the spike (S) protein receptor-binding domain (RBD). The RBD contains critical neutralizing epitopes and serves as an important vaccine target. Since RBD mutations occur in different MERS-CoV isolates and antibody escape mutants, cross-neutralization of divergent MERS-CoV strains by RBD-induced antibodies remains unknown. Here, we constructed four recombinant RBD (rRBD) proteins with single or multiple mutations detected in representative human MERS-CoV strains from the 2012, 2013, 2014, and 2015 outbreaks, respectively, and one rRBD protein with multiple changes derived from camel MERS-CoV strains. Like the RBD of prototype EMC2012 (EMC-RBD), all five RBDs maintained good antigenicity and functionality, the ability to bind RBD-specific neutralizing monoclonal antibodies (MAbs) and the DPP4 receptor, and high immunogenicity, able to elicit S-specific antibodies. They induced potent neutralizing antibodies cross-neutralizing 17 MERS pseudoViruses expressing S proteins of representative human and camel MERS-CoV strains identified during the 2012-2015 outbreaks, 5 MAb escape MERS-CoV mutants, and 2 live human MERS-CoV strains. We then constructed two RBDs mutated in multiple key residues in the receptor-binding motif (RBM) of RBD and demonstrated their strong cross-reactivity with anti-EMC-RBD antibodies. These RBD mutants with diminished DPP4 binding also led to Virus Attenuation, suggesting that immunoevasion after RBD immunization is accompanied by loss of viral fitness. Therefore, this study demonstrates that MERS-CoV RBD is an important vaccine target able to induce highly potent and broad-spectrum neutralizing antibodies against infection by divergent circulating human and camel MERS-CoV strains. Importance MERS-CoV was first identified in June 2012 and has since spread in humans and camels. Mutations in its spike (S) protein receptor-binding domain (RBD), a key vaccine target, have been identified, raising concerns over the efficacy of RBD-based MERS vaccines against circulating human and camel MERS-CoV strains. Here, we constructed five vaccine candidates, designated 2012-RBD, 2013-RBD, 2014-RBD, 2015-RBD, and Camel-RBD, containing single or multiple mutations in the RBD of representative human and camel MERS-CoV strains during the 2012-2015 outbreaks. These RBD-based vaccine candidates maintained good functionality, antigenicity, and immunogenicity, and they induced strong cross-neutralizing antibodies against infection by divergent pseudotyped and live MERS-CoV strains, as well as antibody escape MERS-CoV mutants. This study provides impetus for further development of a safe, highly effective, and broad-spectrum RBD-based subunit vaccine to prevent MERS-CoV infection.
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The Conserved CoronaVirus Macrodomain Promotes Virulence and Suppresses the Innate Immune Response during Severe Acute Respiratory Syndrome CoronaVirus Infection.
Mbio, 2016Co-Authors: Anthony R Fehr, Gytis Jankevicius, Ivan Ahel, Rudragouda Channappanavar, Craig Fett, Jincun Zhao, Jeremiah Athmer, David K. Meyerholz, Stanley PerlmanAbstract:ABSTRACT ADP-ribosylation is a common posttranslational modification that may have antiviral properties and impact innate immunity. To regulate this activity, macrodomain proteins enzymatically remove covalently attached ADP-ribose from protein targets. All members of the Coronavirinae, a subfamily of positive-sense RNA Viruses, contain a highly conserved macrodomain within nonstructural protein 3 (nsp3). However, its function or targets during infection remain unknown. We identified several macrodomain mutations that greatly reduced nsp3’s de-ADP-ribosylation activity in vitro. Next, we created recombinant severe acute respiratory syndrome coronaVirus (SARS-CoV) strains with these mutations. These mutations led to Virus Attenuation and a modest reduction of viral loads in infected mice, despite normal replication in cell culture. Further, macrodomain mutant Virus elicited an early, enhanced interferon (IFN), interferon-stimulated gene (ISG), and proinflammatory cytokine response in mice and in a human bronchial epithelial cell line. Using a coinfection assay, we found that inclusion of mutant Virus in the inoculum protected mice from an otherwise lethal SARS-CoV infection without reducing Virus loads, indicating that the changes in innate immune response were physiologically significant. In conclusion, we have established a novel function for the SARS-CoV macrodomain that implicates ADP-ribose in the regulation of the innate immune response and helps to demonstrate why this domain is conserved in CoVs. IMPORTANCE The macrodomain is a ubiquitous structural domain that removes ADP-ribose from proteins, reversing the activity of ADP-ribosyltransferases. All coronaViruses contain a macrodomain, suggesting that ADP-ribosylation impacts coronaVirus infection. However, its function during infection remains unknown. Here, we found that the macrodomain is an important virulence factor for a highly pathogenic human CoV, SARS-CoV. Viruses with macrodomain mutations that abrogate its ability to remove ADP-ribose from protein were unable to cause lethal disease in mice. Importantly, the SARS-CoV macrodomain suppressed the innate immune response during infection. Our data suggest that an early innate immune response can protect mice from lethal disease. Understanding the mechanism used by this enzyme to promote disease will open up novel avenues for coronaVirus therapies and give further insight into the role of macrodomains in viral pathogenesis.
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The Conserved CoronaVirus Macrodomain Promotes Virulence and Suppresses the Innate Immune Response during Severe Acute Respiratory Syndrome CoronaVirus Infection
American Society for Microbiology, 2016Co-Authors: Anthony R Fehr, Gytis Jankevicius, Ivan Ahel, Rudragouda Channappanavar, Craig Fett, Jincun Zhao, Jeremiah Athmer, David K. Meyerholz, Stanley PerlmanAbstract:ADP-ribosylation is a common posttranslational modification that may have antiviral properties and impact innate immunity. To regulate this activity, macrodomain proteins enzymatically remove covalently attached ADP-ribose from protein targets. All members of the Coronavirinae, a subfamily of positive-sense RNA Viruses, contain a highly conserved macrodomain within nonstructural protein 3 (nsp3). However, its function or targets during infection remain unknown. We identified several macrodomain mutations that greatly reduced nsp3’s de-ADP-ribosylation activity in vitro. Next, we created recombinant severe acute respiratory syndrome coronaVirus (SARS-CoV) strains with these mutations. These mutations led to Virus Attenuation and a modest reduction of viral loads in infected mice, despite normal replication in cell culture. Further, macrodomain mutant Virus elicited an early, enhanced interferon (IFN), interferon-stimulated gene (ISG), and proinflammatory cytokine response in mice and in a human bronchial epithelial cell line. Using a coinfection assay, we found that inclusion of mutant Virus in the inoculum protected mice from an otherwise lethal SARS-CoV infection without reducing Virus loads, indicating that the changes in innate immune response were physiologically significant. In conclusion, we have established a novel function for the SARS-CoV macrodomain that implicates ADP-ribose in the regulation of the innate immune response and helps to demonstrate why this domain is conserved in CoVs
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severe acute respiratory syndrome coronaViruses with mutations in the e protein are attenuated and promising vaccine candidates
Journal of Virology, 2015Co-Authors: Jose Angel Reglanava, Stanley Perlman, Craig Fett, Jose M Jimenezguardeno, Jose L Nietotorres, Raul Fernandezdelgado, Carlos Castanorodriguez, Luis Enjuanes, Marta L DediegoAbstract:ABSTRACT Severe acute respiratory syndrome coronaVirus (SARS-CoV) causes a respiratory disease with a mortality rate of 10%. A mouse-adapted SARS-CoV (SARS-CoV-MA15) lacking the envelope (E) protein (rSARS-CoV-MA15-ΔE) is attenuated in vivo . To identify E protein regions and host responses that contribute to rSARS-CoV-MA15-ΔE Attenuation, several mutants (rSARS-CoV-MA15-E*) containing point mutations or deletions in the amino-terminal or the carboxy-terminal regions of the E protein were generated. Amino acid substitutions in the amino terminus, or deletion of regions in the internal carboxy-terminal region of E protein, led to Virus Attenuation. Attenuated Viruses induced minimal lung injury, diminished limited neutrophil influx, and increased CD4 + and CD8 + T cell counts in the lungs of BALB/c mice, compared to mice infected with the wild-type Virus. To analyze the host responses leading to rSARS-CoV-MA15-E* Attenuation, differences in gene expression elicited by the native and mutant Viruses in the lungs of infected mice were determined. Expression levels of a large number of proinflammatory cytokines associated with lung injury were reduced in the lungs of rSARS-CoV-MA15-E*-infected mice, whereas the levels of anti-inflammatory cytokines were increased, both at the mRNA and protein levels. These results suggested that the reduction in lung inflammation together with a more robust antiviral T cell response contributed to rSARS-CoV-MA15-E* Attenuation. The attenuated Viruses completely protected mice against challenge with the lethal parental Virus, indicating that these Viruses are promising vaccine candidates. IMPORTANCE Human coronaViruses are important zoonotic pathogens. SARS-CoV caused a worldwide epidemic infecting more than 8,000 people with a mortality of around 10%. Therefore, understanding the virulence mechanisms of this pathogen and developing efficacious vaccines are of high importance to prevent epidemics from this and other human coronaViruses. Previously, we demonstrated that a SARS-CoV lacking the E protein was attenuated in vivo . Here, we show that small deletions and modifications within the E protein led to Virus Attenuation, manifested by minimal lung injury, limited neutrophil influx to the lungs, reduced expression of proinflammatory cytokines, increased anti-inflammatory cytokine levels, and enhanced CD4 + and CD8 + T cell counts in vivo , suggesting that these phenomena contribute to Virus Attenuation. The attenuated mutants fully protected mice from challenge with virulent Virus. These studies show that mutations in the E protein are not well tolerated and indicate that this protein is an excellent target for vaccine development.
Negar Makhsous - One of the best experts on this subject based on the ideXlab platform.
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the unstructured paramyxoVirus nucleocapsid protein tail domain modulates viral pathogenesis through regulation of transcriptase activity
Journal of Virology, 2018Co-Authors: Vidhi D Thakkar, Bevan Sawatsky, Renata Da Fontoura Budaszewski, Julien Sourimant, Katrin Wabbel, Veronika Von Messling, Negar Makhsous, Alexander L. Greninger, Richard K. PlemperAbstract:The paramyxoVirus replication machinery comprises the viral large (L) protein and phosphoprotein (P-protein) in addition to the nucleocapsid (N) protein, which encapsidates the single-stranded RNA genome. Common to paramyxoVirus N proteins is a C-terminal tail (Ntail). The mechanistic role and relevance for Virus replication of the structurally disordered central Ntail section are unknown. Focusing initially on members of the MorbilliVirus genus, a series of measles Virus (MeV) and canine distemper Virus (CDV) N proteins were generated with internal deletions in the unstructured tail section. N proteins with large tail truncations remained bioactive in mono- and polycistronic minireplicon assays and supported efficient replication of recombinant Viruses. Bioactivity of Ntail mutants extended to N proteins derived from highly pathogenic Nipah Virus. To probe an effect of Ntail truncations on viral pathogenesis, recombinant CDVs were analyzed in a lethal CDV/ferret model of morbilliVirus disease. The recombinant Viruses displayed different stages of Attenuation ranging from ameliorated clinical symptoms to complete survival of infected animals, depending on the molecular nature of the Ntail truncation. Reinfection of surviving animals with pathogenic CDV revealed robust protection against a lethal challenge. The highly attenuated Virus was genetically stable after ex vivo passaging and recovery from infected animals. Mechanistically, gradual viral Attenuation coincided with stepwise altered viral transcriptase activity in infected cells. These results identify the central Ntail section as a determinant for viral pathogenesis and establish a novel platform to engineer gradual Virus Attenuation for next-generation paramyxoVirus vaccine design. IMPORTANCE Investigating the role of the paramyxoVirus N protein tail domain (Ntail) in Virus replication, we demonstrated in this study that the structurally disordered central Ntail region is a determinant for viral pathogenesis. We show that internal deletions in this Ntail region of up to 55 amino acids in length are compatible with efficient replication of recombinant Viruses in cell culture but result in gradual viral Attenuation in a lethal canine distemper Virus (CDV)/ferret model. Mechanistically, we demonstrate a role of the intact Ntail region in the regulation of viral transcriptase activity. Recombinant Viruses with Ntail truncations induce protective immunity against lethal challenge of ferrets with pathogenic CDV. This identification of the unstructured central Ntail domain as a nonessential paramyxoVirus pathogenesis factor establishes a foundation for harnessing Ntail truncations for vaccine engineering against emerging and reemerging members of the paramyxoVirus family.
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the unstructured paramyxoVirus nucleocapsid protein tail domain modulates viral pathogenesis through regulation of transcriptase activity
Journal of Virology, 2018Co-Authors: Vidhi D Thakkar, Bevan Sawatsky, Renata Da Fontoura Budaszewski, Julien Sourimant, Katrin Wabbel, Veronika Von Messling, Negar Makhsous, Alexander L. Greninger, Richard K. PlemperAbstract:The paramyxoVirus replication machinery comprises the viral large (L) protein and phosphoprotein (P-protein) in addition to the nucleocapsid (N) protein, which encapsidates the single-stranded RNA genome. Common to paramyxoVirus N proteins is a C-terminal tail (Ntail). The mechanistic role and relevance for Virus replication of the structurally disordered central Ntail section are unknown. Focusing initially on members of the MorbilliVirus genus, a series of measles Virus (MeV) and canine distemper Virus (CDV) N proteins were generated with internal deletions in the unstructured tail section. N proteins with large tail truncations remained bioactive in mono- and polycistronic minireplicon assays and supported efficient replication of recombinant Viruses. Bioactivity of Ntail mutants extended to N proteins derived from highly pathogenic Nipah Virus. To probe an effect of Ntail truncations on viral pathogenesis, recombinant CDVs were analyzed in a lethal CDV/ferret model of morbilliVirus disease. The recombinant Viruses displayed different stages of Attenuation ranging from ameliorated clinical symptoms to complete survival of infected animals, depending on the molecular nature of the Ntail truncation. Reinfection of surviving animals with pathogenic CDV revealed robust protection against a lethal challenge. The highly attenuated Virus was genetically stable after ex vivo passaging and recovery from infected animals. Mechanistically, gradual viral Attenuation coincided with stepwise altered viral transcriptase activity in infected cells. These results identify the central Ntail section as a determinant for viral pathogenesis and establish a novel platform to engineer gradual Virus Attenuation for next-generation paramyxoVirus vaccine design. IMPORTANCE Investigating the role of the paramyxoVirus N protein tail domain (Ntail) in Virus replication, we demonstrated in this study that the structurally disordered central Ntail region is a determinant for viral pathogenesis. We show that internal deletions in this Ntail region of up to 55 amino acids in length are compatible with efficient replication of recombinant Viruses in cell culture but result in gradual viral Attenuation in a lethal canine distemper Virus (CDV)/ferret model. Mechanistically, we demonstrate a role of the intact Ntail region in the regulation of viral transcriptase activity. Recombinant Viruses with Ntail truncations induce protective immunity against lethal challenge of ferrets with pathogenic CDV. This identification of the unstructured central Ntail domain as a nonessential paramyxoVirus pathogenesis factor establishes a foundation for harnessing Ntail truncations for vaccine engineering against emerging and reemerging members of the paramyxoVirus family.
Bevan Sawatsky - One of the best experts on this subject based on the ideXlab platform.
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the unstructured paramyxoVirus nucleocapsid protein tail domain modulates viral pathogenesis through regulation of transcriptase activity
Journal of Virology, 2018Co-Authors: Vidhi D Thakkar, Bevan Sawatsky, Renata Da Fontoura Budaszewski, Julien Sourimant, Katrin Wabbel, Veronika Von Messling, Negar Makhsous, Alexander L. Greninger, Richard K. PlemperAbstract:The paramyxoVirus replication machinery comprises the viral large (L) protein and phosphoprotein (P-protein) in addition to the nucleocapsid (N) protein, which encapsidates the single-stranded RNA genome. Common to paramyxoVirus N proteins is a C-terminal tail (Ntail). The mechanistic role and relevance for Virus replication of the structurally disordered central Ntail section are unknown. Focusing initially on members of the MorbilliVirus genus, a series of measles Virus (MeV) and canine distemper Virus (CDV) N proteins were generated with internal deletions in the unstructured tail section. N proteins with large tail truncations remained bioactive in mono- and polycistronic minireplicon assays and supported efficient replication of recombinant Viruses. Bioactivity of Ntail mutants extended to N proteins derived from highly pathogenic Nipah Virus. To probe an effect of Ntail truncations on viral pathogenesis, recombinant CDVs were analyzed in a lethal CDV/ferret model of morbilliVirus disease. The recombinant Viruses displayed different stages of Attenuation ranging from ameliorated clinical symptoms to complete survival of infected animals, depending on the molecular nature of the Ntail truncation. Reinfection of surviving animals with pathogenic CDV revealed robust protection against a lethal challenge. The highly attenuated Virus was genetically stable after ex vivo passaging and recovery from infected animals. Mechanistically, gradual viral Attenuation coincided with stepwise altered viral transcriptase activity in infected cells. These results identify the central Ntail section as a determinant for viral pathogenesis and establish a novel platform to engineer gradual Virus Attenuation for next-generation paramyxoVirus vaccine design. IMPORTANCE Investigating the role of the paramyxoVirus N protein tail domain (Ntail) in Virus replication, we demonstrated in this study that the structurally disordered central Ntail region is a determinant for viral pathogenesis. We show that internal deletions in this Ntail region of up to 55 amino acids in length are compatible with efficient replication of recombinant Viruses in cell culture but result in gradual viral Attenuation in a lethal canine distemper Virus (CDV)/ferret model. Mechanistically, we demonstrate a role of the intact Ntail region in the regulation of viral transcriptase activity. Recombinant Viruses with Ntail truncations induce protective immunity against lethal challenge of ferrets with pathogenic CDV. This identification of the unstructured central Ntail domain as a nonessential paramyxoVirus pathogenesis factor establishes a foundation for harnessing Ntail truncations for vaccine engineering against emerging and reemerging members of the paramyxoVirus family.
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the unstructured paramyxoVirus nucleocapsid protein tail domain modulates viral pathogenesis through regulation of transcriptase activity
Journal of Virology, 2018Co-Authors: Vidhi D Thakkar, Bevan Sawatsky, Renata Da Fontoura Budaszewski, Julien Sourimant, Katrin Wabbel, Veronika Von Messling, Negar Makhsous, Alexander L. Greninger, Richard K. PlemperAbstract:The paramyxoVirus replication machinery comprises the viral large (L) protein and phosphoprotein (P-protein) in addition to the nucleocapsid (N) protein, which encapsidates the single-stranded RNA genome. Common to paramyxoVirus N proteins is a C-terminal tail (Ntail). The mechanistic role and relevance for Virus replication of the structurally disordered central Ntail section are unknown. Focusing initially on members of the MorbilliVirus genus, a series of measles Virus (MeV) and canine distemper Virus (CDV) N proteins were generated with internal deletions in the unstructured tail section. N proteins with large tail truncations remained bioactive in mono- and polycistronic minireplicon assays and supported efficient replication of recombinant Viruses. Bioactivity of Ntail mutants extended to N proteins derived from highly pathogenic Nipah Virus. To probe an effect of Ntail truncations on viral pathogenesis, recombinant CDVs were analyzed in a lethal CDV/ferret model of morbilliVirus disease. The recombinant Viruses displayed different stages of Attenuation ranging from ameliorated clinical symptoms to complete survival of infected animals, depending on the molecular nature of the Ntail truncation. Reinfection of surviving animals with pathogenic CDV revealed robust protection against a lethal challenge. The highly attenuated Virus was genetically stable after ex vivo passaging and recovery from infected animals. Mechanistically, gradual viral Attenuation coincided with stepwise altered viral transcriptase activity in infected cells. These results identify the central Ntail section as a determinant for viral pathogenesis and establish a novel platform to engineer gradual Virus Attenuation for next-generation paramyxoVirus vaccine design. IMPORTANCE Investigating the role of the paramyxoVirus N protein tail domain (Ntail) in Virus replication, we demonstrated in this study that the structurally disordered central Ntail region is a determinant for viral pathogenesis. We show that internal deletions in this Ntail region of up to 55 amino acids in length are compatible with efficient replication of recombinant Viruses in cell culture but result in gradual viral Attenuation in a lethal canine distemper Virus (CDV)/ferret model. Mechanistically, we demonstrate a role of the intact Ntail region in the regulation of viral transcriptase activity. Recombinant Viruses with Ntail truncations induce protective immunity against lethal challenge of ferrets with pathogenic CDV. This identification of the unstructured central Ntail domain as a nonessential paramyxoVirus pathogenesis factor establishes a foundation for harnessing Ntail truncations for vaccine engineering against emerging and reemerging members of the paramyxoVirus family.
