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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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rMARVPSAPmut exhibits delayed growth kinetics.Vero E6 cells were inoculated with either rMARVPSAPmut or rMARVwt. Supernatants and cell lysates were collected at indicated time points p.i. and viral titres determined by TCID50 assay or subjected to Western blot analysis. (A) Growth kinetics of rMARVPSAPmut (grey circle) or rMARVwt (black square) at MOI of 0.01. (B) Western Blot analysis of viral protein levels during an infection at MOI of 0.01. Cell lysates and culture supernatants were collected at indicated time points and were analyzed by SDS-PAGE and Western Blotting using NP- and VP40-specific antibodies. (C) Growth kinetics of rMARVPSAPmut (grey circle) or rMARVwt (black square) at MOI of 0.1. (D) rMARVPSAPmut- or rMARVwt– or mock-infected cells were analyzed for CPE formation during infection at MOI of 0.1 at 3 days p.i. (E) Western Blot analysis of viral protein levels during an infection at MOI of 0.1. Cell lysates and culture supernatants were collected at indicated time points and were analyzed as described in Fig. 1B. P-values are indicated (*, P≤0.05; **, P≤0.001; ***, P≤0.0001).
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ppat-1004463-g002: rMARVPSAPmut exhibits delayed growth kinetics.Vero E6 cells were inoculated with either rMARVPSAPmut or rMARVwt. Supernatants and cell lysates were collected at indicated time points p.i. and viral titres determined by TCID50 assay or subjected to Western blot analysis. (A) Growth kinetics of rMARVPSAPmut (grey circle) or rMARVwt (black square) at MOI of 0.01. (B) Western Blot analysis of viral protein levels during an infection at MOI of 0.01. Cell lysates and culture supernatants were collected at indicated time points and were analyzed by SDS-PAGE and Western Blotting using NP- and VP40-specific antibodies. (C) Growth kinetics of rMARVPSAPmut (grey circle) or rMARVwt (black square) at MOI of 0.1. (D) rMARVPSAPmut- or rMARVwt– or mock-infected cells were analyzed for CPE formation during infection at MOI of 0.1 at 3 days p.i. (E) Western Blot analysis of viral protein levels during an infection at MOI of 0.1. Cell lysates and culture supernatants were collected at indicated time points and were analyzed as described in Fig. 1B. P-values are indicated (*, P≤0.05; **, P≤0.001; ***, P≤0.0001).

Mentions: Because Tsg101 is a multifunctional protein that is involved in several cellular pathways, its down-regulation may have multiple effects on viral replication that are not directly related to its interaction with MARV proteins [32]. We therefore specifically inhibited the interaction of Tsg101 with NP by mutating the C-terminal PSAP motif of NP in the MARV genome using reverse genetics [33]. Our previous studies revealed that this mutation significantly inhibited the interaction between NP and Tsg101 [31]. The mutated virus (rMARVPSAPmut) was rescued, and its phenotype was analyzed (Fig. 2). The rMARVPSAPmut growth kinetics at a low MOI (0.01) were reduced, and the measured TCID50 titers were between one and two logs lower than for rMARVwt (Fig. 2A). These differences were reflected in the amount of viral protein in the supernatant of infected cells. At all tested time-points post-infection (p.i.), the rMARVPSAPmut-infected cells released less VP40 and NP than the rMARVwt-infected cells, whereas the cellular amounts of viral proteins remained similar, indicating that rMARVPSAPmut and rMARVwt had similar RNA synthesis and translation rates (Fig. 2B, left). These results were in line with previous results showing that the PSAP mutation does not affect NP activity in transcribing/replicating a MARV-specific minigenome [31]. A delay in the release of rMARVPSAPmut particles was also observed at a higher MOI (0.1). At three days p.i., cytopathic effects were less pronounced in the cells infected with rMARVPSAPmut than with rMARVwt (Fig. 2C–D). We then analyzed the supernatants of rMARVPSAPmut-infected cells and found that although the release of VP40 was comparable with rMARVwt, the NP levels were severely reduced (Fig. 2E). This result suggests that the budding activity of VP40 is most likely not affected by mutation of the PSAP motif in NP. In contrast, NP incorporation into particles is reduced, resulting in the release of less infectious particles (Fig. 2E). Together, these results suggested a defect in the release of infectious rMARVPSAPmut particles. This idea was supported by the analysis of plaque sizes formed by the different viruses. Plaques produced by rMARVPSAPmut were 5-fold smaller than those produced by rMARVwt (Fig. 3A–B). A comparison of the released viral particles by electron microscopy showed no morphological difference between rMARVPSAPmut and rMARVwt virions (Fig. 3C).


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)

rMARVPSAPmut exhibits delayed growth kinetics.Vero E6 cells were inoculated with either rMARVPSAPmut or rMARVwt. Supernatants and cell lysates were collected at indicated time points p.i. and viral titres determined by TCID50 assay or subjected to Western blot analysis. (A) Growth kinetics of rMARVPSAPmut (grey circle) or rMARVwt (black square) at MOI of 0.01. (B) Western Blot analysis of viral protein levels during an infection at MOI of 0.01. Cell lysates and culture supernatants were collected at indicated time points and were analyzed by SDS-PAGE and Western Blotting using NP- and VP40-specific antibodies. (C) Growth kinetics of rMARVPSAPmut (grey circle) or rMARVwt (black square) at MOI of 0.1. (D) rMARVPSAPmut- or rMARVwt– or mock-infected cells were analyzed for CPE formation during infection at MOI of 0.1 at 3 days p.i. (E) Western Blot analysis of viral protein levels during an infection at MOI of 0.1. Cell lysates and culture supernatants were collected at indicated time points and were analyzed as described in Fig. 1B. P-values are indicated (*, P≤0.05; **, P≤0.001; ***, P≤0.0001).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004463-g002: rMARVPSAPmut exhibits delayed growth kinetics.Vero E6 cells were inoculated with either rMARVPSAPmut or rMARVwt. Supernatants and cell lysates were collected at indicated time points p.i. and viral titres determined by TCID50 assay or subjected to Western blot analysis. (A) Growth kinetics of rMARVPSAPmut (grey circle) or rMARVwt (black square) at MOI of 0.01. (B) Western Blot analysis of viral protein levels during an infection at MOI of 0.01. Cell lysates and culture supernatants were collected at indicated time points and were analyzed by SDS-PAGE and Western Blotting using NP- and VP40-specific antibodies. (C) Growth kinetics of rMARVPSAPmut (grey circle) or rMARVwt (black square) at MOI of 0.1. (D) rMARVPSAPmut- or rMARVwt– or mock-infected cells were analyzed for CPE formation during infection at MOI of 0.1 at 3 days p.i. (E) Western Blot analysis of viral protein levels during an infection at MOI of 0.1. Cell lysates and culture supernatants were collected at indicated time points and were analyzed as described in Fig. 1B. P-values are indicated (*, P≤0.05; **, P≤0.001; ***, P≤0.0001).
Mentions: Because Tsg101 is a multifunctional protein that is involved in several cellular pathways, its down-regulation may have multiple effects on viral replication that are not directly related to its interaction with MARV proteins [32]. We therefore specifically inhibited the interaction of Tsg101 with NP by mutating the C-terminal PSAP motif of NP in the MARV genome using reverse genetics [33]. Our previous studies revealed that this mutation significantly inhibited the interaction between NP and Tsg101 [31]. The mutated virus (rMARVPSAPmut) was rescued, and its phenotype was analyzed (Fig. 2). The rMARVPSAPmut growth kinetics at a low MOI (0.01) were reduced, and the measured TCID50 titers were between one and two logs lower than for rMARVwt (Fig. 2A). These differences were reflected in the amount of viral protein in the supernatant of infected cells. At all tested time-points post-infection (p.i.), the rMARVPSAPmut-infected cells released less VP40 and NP than the rMARVwt-infected cells, whereas the cellular amounts of viral proteins remained similar, indicating that rMARVPSAPmut and rMARVwt had similar RNA synthesis and translation rates (Fig. 2B, left). These results were in line with previous results showing that the PSAP mutation does not affect NP activity in transcribing/replicating a MARV-specific minigenome [31]. A delay in the release of rMARVPSAPmut particles was also observed at a higher MOI (0.1). At three days p.i., cytopathic effects were less pronounced in the cells infected with rMARVPSAPmut than with rMARVwt (Fig. 2C–D). We then analyzed the supernatants of rMARVPSAPmut-infected cells and found that although the release of VP40 was comparable with rMARVwt, the NP levels were severely reduced (Fig. 2E). This result suggests that the budding activity of VP40 is most likely not affected by mutation of the PSAP motif in NP. In contrast, NP incorporation into particles is reduced, resulting in the release of less infectious particles (Fig. 2E). Together, these results suggested a defect in the release of infectious rMARVPSAPmut particles. This idea was supported by the analysis of plaque sizes formed by the different viruses. Plaques produced by rMARVPSAPmut were 5-fold smaller than those produced by rMARVwt (Fig. 3A–B). A comparison of the released viral particles by electron microscopy showed no morphological difference between rMARVPSAPmut and rMARVwt virions (Fig. 3C).

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