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Nucleocapsid formation and RNA synthesis of Marburg virus is dependent on two coiled coil motifs in the nucleoprotein.

DiCarlo A, Möller P, Lander A, Kolesnikova L, Becker S - Virol. J. (2007)

Bottom Line: In the present study, a conserved coiled coil motif in the central part of MARV NP was shown to be an important element for the interactions of NP with itself and VP35, the viral polymerase cofactor.Additionally, the coiled coil motif was essential for the formation of NP-induced intracellular inclusions and for the function of NP in the process of transcription and replication of viral RNA in a minigenome system.The coiled coil motif is bipartite, constituted by two coiled coils which are separated by a flexible linker.

View Article: PubMed Central - HTML - PubMed

Affiliation: Philipps-Universität Marburg, Institut für Virologie, Hans Meerwein-Str, 2, 35032 Marburg, Germany. andrea.dicarlo@promega.com

ABSTRACT
The nucleoprotein (NP) of Marburg virus (MARV) is responsible for the encapsidation of viral genomic RNA and the formation of the helical nucleocapsid precursors that accumulate in intracellular inclusions in infected cells. To form the large helical MARV nucleocapsid, NP needs to interact with itself and the viral proteins VP30, VP35 and L, which are also part of the MARV nucleocapsid. In the present study, a conserved coiled coil motif in the central part of MARV NP was shown to be an important element for the interactions of NP with itself and VP35, the viral polymerase cofactor. Additionally, the coiled coil motif was essential for the formation of NP-induced intracellular inclusions and for the function of NP in the process of transcription and replication of viral RNA in a minigenome system. Transfer of the coiled coil motif to a reporter protein was sufficient to mediate interaction of the constructed fusion protein with the N-terminus of NP. The coiled coil motif is bipartite, constituted by two coiled coils which are separated by a flexible linker.

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Role of the coiled coil region in NP for NP self interaction, inclusion body formation and MARV-specific transcription/replication. (A) In silico analysis of NP predicted two coiled coil motifs at aa position 315 to 400 which are separated by a linker of 23 aa. (B) Schematic presentation of the constructed mutants of NP with deletions in the coiled coil region. (C) The constructed plasmids were in vitro translated, metabolically labeled, separated by SDS-PAGE and analyzed using a BioImager. (D) In vitro translated and metabolically labeled mutants of NP were incubated with bacterially expressed GST-NP or GST (negative control). Complexes were pulled down with glutathion-sepharose, separated on SDS-PAGE and analyzed using a BioImager. Binding of NP to GST-NP was set to 100%. (E) Quantification of 3 separate experiments as shown under (D). (F) Intracellular distribution of NP and NPΔC1. HUH7 cells were transfected with plasmids encoding NP or NPΔC1 together with a plasmid encoding T7 polymerase. Cells were fixed with 4% paraformaldehyde at 18 h post transfection and incubated with a rabbit anti-NP antiserum. Bound antibodies were detected with a rhodamine-coupled donkey anti-rabbit antibody (NP), and a FITC-coupled donkey anti-rabbit antibody (NPΔC1). (G) Impact of coiled coils on the function of NP in a MARV-specific minigenome transcription/replication system. MARV nucleocapsid proteins were expressed in HUHT7 cells together with a MARV specific minigenome. NP was replaced by the indicated mutants of NP and reporter gene activity (CAT) was measured. Below the CAT assay, expression of NP and the NP mutants was confirmed in Western Blot analysis. +: presence of L. -: absence of L (negative control). (H) Results of a transcription/replication analysis using different internal deletion mutants of NP in a minigenome-based assay (G). +: minigenome system active (transcription and replication monitored by CAT assay). -: minigenome system inactive.
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Figure 1: Role of the coiled coil region in NP for NP self interaction, inclusion body formation and MARV-specific transcription/replication. (A) In silico analysis of NP predicted two coiled coil motifs at aa position 315 to 400 which are separated by a linker of 23 aa. (B) Schematic presentation of the constructed mutants of NP with deletions in the coiled coil region. (C) The constructed plasmids were in vitro translated, metabolically labeled, separated by SDS-PAGE and analyzed using a BioImager. (D) In vitro translated and metabolically labeled mutants of NP were incubated with bacterially expressed GST-NP or GST (negative control). Complexes were pulled down with glutathion-sepharose, separated on SDS-PAGE and analyzed using a BioImager. Binding of NP to GST-NP was set to 100%. (E) Quantification of 3 separate experiments as shown under (D). (F) Intracellular distribution of NP and NPΔC1. HUH7 cells were transfected with plasmids encoding NP or NPΔC1 together with a plasmid encoding T7 polymerase. Cells were fixed with 4% paraformaldehyde at 18 h post transfection and incubated with a rabbit anti-NP antiserum. Bound antibodies were detected with a rhodamine-coupled donkey anti-rabbit antibody (NP), and a FITC-coupled donkey anti-rabbit antibody (NPΔC1). (G) Impact of coiled coils on the function of NP in a MARV-specific minigenome transcription/replication system. MARV nucleocapsid proteins were expressed in HUHT7 cells together with a MARV specific minigenome. NP was replaced by the indicated mutants of NP and reporter gene activity (CAT) was measured. Below the CAT assay, expression of NP and the NP mutants was confirmed in Western Blot analysis. +: presence of L. -: absence of L (negative control). (H) Results of a transcription/replication analysis using different internal deletion mutants of NP in a minigenome-based assay (G). +: minigenome system active (transcription and replication monitored by CAT assay). -: minigenome system inactive.

Mentions: A prerequisite for the formation of filoviral nucleocapsids is the homooligomerization of NP, which self-assembles into helical tubules, which are 17 nm in diameter, observed both in cells expressing recombinant NP and in MARV infected cells [8,9]. The formation of the large NP-induced tubules, which are composed of several hundred NP molecules, most likely requires several homooligomerization domains on NP mediating the helical arrangement and the accumulation of the individual tubules into inclusion bodies. In silico analyses of NP (accession number: Z12132) revealed two stretches of 27 aa in the central part of NP (aa 315 to 400) with a high probability to form coiled coils (Fig. 1A, [15]). The two coiled coil motifs are separated by a spacer of 23 aa. Since coiled coils represent a common protein-protein interaction module, we analyzed whether deletion of the individual coiled coil motifs altered the ability of NP to homooligomerize [16,17]. To this end, sequences encoding either the first (Fig. 1B; ΔC1, aa 320–348), the second (ΔC2, aa 371–400), or both coiled coils (ΔC1C2, aa 320 – 400) were removed from the NP-encoding plasmid pT-NP. The resulting mutants were in vitro translated (Fig. 1C) and were subsequently employed in a GST pull-down assay using NP fused to GST (GST-NP). We found that removal of coiled coil 1 (ΔC1) had a significant impact on the binding of NP to itself (Figs. 1D and 1E, ΔC1). The binding strength was also decreased when both coiled coil motifs were deleted (Figs. 1D and 1E, ΔC1C2). Deletion of C2 did not significantly impair the homooligomerization of NP (Figs. 1D and 1E, ΔC2), suggesting that C1, but not C2 is essential for intermolecular interaction between NP molecules.


Nucleocapsid formation and RNA synthesis of Marburg virus is dependent on two coiled coil motifs in the nucleoprotein.

DiCarlo A, Möller P, Lander A, Kolesnikova L, Becker S - Virol. J. (2007)

Role of the coiled coil region in NP for NP self interaction, inclusion body formation and MARV-specific transcription/replication. (A) In silico analysis of NP predicted two coiled coil motifs at aa position 315 to 400 which are separated by a linker of 23 aa. (B) Schematic presentation of the constructed mutants of NP with deletions in the coiled coil region. (C) The constructed plasmids were in vitro translated, metabolically labeled, separated by SDS-PAGE and analyzed using a BioImager. (D) In vitro translated and metabolically labeled mutants of NP were incubated with bacterially expressed GST-NP or GST (negative control). Complexes were pulled down with glutathion-sepharose, separated on SDS-PAGE and analyzed using a BioImager. Binding of NP to GST-NP was set to 100%. (E) Quantification of 3 separate experiments as shown under (D). (F) Intracellular distribution of NP and NPΔC1. HUH7 cells were transfected with plasmids encoding NP or NPΔC1 together with a plasmid encoding T7 polymerase. Cells were fixed with 4% paraformaldehyde at 18 h post transfection and incubated with a rabbit anti-NP antiserum. Bound antibodies were detected with a rhodamine-coupled donkey anti-rabbit antibody (NP), and a FITC-coupled donkey anti-rabbit antibody (NPΔC1). (G) Impact of coiled coils on the function of NP in a MARV-specific minigenome transcription/replication system. MARV nucleocapsid proteins were expressed in HUHT7 cells together with a MARV specific minigenome. NP was replaced by the indicated mutants of NP and reporter gene activity (CAT) was measured. Below the CAT assay, expression of NP and the NP mutants was confirmed in Western Blot analysis. +: presence of L. -: absence of L (negative control). (H) Results of a transcription/replication analysis using different internal deletion mutants of NP in a minigenome-based assay (G). +: minigenome system active (transcription and replication monitored by CAT assay). -: minigenome system inactive.
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Figure 1: Role of the coiled coil region in NP for NP self interaction, inclusion body formation and MARV-specific transcription/replication. (A) In silico analysis of NP predicted two coiled coil motifs at aa position 315 to 400 which are separated by a linker of 23 aa. (B) Schematic presentation of the constructed mutants of NP with deletions in the coiled coil region. (C) The constructed plasmids were in vitro translated, metabolically labeled, separated by SDS-PAGE and analyzed using a BioImager. (D) In vitro translated and metabolically labeled mutants of NP were incubated with bacterially expressed GST-NP or GST (negative control). Complexes were pulled down with glutathion-sepharose, separated on SDS-PAGE and analyzed using a BioImager. Binding of NP to GST-NP was set to 100%. (E) Quantification of 3 separate experiments as shown under (D). (F) Intracellular distribution of NP and NPΔC1. HUH7 cells were transfected with plasmids encoding NP or NPΔC1 together with a plasmid encoding T7 polymerase. Cells were fixed with 4% paraformaldehyde at 18 h post transfection and incubated with a rabbit anti-NP antiserum. Bound antibodies were detected with a rhodamine-coupled donkey anti-rabbit antibody (NP), and a FITC-coupled donkey anti-rabbit antibody (NPΔC1). (G) Impact of coiled coils on the function of NP in a MARV-specific minigenome transcription/replication system. MARV nucleocapsid proteins were expressed in HUHT7 cells together with a MARV specific minigenome. NP was replaced by the indicated mutants of NP and reporter gene activity (CAT) was measured. Below the CAT assay, expression of NP and the NP mutants was confirmed in Western Blot analysis. +: presence of L. -: absence of L (negative control). (H) Results of a transcription/replication analysis using different internal deletion mutants of NP in a minigenome-based assay (G). +: minigenome system active (transcription and replication monitored by CAT assay). -: minigenome system inactive.
Mentions: A prerequisite for the formation of filoviral nucleocapsids is the homooligomerization of NP, which self-assembles into helical tubules, which are 17 nm in diameter, observed both in cells expressing recombinant NP and in MARV infected cells [8,9]. The formation of the large NP-induced tubules, which are composed of several hundred NP molecules, most likely requires several homooligomerization domains on NP mediating the helical arrangement and the accumulation of the individual tubules into inclusion bodies. In silico analyses of NP (accession number: Z12132) revealed two stretches of 27 aa in the central part of NP (aa 315 to 400) with a high probability to form coiled coils (Fig. 1A, [15]). The two coiled coil motifs are separated by a spacer of 23 aa. Since coiled coils represent a common protein-protein interaction module, we analyzed whether deletion of the individual coiled coil motifs altered the ability of NP to homooligomerize [16,17]. To this end, sequences encoding either the first (Fig. 1B; ΔC1, aa 320–348), the second (ΔC2, aa 371–400), or both coiled coils (ΔC1C2, aa 320 – 400) were removed from the NP-encoding plasmid pT-NP. The resulting mutants were in vitro translated (Fig. 1C) and were subsequently employed in a GST pull-down assay using NP fused to GST (GST-NP). We found that removal of coiled coil 1 (ΔC1) had a significant impact on the binding of NP to itself (Figs. 1D and 1E, ΔC1). The binding strength was also decreased when both coiled coil motifs were deleted (Figs. 1D and 1E, ΔC1C2). Deletion of C2 did not significantly impair the homooligomerization of NP (Figs. 1D and 1E, ΔC2), suggesting that C1, but not C2 is essential for intermolecular interaction between NP molecules.

Bottom Line: In the present study, a conserved coiled coil motif in the central part of MARV NP was shown to be an important element for the interactions of NP with itself and VP35, the viral polymerase cofactor.Additionally, the coiled coil motif was essential for the formation of NP-induced intracellular inclusions and for the function of NP in the process of transcription and replication of viral RNA in a minigenome system.The coiled coil motif is bipartite, constituted by two coiled coils which are separated by a flexible linker.

View Article: PubMed Central - HTML - PubMed

Affiliation: Philipps-Universität Marburg, Institut für Virologie, Hans Meerwein-Str, 2, 35032 Marburg, Germany. andrea.dicarlo@promega.com

ABSTRACT
The nucleoprotein (NP) of Marburg virus (MARV) is responsible for the encapsidation of viral genomic RNA and the formation of the helical nucleocapsid precursors that accumulate in intracellular inclusions in infected cells. To form the large helical MARV nucleocapsid, NP needs to interact with itself and the viral proteins VP30, VP35 and L, which are also part of the MARV nucleocapsid. In the present study, a conserved coiled coil motif in the central part of MARV NP was shown to be an important element for the interactions of NP with itself and VP35, the viral polymerase cofactor. Additionally, the coiled coil motif was essential for the formation of NP-induced intracellular inclusions and for the function of NP in the process of transcription and replication of viral RNA in a minigenome system. Transfer of the coiled coil motif to a reporter protein was sufficient to mediate interaction of the constructed fusion protein with the N-terminus of NP. The coiled coil motif is bipartite, constituted by two coiled coils which are separated by a flexible linker.

Show MeSH
Related in: MedlinePlus