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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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Inclusions in rMARVPSAPmut–infected cells are more densely packed with nucleocapsids than inclusions in rMARVwt–infected cells.Huh-7 cells were infected with rMARVwt or rMARVPSAPmut. At 28 h p.i., cells were processed in two ways (i) fixed, scraped, pelleted and then embedded in Epoxy resin (A and C); or (ii) fixed and embedded in Epoxy resin on Thermanox slides (B and D). Ultrathin sections were stained with uranyl acetate and subjected to electron microscopy. (A–B) rMARVwt–infected cells, (C–D) rMARVPSAPmut–infected cells. Bars, 500 nm. (E) Morphometric analysis of inclusions. Volume density of nucleocapsids inside inclusions is shown, p-value (***, p≤0.0001). (F) Amount of electron dense (mature) nucleocapsids inside inclusions (see black arrows Fig. 6B und D) determined per 2.5 µm2 of inclusion at electron micrographs, p-value (*, p≤0.05).
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ppat-1004463-g006: Inclusions in rMARVPSAPmut–infected cells are more densely packed with nucleocapsids than inclusions in rMARVwt–infected cells.Huh-7 cells were infected with rMARVwt or rMARVPSAPmut. At 28 h p.i., cells were processed in two ways (i) fixed, scraped, pelleted and then embedded in Epoxy resin (A and C); or (ii) fixed and embedded in Epoxy resin on Thermanox slides (B and D). Ultrathin sections were stained with uranyl acetate and subjected to electron microscopy. (A–B) rMARVwt–infected cells, (C–D) rMARVPSAPmut–infected cells. Bars, 500 nm. (E) Morphometric analysis of inclusions. Volume density of nucleocapsids inside inclusions is shown, p-value (***, p≤0.0001). (F) Amount of electron dense (mature) nucleocapsids inside inclusions (see black arrows Fig. 6B und D) determined per 2.5 µm2 of inclusion at electron micrographs, p-value (*, p≤0.05).

Mentions: Using electron microscopy, we further analyzed how mutation of the PSAP motif affected the morphology of viral inclusions. These analyses confirmed that inclusions in rMARVwt-infected cells primarily appeared as a disperse pleomorphic viroplasm in which nucleocapsids were packed with variable density, similar to the MARV inclusions described previously (Fig. 6A–B) [21], [22], [34]. In contrast, the majority of viral inclusions in rMARVPSAPmut-infected cells had a compact and spherical appearance, and they always contained densely packed nucleocapsids (Fig. 6C–D). Using the stereological morphometry of electron micrographs, we quantitatively determined the volume density of nucleocapsids. Packing of nucleocapsids was 1.7-fold higher in inclusions from rMARVPSAPmut-infected cells than in those from rMARVwt-infected cells (75%±6% and 44%±8%, respectively, Fig. 6E). Electron-dense nucleocapsids were detected 3.3-fold more frequently in inclusions from rMARVPSAPmut-infected cells compared with rMARVwt-infected cells (8.6±5 and 2.6±2 per 2.5 µm2, respectively, Fig. 6F). These results confirmed the immunofluorescence analyses and indicated that the missing interaction between Tsg101 and NP modifies the morphodynamics of viral inclusions.


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)

Inclusions in rMARVPSAPmut–infected cells are more densely packed with nucleocapsids than inclusions in rMARVwt–infected cells.Huh-7 cells were infected with rMARVwt or rMARVPSAPmut. At 28 h p.i., cells were processed in two ways (i) fixed, scraped, pelleted and then embedded in Epoxy resin (A and C); or (ii) fixed and embedded in Epoxy resin on Thermanox slides (B and D). Ultrathin sections were stained with uranyl acetate and subjected to electron microscopy. (A–B) rMARVwt–infected cells, (C–D) rMARVPSAPmut–infected cells. Bars, 500 nm. (E) Morphometric analysis of inclusions. Volume density of nucleocapsids inside inclusions is shown, p-value (***, p≤0.0001). (F) Amount of electron dense (mature) nucleocapsids inside inclusions (see black arrows Fig. 6B und D) determined per 2.5 µm2 of inclusion at electron micrographs, p-value (*, p≤0.05).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004463-g006: Inclusions in rMARVPSAPmut–infected cells are more densely packed with nucleocapsids than inclusions in rMARVwt–infected cells.Huh-7 cells were infected with rMARVwt or rMARVPSAPmut. At 28 h p.i., cells were processed in two ways (i) fixed, scraped, pelleted and then embedded in Epoxy resin (A and C); or (ii) fixed and embedded in Epoxy resin on Thermanox slides (B and D). Ultrathin sections were stained with uranyl acetate and subjected to electron microscopy. (A–B) rMARVwt–infected cells, (C–D) rMARVPSAPmut–infected cells. Bars, 500 nm. (E) Morphometric analysis of inclusions. Volume density of nucleocapsids inside inclusions is shown, p-value (***, p≤0.0001). (F) Amount of electron dense (mature) nucleocapsids inside inclusions (see black arrows Fig. 6B und D) determined per 2.5 µm2 of inclusion at electron micrographs, p-value (*, p≤0.05).
Mentions: Using electron microscopy, we further analyzed how mutation of the PSAP motif affected the morphology of viral inclusions. These analyses confirmed that inclusions in rMARVwt-infected cells primarily appeared as a disperse pleomorphic viroplasm in which nucleocapsids were packed with variable density, similar to the MARV inclusions described previously (Fig. 6A–B) [21], [22], [34]. In contrast, the majority of viral inclusions in rMARVPSAPmut-infected cells had a compact and spherical appearance, and they always contained densely packed nucleocapsids (Fig. 6C–D). Using the stereological morphometry of electron micrographs, we quantitatively determined the volume density of nucleocapsids. Packing of nucleocapsids was 1.7-fold higher in inclusions from rMARVPSAPmut-infected cells than in those from rMARVwt-infected cells (75%±6% and 44%±8%, respectively, Fig. 6E). Electron-dense nucleocapsids were detected 3.3-fold more frequently in inclusions from rMARVPSAPmut-infected cells compared with rMARVwt-infected cells (8.6±5 and 2.6±2 per 2.5 µm2, respectively, Fig. 6F). These results confirmed the immunofluorescence analyses and indicated that the missing interaction between Tsg101 and NP modifies the morphodynamics of viral inclusions.

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