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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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Tsg101 knockdown in MARV-infected cells results in reduced particle release.MARV-infected Huh-7 cells (MOI of 1) were transfected twice with Tsg101-specific siRNA or control siRNA (1 h and 18 h p.i.). Cells and virus particles were harvested at 48 h p.i. (A) Quantification of Tsg101 protein level was performed by Western Blot in cells transfected with Tsg101-specific siRNA and control siRNA. Tsg101 levels in cells transfected with control siRNA (ctr.) were set to 100%. (B) Lysates and supernatants of MARV infected cells transfected with Tsg101-specific or control siRNA were subjected to Western Blot and analyzed for the presence of viral proteins NP and VP40 and Tsg101. (C) The 65 kDa form of Tsg101 is ubiquitinated. HEK293 cells were transfected with Tsg101-Flag and HA-Ubiquitin expression plasmids. At 48 h p.tr., cell lysates were subjected to immunoprecipitation with anti-HA-agarose. Cell lysates and precipitates were separated by SDS-PAGE and analyzed by Western Blot using HA- and Tsg101-specific antibodies. The position of the ubiquitinated Tsg101 (Ub-Tsg101) band is indicated by an arrow between 55 and 70 kDa. (D) Virus titers in the supernatants of MARV infected cells transfected with Tsg101-specific or control siRNA were determined by TCID50 titration, p-value (*, P≤0.05). (E) Phenotype of Tsg101 knockdown in MARV-infected cells. Huh-7 cells were infected with MARV and treated with Tsg101 specific or control siRNA and subjected to immunofluorescence analysis using a guinea pig anti-NP and secondary goat anti-guinea pig FITC-conjugated antibody. Grey boxes indicate marginal region of cells. Lower panels show higher magnification of boxed area, arrows indicate accumulation of nucleocapsids upon Tsg101 knockdown. Bars, 10 µm. (F) Western blot analysis of Tsg101 knockdown. Cells transfected with Tsg101-specific or control siRNA were analyzed by Western Blot using Tsg101- and tubulin-specific antibodies.
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ppat-1004463-g001: Tsg101 knockdown in MARV-infected cells results in reduced particle release.MARV-infected Huh-7 cells (MOI of 1) were transfected twice with Tsg101-specific siRNA or control siRNA (1 h and 18 h p.i.). Cells and virus particles were harvested at 48 h p.i. (A) Quantification of Tsg101 protein level was performed by Western Blot in cells transfected with Tsg101-specific siRNA and control siRNA. Tsg101 levels in cells transfected with control siRNA (ctr.) were set to 100%. (B) Lysates and supernatants of MARV infected cells transfected with Tsg101-specific or control siRNA were subjected to Western Blot and analyzed for the presence of viral proteins NP and VP40 and Tsg101. (C) The 65 kDa form of Tsg101 is ubiquitinated. HEK293 cells were transfected with Tsg101-Flag and HA-Ubiquitin expression plasmids. At 48 h p.tr., cell lysates were subjected to immunoprecipitation with anti-HA-agarose. Cell lysates and precipitates were separated by SDS-PAGE and analyzed by Western Blot using HA- and Tsg101-specific antibodies. The position of the ubiquitinated Tsg101 (Ub-Tsg101) band is indicated by an arrow between 55 and 70 kDa. (D) Virus titers in the supernatants of MARV infected cells transfected with Tsg101-specific or control siRNA were determined by TCID50 titration, p-value (*, P≤0.05). (E) Phenotype of Tsg101 knockdown in MARV-infected cells. Huh-7 cells were infected with MARV and treated with Tsg101 specific or control siRNA and subjected to immunofluorescence analysis using a guinea pig anti-NP and secondary goat anti-guinea pig FITC-conjugated antibody. Grey boxes indicate marginal region of cells. Lower panels show higher magnification of boxed area, arrows indicate accumulation of nucleocapsids upon Tsg101 knockdown. Bars, 10 µm. (F) Western blot analysis of Tsg101 knockdown. Cells transfected with Tsg101-specific or control siRNA were analyzed by Western Blot using Tsg101- and tubulin-specific antibodies.

Mentions: Tsg101 interacts with the MARV NP through a C-terminal PSAP late domain. The mutation of this domain impairs binding of Tsg101 and simultaneously abolishes the positive impact of NP on the release of VLPs induced by the MARV matrix protein VP40 [31]. To further analyze the function of Tsg101 during MARV infection, we down-regulated Tsg101 expression in MARV-infected cells using small interfering RNA (siRNA) technology. Tsg101-specific siRNA reduced the levels of Tsg101 expression to 30% compared with control siRNA (Fig. 1A). Western blot analysis of the cell lysates detected two prominent forms of Tsg101 at 46 and 65 kDa, respectively (Fig. 1B, lane 1). Transfection of Tsg101-specific siRNA reduced the levels of both forms of Tsg101, and Tsg101 incorporation into viral particles was reduced to undetectable levels (Fig. 1B, lanes 3 and 4). Because Tsg101 (46 kDa) can be multi-monoubiquitinated, it was presumed that the 65-kDa form represented ubiquitinated Tsg101 [10], [12]–[14]. To confirm this presumption, Flag-tagged Tsg101 and HA-tagged ubiquitin (HA-Ub) were co-expressed in HEK293 cells, and the cell lysates were used for immunoprecipitation with anti-HA agarose. Western blot analysis of the cell lysates using an anti-Tsg101 antibody mainly revealed the expected 46-kDa form of Tsg101 (Fig. 1C, lanes 1 and 2, upper panel). Immunoprecipitation of HA-tagged ubiquitinated cellular proteins and staining of the precipitates with anti-Tsg101 antibody revealed Tsg101-specific proteins with a major signal at approximately 65 kDa, corresponding to multi-ubiquitinated Tsg101 (Fig. 1C, lane 2, lower panel). Although the intracellular amounts of viral proteins remained unaffected by Tsg101 knockdown (Fig. 1B, lane 2), the release of viral proteins was reduced to 32%±18.6% for NP and 44%±10.4% for VP40 (n = 3; Fig. 1B, lane 4). Corresponding with this impaired release of viral proteins, the viral titers in the supernatants of Tsg101-depleted cells were 3- to 4-fold lower than in the control cells (Fig. 1D). Additionally, Tsg101 depletion resulted in the accumulation of nucleocapsids in the cytoplasm. Although 15% of the infected and Tsg101-depleted cells showed intracytoplasmic accumulation of nucleocapsids, only 1.5% of the infected cells treated with the control siRNA showed a similar phenotype (Fig. 1E–F, white arrows, and lower panel right). Together, these results support the hypothesis that Tsg101 is needed for the efficient release of MARV nucleocapsids, and correspondingly, a lack of Tsg101 leads to the intracellular accumulation of nucleocapsids.


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)

Tsg101 knockdown in MARV-infected cells results in reduced particle release.MARV-infected Huh-7 cells (MOI of 1) were transfected twice with Tsg101-specific siRNA or control siRNA (1 h and 18 h p.i.). Cells and virus particles were harvested at 48 h p.i. (A) Quantification of Tsg101 protein level was performed by Western Blot in cells transfected with Tsg101-specific siRNA and control siRNA. Tsg101 levels in cells transfected with control siRNA (ctr.) were set to 100%. (B) Lysates and supernatants of MARV infected cells transfected with Tsg101-specific or control siRNA were subjected to Western Blot and analyzed for the presence of viral proteins NP and VP40 and Tsg101. (C) The 65 kDa form of Tsg101 is ubiquitinated. HEK293 cells were transfected with Tsg101-Flag and HA-Ubiquitin expression plasmids. At 48 h p.tr., cell lysates were subjected to immunoprecipitation with anti-HA-agarose. Cell lysates and precipitates were separated by SDS-PAGE and analyzed by Western Blot using HA- and Tsg101-specific antibodies. The position of the ubiquitinated Tsg101 (Ub-Tsg101) band is indicated by an arrow between 55 and 70 kDa. (D) Virus titers in the supernatants of MARV infected cells transfected with Tsg101-specific or control siRNA were determined by TCID50 titration, p-value (*, P≤0.05). (E) Phenotype of Tsg101 knockdown in MARV-infected cells. Huh-7 cells were infected with MARV and treated with Tsg101 specific or control siRNA and subjected to immunofluorescence analysis using a guinea pig anti-NP and secondary goat anti-guinea pig FITC-conjugated antibody. Grey boxes indicate marginal region of cells. Lower panels show higher magnification of boxed area, arrows indicate accumulation of nucleocapsids upon Tsg101 knockdown. Bars, 10 µm. (F) Western blot analysis of Tsg101 knockdown. Cells transfected with Tsg101-specific or control siRNA were analyzed by Western Blot using Tsg101- and tubulin-specific antibodies.
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ppat-1004463-g001: Tsg101 knockdown in MARV-infected cells results in reduced particle release.MARV-infected Huh-7 cells (MOI of 1) were transfected twice with Tsg101-specific siRNA or control siRNA (1 h and 18 h p.i.). Cells and virus particles were harvested at 48 h p.i. (A) Quantification of Tsg101 protein level was performed by Western Blot in cells transfected with Tsg101-specific siRNA and control siRNA. Tsg101 levels in cells transfected with control siRNA (ctr.) were set to 100%. (B) Lysates and supernatants of MARV infected cells transfected with Tsg101-specific or control siRNA were subjected to Western Blot and analyzed for the presence of viral proteins NP and VP40 and Tsg101. (C) The 65 kDa form of Tsg101 is ubiquitinated. HEK293 cells were transfected with Tsg101-Flag and HA-Ubiquitin expression plasmids. At 48 h p.tr., cell lysates were subjected to immunoprecipitation with anti-HA-agarose. Cell lysates and precipitates were separated by SDS-PAGE and analyzed by Western Blot using HA- and Tsg101-specific antibodies. The position of the ubiquitinated Tsg101 (Ub-Tsg101) band is indicated by an arrow between 55 and 70 kDa. (D) Virus titers in the supernatants of MARV infected cells transfected with Tsg101-specific or control siRNA were determined by TCID50 titration, p-value (*, P≤0.05). (E) Phenotype of Tsg101 knockdown in MARV-infected cells. Huh-7 cells were infected with MARV and treated with Tsg101 specific or control siRNA and subjected to immunofluorescence analysis using a guinea pig anti-NP and secondary goat anti-guinea pig FITC-conjugated antibody. Grey boxes indicate marginal region of cells. Lower panels show higher magnification of boxed area, arrows indicate accumulation of nucleocapsids upon Tsg101 knockdown. Bars, 10 µm. (F) Western blot analysis of Tsg101 knockdown. Cells transfected with Tsg101-specific or control siRNA were analyzed by Western Blot using Tsg101- and tubulin-specific antibodies.
Mentions: Tsg101 interacts with the MARV NP through a C-terminal PSAP late domain. The mutation of this domain impairs binding of Tsg101 and simultaneously abolishes the positive impact of NP on the release of VLPs induced by the MARV matrix protein VP40 [31]. To further analyze the function of Tsg101 during MARV infection, we down-regulated Tsg101 expression in MARV-infected cells using small interfering RNA (siRNA) technology. Tsg101-specific siRNA reduced the levels of Tsg101 expression to 30% compared with control siRNA (Fig. 1A). Western blot analysis of the cell lysates detected two prominent forms of Tsg101 at 46 and 65 kDa, respectively (Fig. 1B, lane 1). Transfection of Tsg101-specific siRNA reduced the levels of both forms of Tsg101, and Tsg101 incorporation into viral particles was reduced to undetectable levels (Fig. 1B, lanes 3 and 4). Because Tsg101 (46 kDa) can be multi-monoubiquitinated, it was presumed that the 65-kDa form represented ubiquitinated Tsg101 [10], [12]–[14]. To confirm this presumption, Flag-tagged Tsg101 and HA-tagged ubiquitin (HA-Ub) were co-expressed in HEK293 cells, and the cell lysates were used for immunoprecipitation with anti-HA agarose. Western blot analysis of the cell lysates using an anti-Tsg101 antibody mainly revealed the expected 46-kDa form of Tsg101 (Fig. 1C, lanes 1 and 2, upper panel). Immunoprecipitation of HA-tagged ubiquitinated cellular proteins and staining of the precipitates with anti-Tsg101 antibody revealed Tsg101-specific proteins with a major signal at approximately 65 kDa, corresponding to multi-ubiquitinated Tsg101 (Fig. 1C, lane 2, lower panel). Although the intracellular amounts of viral proteins remained unaffected by Tsg101 knockdown (Fig. 1B, lane 2), the release of viral proteins was reduced to 32%±18.6% for NP and 44%±10.4% for VP40 (n = 3; Fig. 1B, lane 4). Corresponding with this impaired release of viral proteins, the viral titers in the supernatants of Tsg101-depleted cells were 3- to 4-fold lower than in the control cells (Fig. 1D). Additionally, Tsg101 depletion resulted in the accumulation of nucleocapsids in the cytoplasm. Although 15% of the infected and Tsg101-depleted cells showed intracytoplasmic accumulation of nucleocapsids, only 1.5% of the infected cells treated with the control siRNA showed a similar phenotype (Fig. 1E–F, white arrows, and lower panel right). Together, these results support the hypothesis that Tsg101 is needed for the efficient release of MARV nucleocapsids, and correspondingly, a lack of Tsg101 leads to the intracellular accumulation of nucleocapsids.

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