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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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Mapping of regions on NP involved in binding to VP35. (A) Schematic presentation of the constructed mutants of NP with C-terminal truncations. (B) The constructed plasmids were in vitro translated, metabolically labeled using [35S]ProMix, separated by SDS-PAGE and analyzed using a BioImager. (C) In vitro translated and metabolically labeled mutants of NP were incubated with bacterially expressed GST-VP35 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-VP35 was set to 100%. (D) Quantification of 3 separate experiments as shown under (C).
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Figure 4: Mapping of regions on NP involved in binding to VP35. (A) Schematic presentation of the constructed mutants of NP with C-terminal truncations. (B) The constructed plasmids were in vitro translated, metabolically labeled using [35S]ProMix, separated by SDS-PAGE and analyzed using a BioImager. (C) In vitro translated and metabolically labeled mutants of NP were incubated with bacterially expressed GST-VP35 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-VP35 was set to 100%. (D) Quantification of 3 separate experiments as shown under (C).

Mentions: To address the question of whether the interaction domain for VP35 is present in the coiled coil region itself or whether the coiled coil region is necessary to support the integrity of the VP35 binding site, we analyzed the interaction between VP35 and C-terminally truncated NP mutants (Fig. 4A). NP mutants were in vitro translated (Fig. 4B) and incubated with the bacterially expressed fusion protein of VP35 and GST. The resultant complexes were precipitated with glutathione-sepharose and the amounts of precipitated NP mutants were quantified by BioImaging (Figs. 4C and 4D). NP mutants containing the N-terminal amino acids 1–330, 1–389 and 1–440 showed only weak binding to GST-VP35 (Fig. 4C. lanes 1, 3, 5, 7 and Fig. 4D). The inclusion of the next 40 amino acids, which resulted in NP mutant 1–480, increased binding to GST-VP35 significantly (Fig. 4C, lane 9 and 3D). Further elongation of the protein, however, diminished binding of the respective NP mutant to GST-VP35. These results suggested that the interaction between NP and VP35 is dependent on the coiled coil region and amino acids 440 to 480.


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)

Mapping of regions on NP involved in binding to VP35. (A) Schematic presentation of the constructed mutants of NP with C-terminal truncations. (B) The constructed plasmids were in vitro translated, metabolically labeled using [35S]ProMix, separated by SDS-PAGE and analyzed using a BioImager. (C) In vitro translated and metabolically labeled mutants of NP were incubated with bacterially expressed GST-VP35 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-VP35 was set to 100%. (D) Quantification of 3 separate experiments as shown under (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Mapping of regions on NP involved in binding to VP35. (A) Schematic presentation of the constructed mutants of NP with C-terminal truncations. (B) The constructed plasmids were in vitro translated, metabolically labeled using [35S]ProMix, separated by SDS-PAGE and analyzed using a BioImager. (C) In vitro translated and metabolically labeled mutants of NP were incubated with bacterially expressed GST-VP35 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-VP35 was set to 100%. (D) Quantification of 3 separate experiments as shown under (C).
Mentions: To address the question of whether the interaction domain for VP35 is present in the coiled coil region itself or whether the coiled coil region is necessary to support the integrity of the VP35 binding site, we analyzed the interaction between VP35 and C-terminally truncated NP mutants (Fig. 4A). NP mutants were in vitro translated (Fig. 4B) and incubated with the bacterially expressed fusion protein of VP35 and GST. The resultant complexes were precipitated with glutathione-sepharose and the amounts of precipitated NP mutants were quantified by BioImaging (Figs. 4C and 4D). NP mutants containing the N-terminal amino acids 1–330, 1–389 and 1–440 showed only weak binding to GST-VP35 (Fig. 4C. lanes 1, 3, 5, 7 and Fig. 4D). The inclusion of the next 40 amino acids, which resulted in NP mutant 1–480, increased binding to GST-VP35 significantly (Fig. 4C, lane 9 and 3D). Further elongation of the protein, however, diminished binding of the respective NP mutant to GST-VP35. These results suggested that the interaction between NP and VP35 is dependent on the coiled coil region and amino acids 440 to 480.

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