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Stephan Becker - One of the best experts on this subject based on the ideXlab platform.
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Ebola virus Proteins NP, VP35, and VP24 are essential and sufficient to mediate nucleocapsid transport
Proceedings of the National Academy of Sciences, 2018Co-Authors: Yuki Takamatsu, Larissa Kolesnikova, Stephan BeckerAbstract:The intracytoplasmic movement of nucleocapsids is a crucial step in the life cycle of enveloped viruses. Determination of the Viral components necessary for Viral nucleocapsid transport competency is complicated by the dynamic and complex nature of nucleocapsid assembly and the lack of appropriate model systems. Here, we established a live-cell imaging system based on the ectopic expression of fluorescent Ebola virus (EBOV) fusion Proteins, allowing the visualization and analysis of the movement of EBOV nucleocapsid-like structures with different Protein compositions. Only three of the five EBOV nucleocapsid Proteins—nucleoProtein, VP35, and VP24—were necessary and sufficient to form transport-competent nucleocapsid-like structures. The transport of these structures was found to be dependent on actin polymerization and to have dynamics that were undistinguishable from those of nucleocapsids in EBOV-infected cells. The intracytoplasmic movement of nucleocapsid-like structures was completely independent of the Viral Matrix Protein VP40 and the Viral surface glycoProtein GP. However, VP40 greatly enhanced the efficiency of nucleocapsid recruitment into filopodia, the sites of EBOV budding.
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A Single Amino Acid Change in the Marburg Virus Matrix Protein VP40 Provides a Replicative Advantage in a Species-Specific Manner.
Journal of virology, 2015Co-Authors: Alexander Koehler, Gordian Schudt, Larissa Kolesnikova, Ulla Welzel, Astrid Herwig, Stephan BeckerAbstract:UNLABELLED Marburg virus (MARV) induces severe hemorrhagic fever in humans and nonhuman primates but only transient nonlethal disease in rodents. However, sequential passages of MARV in rodents boosts infection leading to lethal disease. Guinea pig-adapted MARV contains one mutation in the Viral Matrix Protein VP40 at position 184 (VP40D184N). The contribution of the D184N mutation to the efficacy of replication in a new host is unknown. In the present study, we demonstrated that recombinant MARV containing the D184N mutation in VP40 [rMARVVP40(D184N)] grew to higher titers than wild-type recombinant MARV (rMARVWT) in guinea pig cells. Moreover, rMARVVP40(D184N) displayed higher infectivity in guinea pig cells. Comparative analysis of VP40 functions indicated that neither the interferon (IFN)-antagonistic function nor the membrane binding capabilities of VP40 were affected by the D184N mutation. However, the production of VP40-induced virus-like particles (VLPs) and the recruitment of other Viral Proteins to the budding site was improved by the D184N mutation in guinea pig cells, which resulted in the higher infectivity of VP40D184N-induced infectious VLPs (iVLPs) compared to that of VP40-induced iVLPs. In addition, the function of VP40 in suppressing Viral RNA synthesis was influenced by the D184N mutation specifically in guinea pig cells, thus allowing greater rates of transcription and replication. Our results showed that the improved Viral fitness of rMARVVP40(D184N) in guinea pig cells was due to the better Viral assembly function of VP40D184N and its lower inhibitory effect on Viral transcription and replication rather than modulation of the VP40-mediated suppression of IFN signaling. IMPORTANCE The increased virulence achieved by virus passaging in a new host was accompanied by mutations in the Viral genome. Analyzing how these mutations affect the functions of Viral Proteins and the ability of the virus to grow within new host cells helps in the understanding of the molecular mechanisms increasing virulence. Using a reverse genetics approach, we demonstrated that a single mutation in MARV VP40 detected in a guinea pig-adapted MARV provided a replicative advantage of rMARVVP40(D184N) in guinea pig cells. Our studies show that this replicative advantage of rMARV VP40D184N was based on the improved functions of VP40 in iVLP assembly and in the regulation of transcription and replication rather than on the ability of VP40 to combat the host innate immunity.
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structural dissection of ebola virus and its assembly determinants using cryo electron tomography
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Tanmay A M Bharat, Larissa Kolesnikova, Stephan Becker, James D. Riches, Verena Kraehling, John A. G. BriggsAbstract:Ebola virus is a highly pathogenic filovirus causing severe hemorrhagic fever with high mortality rates. It assembles heterogenous, filamentous, enveloped virus particles containing a negative-sense, single-stranded RNA genome packaged within a helical nucleocapsid (NC). We have used cryo-electron microscopy and tomography to visualize Ebola virus particles, as well as Ebola virus-like particles, in three dimensions in a near-native state. The NC within the virion forms a left-handed helix with an inner nucleoProtein layer decorated with protruding arms composed of VP24 and VP35. A comparison with the closely related Marburg virus shows that the N-terminal region of nucleoProtein defines the inner diameter of the Ebola virus NC, whereas the RNA genome defines its length. Binding of the nucleoProtein to RNA can assemble a loosely coiled NC-like structure; the loose coil can be condensed by binding of the Viral Matrix Protein VP40 to the C terminus of the nucleoProtein, and rigidified by binding of VP24 and VP35 to alternate copies of the nucleoProtein. Four Proteins (NP, VP24, VP35, and VP40) are necessary and sufficient to mediate assembly of an NC with structure, symmetry, variability, and flexibility indistinguishable from that in Ebola virus particles released from infected cells. Together these data provide a structural and architectural description of Ebola virus and define the roles of Viral Proteins in its structure and assembly.
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cryo electron tomography of marburg virus particles and their morphogenesis within infected cells
PLOS Biology, 2011Co-Authors: Tanmay A M Bharat, Larissa Kolesnikova, Stephan Becker, James D. Riches, Sonja Welsch, Verena Krahling, Norman E Davey, Marielaure Parsy, John A. G. BriggsAbstract:Several major human pathogens, including the filoviruses, paramyxoviruses, and rhabdoviruses, package their single-stranded RNA genomes within helical nucleocapsids, which bud through the plasma membrane of the infected cell to release enveloped virions. The virions are often heterogeneous in shape, which makes it difficult to study their structure and assembly mechanisms. We have applied cryo-electron tomography and sub-tomogram averaging methods to derive structures of Marburg virus, a highly pathogenic filovirus, both after release and during assembly within infected cells. The data demonstrate the potential of cryo-electron tomography methods to derive detailed structural information for intermediate steps in biological pathways within intact cells. We describe the location and arrangement of the Viral Proteins within the virion. We show that the N-terminal domain of the nucleoProtein contains the minimal assembly determinants for a helical nucleocapsid with variable number of Proteins per turn. Lobes protruding from alternate interfaces between each nucleoProtein are formed by the C-terminal domain of the nucleoProtein, together with Viral Proteins VP24 and VP35. Each nucleoProtein packages six RNA bases. The nucleocapsid interacts in an unusual, flexible “Velcro-like” manner with the Viral Matrix Protein VP40. Determination of the structures of assembly intermediates showed that the nucleocapsid has a defined orientation during transport and budding. Together the data show striking architectural homology between the nucleocapsid helix of rhabdoviruses and filoviruses, but unexpected, fundamental differences in the mechanisms by which the nucleocapsids are then assembled together with Matrix Proteins and initiate membrane envelopment to release infectious virions, suggesting that the viruses have evolved different solutions to these conserved assembly steps.
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efficient budding of the tacaribe virus Matrix Protein z requires the nucleoProtein
Journal of Virology, 2010Co-Authors: Allison Groseth, Svenja Wolff, Thomas Strecker, Thomas Hoenen, Stephan BeckerAbstract:The Z Protein has been shown for several arenaviruses to serve as the Viral Matrix Protein. As such, Z provides the principal force for the budding of virus particles and is capable of forming virus-like particles (VLPs) when expressed alone. For most arenaviruses, this activity has been shown to be linked to the presence of proline-rich late-domain motifs in the C terminus; however, for the New World arenavirus Tacaribe virus (TCRV), no such motif exists within Z. It was recently demonstrated that while TCRV Z is still capable of functioning as a Matrix Protein to induce the formation of VLPs, neither its ASAP motif, which replaces a canonical PT/SAP motif in related viruses, nor its YxxL motif is involved in budding, leading to the suggestion that TCRV uses a novel budding mechanism. Here we show that in comparison to its closest relative, Junin virus (JUNV), TCRV Z buds only weakly when expressed in isolation. While this budding activity is independent of the ASAP or YxxL motif, it is significantly enhanced by coexpression with the nucleoProtein (NP), an effect not seen with JUNV Z. Interestingly, both the ASAP and YxxL motifs of Z appear to be critical for the recruitment of NP into VLPs, as well as for the enhancement of TCRV Z-mediated budding. While it is known that TCRV budding remains dependent on the endosomal sorting complex required for transport, our findings provide further evidence that TCRV uses a budding mechanism distinct from that of other known arenaviruses and suggest an essential role for NP in this process.
John A. G. Briggs - One of the best experts on this subject based on the ideXlab platform.
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structural dissection of ebola virus and its assembly determinants using cryo electron tomography
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Tanmay A M Bharat, Larissa Kolesnikova, Stephan Becker, James D. Riches, Verena Kraehling, John A. G. BriggsAbstract:Ebola virus is a highly pathogenic filovirus causing severe hemorrhagic fever with high mortality rates. It assembles heterogenous, filamentous, enveloped virus particles containing a negative-sense, single-stranded RNA genome packaged within a helical nucleocapsid (NC). We have used cryo-electron microscopy and tomography to visualize Ebola virus particles, as well as Ebola virus-like particles, in three dimensions in a near-native state. The NC within the virion forms a left-handed helix with an inner nucleoProtein layer decorated with protruding arms composed of VP24 and VP35. A comparison with the closely related Marburg virus shows that the N-terminal region of nucleoProtein defines the inner diameter of the Ebola virus NC, whereas the RNA genome defines its length. Binding of the nucleoProtein to RNA can assemble a loosely coiled NC-like structure; the loose coil can be condensed by binding of the Viral Matrix Protein VP40 to the C terminus of the nucleoProtein, and rigidified by binding of VP24 and VP35 to alternate copies of the nucleoProtein. Four Proteins (NP, VP24, VP35, and VP40) are necessary and sufficient to mediate assembly of an NC with structure, symmetry, variability, and flexibility indistinguishable from that in Ebola virus particles released from infected cells. Together these data provide a structural and architectural description of Ebola virus and define the roles of Viral Proteins in its structure and assembly.
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cryo electron tomography of marburg virus particles and their morphogenesis within infected cells
PLOS Biology, 2011Co-Authors: Tanmay A M Bharat, Larissa Kolesnikova, Stephan Becker, James D. Riches, Sonja Welsch, Verena Krahling, Norman E Davey, Marielaure Parsy, John A. G. BriggsAbstract:Several major human pathogens, including the filoviruses, paramyxoviruses, and rhabdoviruses, package their single-stranded RNA genomes within helical nucleocapsids, which bud through the plasma membrane of the infected cell to release enveloped virions. The virions are often heterogeneous in shape, which makes it difficult to study their structure and assembly mechanisms. We have applied cryo-electron tomography and sub-tomogram averaging methods to derive structures of Marburg virus, a highly pathogenic filovirus, both after release and during assembly within infected cells. The data demonstrate the potential of cryo-electron tomography methods to derive detailed structural information for intermediate steps in biological pathways within intact cells. We describe the location and arrangement of the Viral Proteins within the virion. We show that the N-terminal domain of the nucleoProtein contains the minimal assembly determinants for a helical nucleocapsid with variable number of Proteins per turn. Lobes protruding from alternate interfaces between each nucleoProtein are formed by the C-terminal domain of the nucleoProtein, together with Viral Proteins VP24 and VP35. Each nucleoProtein packages six RNA bases. The nucleocapsid interacts in an unusual, flexible “Velcro-like” manner with the Viral Matrix Protein VP40. Determination of the structures of assembly intermediates showed that the nucleocapsid has a defined orientation during transport and budding. Together the data show striking architectural homology between the nucleocapsid helix of rhabdoviruses and filoviruses, but unexpected, fundamental differences in the mechanisms by which the nucleocapsids are then assembled together with Matrix Proteins and initiate membrane envelopment to release infectious virions, suggesting that the viruses have evolved different solutions to these conserved assembly steps.
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double labelled hiv 1 particles for study of virus cell interaction
Virology, 2007Co-Authors: Marko Lampe, John A. G. Briggs, Thomas Endress, Barbel Glass, Stefan Riegelsberger, Hansgeorg Krausslich, Don C Lamb, Christoph Brauchle, Barbara MullerAbstract:Human immunodeficiency virus (HIV) delivers its genome to a host cell through fusion of the Viral envelope with a cellular membrane. While the Viral and cellular Proteins involved in entry have been analyzed in detail, the dynamics of virus-cell fusion are largely unknown. Single virus tracing (SVT) provides the unique opportunity to visualize Viral particles in real time allowing direct observation of the dynamics of this stochastic process. For this purpose, we developed a double-coloured HIV derivative carrying a green fluorescent label attached to the Viral Matrix Protein combined with a red label fused to the Viral Vpr Protein designed to distinguish between complete virions and subViral particles lacking MA after membrane fusion. We present here a detailed characterization of this novel tool together with exemplary live cell imaging studies, demonstrating its suitability for real-time analyses of HIV-cell interaction.
Larissa Kolesnikova - One of the best experts on this subject based on the ideXlab platform.
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Ebola virus Proteins NP, VP35, and VP24 are essential and sufficient to mediate nucleocapsid transport
Proceedings of the National Academy of Sciences, 2018Co-Authors: Yuki Takamatsu, Larissa Kolesnikova, Stephan BeckerAbstract:The intracytoplasmic movement of nucleocapsids is a crucial step in the life cycle of enveloped viruses. Determination of the Viral components necessary for Viral nucleocapsid transport competency is complicated by the dynamic and complex nature of nucleocapsid assembly and the lack of appropriate model systems. Here, we established a live-cell imaging system based on the ectopic expression of fluorescent Ebola virus (EBOV) fusion Proteins, allowing the visualization and analysis of the movement of EBOV nucleocapsid-like structures with different Protein compositions. Only three of the five EBOV nucleocapsid Proteins—nucleoProtein, VP35, and VP24—were necessary and sufficient to form transport-competent nucleocapsid-like structures. The transport of these structures was found to be dependent on actin polymerization and to have dynamics that were undistinguishable from those of nucleocapsids in EBOV-infected cells. The intracytoplasmic movement of nucleocapsid-like structures was completely independent of the Viral Matrix Protein VP40 and the Viral surface glycoProtein GP. However, VP40 greatly enhanced the efficiency of nucleocapsid recruitment into filopodia, the sites of EBOV budding.
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A Single Amino Acid Change in the Marburg Virus Matrix Protein VP40 Provides a Replicative Advantage in a Species-Specific Manner.
Journal of virology, 2015Co-Authors: Alexander Koehler, Gordian Schudt, Larissa Kolesnikova, Ulla Welzel, Astrid Herwig, Stephan BeckerAbstract:UNLABELLED Marburg virus (MARV) induces severe hemorrhagic fever in humans and nonhuman primates but only transient nonlethal disease in rodents. However, sequential passages of MARV in rodents boosts infection leading to lethal disease. Guinea pig-adapted MARV contains one mutation in the Viral Matrix Protein VP40 at position 184 (VP40D184N). The contribution of the D184N mutation to the efficacy of replication in a new host is unknown. In the present study, we demonstrated that recombinant MARV containing the D184N mutation in VP40 [rMARVVP40(D184N)] grew to higher titers than wild-type recombinant MARV (rMARVWT) in guinea pig cells. Moreover, rMARVVP40(D184N) displayed higher infectivity in guinea pig cells. Comparative analysis of VP40 functions indicated that neither the interferon (IFN)-antagonistic function nor the membrane binding capabilities of VP40 were affected by the D184N mutation. However, the production of VP40-induced virus-like particles (VLPs) and the recruitment of other Viral Proteins to the budding site was improved by the D184N mutation in guinea pig cells, which resulted in the higher infectivity of VP40D184N-induced infectious VLPs (iVLPs) compared to that of VP40-induced iVLPs. In addition, the function of VP40 in suppressing Viral RNA synthesis was influenced by the D184N mutation specifically in guinea pig cells, thus allowing greater rates of transcription and replication. Our results showed that the improved Viral fitness of rMARVVP40(D184N) in guinea pig cells was due to the better Viral assembly function of VP40D184N and its lower inhibitory effect on Viral transcription and replication rather than modulation of the VP40-mediated suppression of IFN signaling. IMPORTANCE The increased virulence achieved by virus passaging in a new host was accompanied by mutations in the Viral genome. Analyzing how these mutations affect the functions of Viral Proteins and the ability of the virus to grow within new host cells helps in the understanding of the molecular mechanisms increasing virulence. Using a reverse genetics approach, we demonstrated that a single mutation in MARV VP40 detected in a guinea pig-adapted MARV provided a replicative advantage of rMARVVP40(D184N) in guinea pig cells. Our studies show that this replicative advantage of rMARV VP40D184N was based on the improved functions of VP40 in iVLP assembly and in the regulation of transcription and replication rather than on the ability of VP40 to combat the host innate immunity.
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structural dissection of ebola virus and its assembly determinants using cryo electron tomography
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Tanmay A M Bharat, Larissa Kolesnikova, Stephan Becker, James D. Riches, Verena Kraehling, John A. G. BriggsAbstract:Ebola virus is a highly pathogenic filovirus causing severe hemorrhagic fever with high mortality rates. It assembles heterogenous, filamentous, enveloped virus particles containing a negative-sense, single-stranded RNA genome packaged within a helical nucleocapsid (NC). We have used cryo-electron microscopy and tomography to visualize Ebola virus particles, as well as Ebola virus-like particles, in three dimensions in a near-native state. The NC within the virion forms a left-handed helix with an inner nucleoProtein layer decorated with protruding arms composed of VP24 and VP35. A comparison with the closely related Marburg virus shows that the N-terminal region of nucleoProtein defines the inner diameter of the Ebola virus NC, whereas the RNA genome defines its length. Binding of the nucleoProtein to RNA can assemble a loosely coiled NC-like structure; the loose coil can be condensed by binding of the Viral Matrix Protein VP40 to the C terminus of the nucleoProtein, and rigidified by binding of VP24 and VP35 to alternate copies of the nucleoProtein. Four Proteins (NP, VP24, VP35, and VP40) are necessary and sufficient to mediate assembly of an NC with structure, symmetry, variability, and flexibility indistinguishable from that in Ebola virus particles released from infected cells. Together these data provide a structural and architectural description of Ebola virus and define the roles of Viral Proteins in its structure and assembly.
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cryo electron tomography of marburg virus particles and their morphogenesis within infected cells
PLOS Biology, 2011Co-Authors: Tanmay A M Bharat, Larissa Kolesnikova, Stephan Becker, James D. Riches, Sonja Welsch, Verena Krahling, Norman E Davey, Marielaure Parsy, John A. G. BriggsAbstract:Several major human pathogens, including the filoviruses, paramyxoviruses, and rhabdoviruses, package their single-stranded RNA genomes within helical nucleocapsids, which bud through the plasma membrane of the infected cell to release enveloped virions. The virions are often heterogeneous in shape, which makes it difficult to study their structure and assembly mechanisms. We have applied cryo-electron tomography and sub-tomogram averaging methods to derive structures of Marburg virus, a highly pathogenic filovirus, both after release and during assembly within infected cells. The data demonstrate the potential of cryo-electron tomography methods to derive detailed structural information for intermediate steps in biological pathways within intact cells. We describe the location and arrangement of the Viral Proteins within the virion. We show that the N-terminal domain of the nucleoProtein contains the minimal assembly determinants for a helical nucleocapsid with variable number of Proteins per turn. Lobes protruding from alternate interfaces between each nucleoProtein are formed by the C-terminal domain of the nucleoProtein, together with Viral Proteins VP24 and VP35. Each nucleoProtein packages six RNA bases. The nucleocapsid interacts in an unusual, flexible “Velcro-like” manner with the Viral Matrix Protein VP40. Determination of the structures of assembly intermediates showed that the nucleocapsid has a defined orientation during transport and budding. Together the data show striking architectural homology between the nucleocapsid helix of rhabdoviruses and filoviruses, but unexpected, fundamental differences in the mechanisms by which the nucleocapsids are then assembled together with Matrix Proteins and initiate membrane envelopment to release infectious virions, suggesting that the viruses have evolved different solutions to these conserved assembly steps.
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Morphology of Marburg Virus NP–RNA
Virology, 2002Co-Authors: Manos Mavrakis, Larissa Kolesnikova, Guy Schoehn, Stephan BeckerAbstract:Abstract When Marburg virus (MBGV) nucleoProtein (NP) is expressed in insect cells, it binds to cellular RNA and forms NP–RNA complexes such as insect cell-expressed nucleoProteins from other nonsegmented negative-strand RNA viruses. Recombinant MBGV NP–RNA forms loose coils that resemble rabies virus N–RNA. MBGV NP monomers are rods that are spaced along the coil similar to the nucleoProtein monomers of the rabies virus N–RNA. High salt treatment induces tight coiling of the MBGV NP–RNA, again a characteristic observed for other nonsegmented negative-strand virus N–RNAs. Electron microscopy of fixed Marburg virus particles shows that the Viral nucleocapsid has a smaller diameter than the free, recombinant NP–RNA. This difference in helical parameters could be caused by the interaction of other Viral Proteins with the NP–RNA. A similar but opposite phenomenon is observed for rhabdovirus nucleocapsids that are condensed by the Viral Matrix Protein upon which they acquire a larger diameter. Finally, there appears to be an extensive and regular Protein scaffold between the Viral nucleocapsid and the membrane that seems not to exist in the other negative-strand RNA viruses.
Tanmay A M Bharat - One of the best experts on this subject based on the ideXlab platform.
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structural dissection of ebola virus and its assembly determinants using cryo electron tomography
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Tanmay A M Bharat, Larissa Kolesnikova, Stephan Becker, James D. Riches, Verena Kraehling, John A. G. BriggsAbstract:Ebola virus is a highly pathogenic filovirus causing severe hemorrhagic fever with high mortality rates. It assembles heterogenous, filamentous, enveloped virus particles containing a negative-sense, single-stranded RNA genome packaged within a helical nucleocapsid (NC). We have used cryo-electron microscopy and tomography to visualize Ebola virus particles, as well as Ebola virus-like particles, in three dimensions in a near-native state. The NC within the virion forms a left-handed helix with an inner nucleoProtein layer decorated with protruding arms composed of VP24 and VP35. A comparison with the closely related Marburg virus shows that the N-terminal region of nucleoProtein defines the inner diameter of the Ebola virus NC, whereas the RNA genome defines its length. Binding of the nucleoProtein to RNA can assemble a loosely coiled NC-like structure; the loose coil can be condensed by binding of the Viral Matrix Protein VP40 to the C terminus of the nucleoProtein, and rigidified by binding of VP24 and VP35 to alternate copies of the nucleoProtein. Four Proteins (NP, VP24, VP35, and VP40) are necessary and sufficient to mediate assembly of an NC with structure, symmetry, variability, and flexibility indistinguishable from that in Ebola virus particles released from infected cells. Together these data provide a structural and architectural description of Ebola virus and define the roles of Viral Proteins in its structure and assembly.
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cryo electron tomography of marburg virus particles and their morphogenesis within infected cells
PLOS Biology, 2011Co-Authors: Tanmay A M Bharat, Larissa Kolesnikova, Stephan Becker, James D. Riches, Sonja Welsch, Verena Krahling, Norman E Davey, Marielaure Parsy, John A. G. BriggsAbstract:Several major human pathogens, including the filoviruses, paramyxoviruses, and rhabdoviruses, package their single-stranded RNA genomes within helical nucleocapsids, which bud through the plasma membrane of the infected cell to release enveloped virions. The virions are often heterogeneous in shape, which makes it difficult to study their structure and assembly mechanisms. We have applied cryo-electron tomography and sub-tomogram averaging methods to derive structures of Marburg virus, a highly pathogenic filovirus, both after release and during assembly within infected cells. The data demonstrate the potential of cryo-electron tomography methods to derive detailed structural information for intermediate steps in biological pathways within intact cells. We describe the location and arrangement of the Viral Proteins within the virion. We show that the N-terminal domain of the nucleoProtein contains the minimal assembly determinants for a helical nucleocapsid with variable number of Proteins per turn. Lobes protruding from alternate interfaces between each nucleoProtein are formed by the C-terminal domain of the nucleoProtein, together with Viral Proteins VP24 and VP35. Each nucleoProtein packages six RNA bases. The nucleocapsid interacts in an unusual, flexible “Velcro-like” manner with the Viral Matrix Protein VP40. Determination of the structures of assembly intermediates showed that the nucleocapsid has a defined orientation during transport and budding. Together the data show striking architectural homology between the nucleocapsid helix of rhabdoviruses and filoviruses, but unexpected, fundamental differences in the mechanisms by which the nucleocapsids are then assembled together with Matrix Proteins and initiate membrane envelopment to release infectious virions, suggesting that the viruses have evolved different solutions to these conserved assembly steps.
Andrea Maisner - One of the best experts on this subject based on the ideXlab platform.
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measles virus nucleocapsid transport to the plasma membrane requires stable expression and surface accumulation of the Viral Matrix Protein
Cellular Microbiology, 2007Co-Authors: Nicole Runkler, Hans-dieter Klenk, Christine Pohl, Sibylle Schneiderschaulies, Andrea MaisnerAbstract:In measles virus (MV)-infected cells the Matrix (M) Protein plays a key role in virus assembly and budding processes at the plasma membrane because it mediates the contact between the Viral surface glycoProteins and the nucleocapsids. By exchanging valine 101, a highly conserved residue among all paramyxoViral M Proteins, we generated a recombinant MV (rMV) from cloned cDNA encoding for a M Protein with an increased intracellular turnover. The mutant rMV was barely released from the infected cells. This assembly defect was not due to a defective M binding to other Matrix- or nucleoProteins, but could rather be assigned to a reduced ability to associate with cellular membranes, and more importantly, to a defective accumulation at the plasma membrane which was accompanied by the deficient transport of nucleocapsids to the cell surface. Thus, we show for the first time that M stability and accumulation at intracellular membranes is a prerequisite for M and nucleocapsid co-transport to the plasma membrane and for subsequent virus assembly and budding processes.
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Measles virus Matrix Protein is not cotransported with the Viral glycoProteins but requires virus infection for efficient surface targeting
Virus research, 2002Co-Authors: Petra Riedl, Markus Moll, Hans-dieter Klenk, Andrea MaisnerAbstract:As we have shown earlier, the measles virus (MV) glycoProteins H and F are expressed on both, the apical and the basolateral membrane of polarized Madin-Darby canine kidney cells. In contrast to the glycoProteins, we found the Viral Matrix Protein (M) to accumulate selectively at the apical plasma membrane of MV-infected cells. M did not colocalize with the glycoProteins at basolateral membranes of polarized cells indicating an independent surface transport mechanism. Analysis of infected cells treated with monensin supported this view. When H and F were retained in the medial Golgi by monensin treatment, M did not accumulate in this cellular compartment. To elucidate the subcellular transport mechanism of the cytosolic M Protein, M was expressed in the absence of other Viral Proteins. Flotation analysis demonstrated that most of the M Protein coflotated in infected or in M-transfected cells with cellular membranes. Thus, the M Protein possesses the intrinsic ability to bind to lipid membranes. Unexpectedly, plasmid-encoded M Protein was rarely found to accumulate at surface membranes. Although cotransport with the Viral glycoProteins was not needed, M transport to the plasma membrane required a component only provided in MV-infected cells.
