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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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The coiled coil region in NP is sufficient to mediate interaction with NP. (A) Schematic presentation of the constructed mutants. The coiled coil region (C1C2, grey boxes) was fused to an unrelated reporter protein (MFlag, striped box). (B) The mutants were expressed using the MVA-T7 system in HeLa cells and metabolically labeled using [35S]ProMix. Cells were lysed at 24 h post transfection and cell lysates precipitated using mouse monoclonal anti-Flag (M2) and/or mouse monoclonal anti NP (2B10) as indicated. Precipitates were separated by SDS-PAGE and analyzed using BioImager. (C) Epitope of the mouse monoclonal antibody 2B10 on NP. Mutants of NP with sequential deletions of 10 aa were expressed in HUHT7 cells and cells were lysed at 24 h post transfection. Cell lysates were separated by SDS-PAGE and gels blotted onto polyvinylidene fluoride membrane. Membrane was subjected to Western Blot analysis using either the anti MARV NP mouse monoclonal antibody 2B10 (2B10) or a rabbit anti MARV nucleocapsid antiserum (α-NC).
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Figure 2: The coiled coil region in NP is sufficient to mediate interaction with NP. (A) Schematic presentation of the constructed mutants. The coiled coil region (C1C2, grey boxes) was fused to an unrelated reporter protein (MFlag, striped box). (B) The mutants were expressed using the MVA-T7 system in HeLa cells and metabolically labeled using [35S]ProMix. Cells were lysed at 24 h post transfection and cell lysates precipitated using mouse monoclonal anti-Flag (M2) and/or mouse monoclonal anti NP (2B10) as indicated. Precipitates were separated by SDS-PAGE and analyzed using BioImager. (C) Epitope of the mouse monoclonal antibody 2B10 on NP. Mutants of NP with sequential deletions of 10 aa were expressed in HUHT7 cells and cells were lysed at 24 h post transfection. Cell lysates were separated by SDS-PAGE and gels blotted onto polyvinylidene fluoride membrane. Membrane was subjected to Western Blot analysis using either the anti MARV NP mouse monoclonal antibody 2B10 (2B10) or a rabbit anti MARV nucleocapsid antiserum (α-NC).

Mentions: Next, we aimed to determine whether the coiled motifs are sufficient to mediate protein-protein interactions. The region of the NP gene encoding amino acids 310 to 400 was cloned in frame with a mutant of Ebola virus VP30 (MFlag; Fig. 2A, [14]). The fusion protein, named C1C2-MFlag, was coexpressed with the N-terminus of NP (NPΔ441–695), which contains the two coiled coil motifs (Fig. 2a, Fig. 2B, lane 1). C1C2-MFlag was then specifically precipitated using an anti-Flag antibody; this antibody did not precipitate the NP N-terminus (Fig. 2B lanes 2 and 11). The precipitation anti-Flag antibody resulted in the cosedimentation of the NP N-terminus, suggesting that both proteins were able to interact with each other. However, when a mouse monoclonal anti-NP antibody (2B10) was employed in the coimmunoprecipitation, only NPΔ441–695 was precipitated, suggesting that this antibody inhibited the interaction of the two proteins (Fig. 2B, lane 3). Control experiments showed that the Flag-tagged mutant of VP30 (MFlag) was unable to interact with the NPΔ441–695 (Fig. 2B, lanes 10 and 11).


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)

The coiled coil region in NP is sufficient to mediate interaction with NP. (A) Schematic presentation of the constructed mutants. The coiled coil region (C1C2, grey boxes) was fused to an unrelated reporter protein (MFlag, striped box). (B) The mutants were expressed using the MVA-T7 system in HeLa cells and metabolically labeled using [35S]ProMix. Cells were lysed at 24 h post transfection and cell lysates precipitated using mouse monoclonal anti-Flag (M2) and/or mouse monoclonal anti NP (2B10) as indicated. Precipitates were separated by SDS-PAGE and analyzed using BioImager. (C) Epitope of the mouse monoclonal antibody 2B10 on NP. Mutants of NP with sequential deletions of 10 aa were expressed in HUHT7 cells and cells were lysed at 24 h post transfection. Cell lysates were separated by SDS-PAGE and gels blotted onto polyvinylidene fluoride membrane. Membrane was subjected to Western Blot analysis using either the anti MARV NP mouse monoclonal antibody 2B10 (2B10) or a rabbit anti MARV nucleocapsid antiserum (α-NC).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2170442&req=5

Figure 2: The coiled coil region in NP is sufficient to mediate interaction with NP. (A) Schematic presentation of the constructed mutants. The coiled coil region (C1C2, grey boxes) was fused to an unrelated reporter protein (MFlag, striped box). (B) The mutants were expressed using the MVA-T7 system in HeLa cells and metabolically labeled using [35S]ProMix. Cells were lysed at 24 h post transfection and cell lysates precipitated using mouse monoclonal anti-Flag (M2) and/or mouse monoclonal anti NP (2B10) as indicated. Precipitates were separated by SDS-PAGE and analyzed using BioImager. (C) Epitope of the mouse monoclonal antibody 2B10 on NP. Mutants of NP with sequential deletions of 10 aa were expressed in HUHT7 cells and cells were lysed at 24 h post transfection. Cell lysates were separated by SDS-PAGE and gels blotted onto polyvinylidene fluoride membrane. Membrane was subjected to Western Blot analysis using either the anti MARV NP mouse monoclonal antibody 2B10 (2B10) or a rabbit anti MARV nucleocapsid antiserum (α-NC).
Mentions: Next, we aimed to determine whether the coiled motifs are sufficient to mediate protein-protein interactions. The region of the NP gene encoding amino acids 310 to 400 was cloned in frame with a mutant of Ebola virus VP30 (MFlag; Fig. 2A, [14]). The fusion protein, named C1C2-MFlag, was coexpressed with the N-terminus of NP (NPΔ441–695), which contains the two coiled coil motifs (Fig. 2a, Fig. 2B, lane 1). C1C2-MFlag was then specifically precipitated using an anti-Flag antibody; this antibody did not precipitate the NP N-terminus (Fig. 2B lanes 2 and 11). The precipitation anti-Flag antibody resulted in the cosedimentation of the NP N-terminus, suggesting that both proteins were able to interact with each other. However, when a mouse monoclonal anti-NP antibody (2B10) was employed in the coimmunoprecipitation, only NPΔ441–695 was precipitated, suggesting that this antibody inhibited the interaction of the two proteins (Fig. 2B, lane 3). Control experiments showed that the Flag-tagged mutant of VP30 (MFlag) was unable to interact with the NPΔ441–695 (Fig. 2B, lanes 10 and 11).

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