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Interaction with Tsg101 is necessary for the efficient transport and release of nucleocapsids in marburg virus-infected cells.

Dolnik O, Kolesnikova L, Welsch S, Strecker T, Schudt G, Becker S - PLoS Pathog. (2014)

Bottom Line: In contrast, rMARV(PSAPmut) nucleocapsids did not display co-localization with Tsg101, had significantly shorter transport trajectories, and migration close to the plasma membrane was severely impaired, resulting in reduced recruitment into filopodia, the major budding sites of MARV.Down regulation of IQGAP1 impaired release of MARV.These results indicate that the PSAP motif in NP, which enables binding to Tsg101, is important for the efficient actin-dependent transport of nucleocapsids to the sites of budding.

View Article: PubMed Central - PubMed

Affiliation: Institut für Virologie, Philipps Universität Marburg, Marburg, Germany.

ABSTRACT
Endosomal sorting complex required for transport (ESCRT) machinery supports the efficient budding of Marburg virus (MARV) and many other enveloped viruses. Interaction between components of the ESCRT machinery and viral proteins is predominantly mediated by short tetrapeptide motifs, known as late domains. MARV contains late domain motifs in the matrix protein VP40 and in the genome-encapsidating nucleoprotein (NP). The PSAP late domain motif of NP recruits the ESCRT-I protein tumor susceptibility gene 101 (Tsg101). Here, we generated a recombinant MARV encoding NP with a mutated PSAP late domain (rMARV(PSAPmut)). rMARV(PSAPmut) was attenuated by up to one log compared with recombinant wild-type MARV (rMARV(wt)), formed smaller plaques and exhibited delayed virus release. Nucleocapsids in rMARV(PSAPmut)-infected cells were more densely packed inside viral inclusions and more abundant in the cytoplasm than in rMARV(wt)-infected cells. A similar phenotype was detected when MARV-infected cells were depleted of Tsg101. Live-cell imaging analyses revealed that Tsg101 accumulated in inclusions of rMARV(wt)-infected cells and was co-transported together with nucleocapsids. In contrast, rMARV(PSAPmut) nucleocapsids did not display co-localization with Tsg101, had significantly shorter transport trajectories, and migration close to the plasma membrane was severely impaired, resulting in reduced recruitment into filopodia, the major budding sites of MARV. We further show that the Tsg101 interacting protein IQGAP1, an actin cytoskeleton regulator, was recruited into inclusions and to individual nucleocapsids together with Tsg101. Moreover, IQGAP1 was detected in a contrail-like structure at the rear end of migrating nucleocapsids. Down regulation of IQGAP1 impaired release of MARV. These results indicate that the PSAP motif in NP, which enables binding to Tsg101, is important for the efficient actin-dependent transport of nucleocapsids to the sites of budding. Thus, the interaction between NP and Tsg101 supports several steps of MARV assembly before virus fission.

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Infection with MARVPSAPmut results in compact inclusion bodies and accumulation of nucleocapsids in the periphery of cells.Huh-7 cells were infected with rMARVwt or rMARVPSAPmut, fixed at 24 h p.i. and subjected to immunofluorescence staining using NP-specific antibodies. Samples were then analyzed by confocal laser scanning microscopy. Left panels: rMARVwt infection. Right panels: rMARVPSAPmut infection. Grey boxes in the upper pictures indicate different regions of the same cell that are shown in higher magnification below. (A) and (B) periphery of cells. (C) and (D) inclusion bodies. Arrows indicate nucleocapsids.
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ppat-1004463-g005: Infection with MARVPSAPmut results in compact inclusion bodies and accumulation of nucleocapsids in the periphery of cells.Huh-7 cells were infected with rMARVwt or rMARVPSAPmut, fixed at 24 h p.i. and subjected to immunofluorescence staining using NP-specific antibodies. Samples were then analyzed by confocal laser scanning microscopy. Left panels: rMARVwt infection. Right panels: rMARVPSAPmut infection. Grey boxes in the upper pictures indicate different regions of the same cell that are shown in higher magnification below. (A) and (B) periphery of cells. (C) and (D) inclusion bodies. Arrows indicate nucleocapsids.

Mentions: We next analyzed which step in the transport and release of nucleocapsids was impaired by the missing interaction with Tsg101. Because depletion of Tsg101 led to an accumulation of cytoplasmic MARV nucleocapsids (Fig. 1E), we tested whether nucleocapsids in rMARVPSAPmut-infected cells exhibited a similar phenotype (Fig. 5). Indeed, indirect immunofluorescence analysis revealed an accumulation of elongated filamentous structures, mostly in the periphery of rMARVPSAPmut-infected cells (Fig. 5A, white arrows). Based on their NP content and dimensions, these structures have recently been identified as nucleocapsids [26]. Cells infected with rMARVwt did not exhibit this accumulation of nucleocapsids (Fig. 5B). In addition, although inclusions in rMARVwt-infected cells were pleomorphic (Fig. 5D), inclusions in rMARVPSAPmut-infected cells were more round and compact and frequently exhibited a bright NP signal at their rim (Fig. 5C).


Interaction with Tsg101 is necessary for the efficient transport and release of nucleocapsids in marburg virus-infected cells.

Dolnik O, Kolesnikova L, Welsch S, Strecker T, Schudt G, Becker S - PLoS Pathog. (2014)

Infection with MARVPSAPmut results in compact inclusion bodies and accumulation of nucleocapsids in the periphery of cells.Huh-7 cells were infected with rMARVwt or rMARVPSAPmut, fixed at 24 h p.i. and subjected to immunofluorescence staining using NP-specific antibodies. Samples were then analyzed by confocal laser scanning microscopy. Left panels: rMARVwt infection. Right panels: rMARVPSAPmut infection. Grey boxes in the upper pictures indicate different regions of the same cell that are shown in higher magnification below. (A) and (B) periphery of cells. (C) and (D) inclusion bodies. Arrows indicate nucleocapsids.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199773&req=5

ppat-1004463-g005: Infection with MARVPSAPmut results in compact inclusion bodies and accumulation of nucleocapsids in the periphery of cells.Huh-7 cells were infected with rMARVwt or rMARVPSAPmut, fixed at 24 h p.i. and subjected to immunofluorescence staining using NP-specific antibodies. Samples were then analyzed by confocal laser scanning microscopy. Left panels: rMARVwt infection. Right panels: rMARVPSAPmut infection. Grey boxes in the upper pictures indicate different regions of the same cell that are shown in higher magnification below. (A) and (B) periphery of cells. (C) and (D) inclusion bodies. Arrows indicate nucleocapsids.
Mentions: We next analyzed which step in the transport and release of nucleocapsids was impaired by the missing interaction with Tsg101. Because depletion of Tsg101 led to an accumulation of cytoplasmic MARV nucleocapsids (Fig. 1E), we tested whether nucleocapsids in rMARVPSAPmut-infected cells exhibited a similar phenotype (Fig. 5). Indeed, indirect immunofluorescence analysis revealed an accumulation of elongated filamentous structures, mostly in the periphery of rMARVPSAPmut-infected cells (Fig. 5A, white arrows). Based on their NP content and dimensions, these structures have recently been identified as nucleocapsids [26]. Cells infected with rMARVwt did not exhibit this accumulation of nucleocapsids (Fig. 5B). In addition, although inclusions in rMARVwt-infected cells were pleomorphic (Fig. 5D), inclusions in rMARVPSAPmut-infected cells were more round and compact and frequently exhibited a bright NP signal at their rim (Fig. 5C).

Bottom Line: In contrast, rMARV(PSAPmut) nucleocapsids did not display co-localization with Tsg101, had significantly shorter transport trajectories, and migration close to the plasma membrane was severely impaired, resulting in reduced recruitment into filopodia, the major budding sites of MARV.Down regulation of IQGAP1 impaired release of MARV.These results indicate that the PSAP motif in NP, which enables binding to Tsg101, is important for the efficient actin-dependent transport of nucleocapsids to the sites of budding.

View Article: PubMed Central - PubMed

Affiliation: Institut für Virologie, Philipps Universität Marburg, Marburg, Germany.

ABSTRACT
Endosomal sorting complex required for transport (ESCRT) machinery supports the efficient budding of Marburg virus (MARV) and many other enveloped viruses. Interaction between components of the ESCRT machinery and viral proteins is predominantly mediated by short tetrapeptide motifs, known as late domains. MARV contains late domain motifs in the matrix protein VP40 and in the genome-encapsidating nucleoprotein (NP). The PSAP late domain motif of NP recruits the ESCRT-I protein tumor susceptibility gene 101 (Tsg101). Here, we generated a recombinant MARV encoding NP with a mutated PSAP late domain (rMARV(PSAPmut)). rMARV(PSAPmut) was attenuated by up to one log compared with recombinant wild-type MARV (rMARV(wt)), formed smaller plaques and exhibited delayed virus release. Nucleocapsids in rMARV(PSAPmut)-infected cells were more densely packed inside viral inclusions and more abundant in the cytoplasm than in rMARV(wt)-infected cells. A similar phenotype was detected when MARV-infected cells were depleted of Tsg101. Live-cell imaging analyses revealed that Tsg101 accumulated in inclusions of rMARV(wt)-infected cells and was co-transported together with nucleocapsids. In contrast, rMARV(PSAPmut) nucleocapsids did not display co-localization with Tsg101, had significantly shorter transport trajectories, and migration close to the plasma membrane was severely impaired, resulting in reduced recruitment into filopodia, the major budding sites of MARV. We further show that the Tsg101 interacting protein IQGAP1, an actin cytoskeleton regulator, was recruited into inclusions and to individual nucleocapsids together with Tsg101. Moreover, IQGAP1 was detected in a contrail-like structure at the rear end of migrating nucleocapsids. Down regulation of IQGAP1 impaired release of MARV. These results indicate that the PSAP motif in NP, which enables binding to Tsg101, is important for the efficient actin-dependent transport of nucleocapsids to the sites of budding. Thus, the interaction between NP and Tsg101 supports several steps of MARV assembly before virus fission.

Show MeSH
Related in: MedlinePlus