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. 2010 May;84(9):4816-20.
doi: 10.1128/JVI.00010-10. Epub 2010 Feb 17.

Role of the GTPase Rab1b in ebolavirus particle formation

Seiya Yamayoshi  1 , Gabriele Neumann, Yoshihiro Kawaoka
Affiliations

Role of the GTPase Rab1b in ebolavirus particle formation

Seiya Yamayoshi et al. J Virol. 2010 May.

Abstract

The Ebolavirus matrix protein VP40 is essential for virion assembly and egress. Recently, we reported that the coat protein complex II (COPII) transport system plays an important role in the transport of VP40 to the plasma membrane. Here, we show that dominant-negative mutants of the GTPase Rab1b interfere with VP40-mediated particle formation. Rab1b activates GBF1 (Golgi-specific BFA [brefeldin A] resistance factor 1), a critical factor in the assembly of COPI vesicles. Activated GBF1 stimulates ARF1 (ADP ribosylation factor 1), which recruits coat protein to cellular membranes for the assembly of COPI vesicles. Here, we demonstrate that GBF1 and ARF1 are involved in Ebolavirus virion formation, suggesting that both the COPII and COPI transport systems play a role in Ebolavirus VP40-mediated particle formation. These findings provide new insights into the cellular pathways employed for Ebolavirus virion formation.

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Figures

FIG. 1.
FIG. 1.
Schematic diagram of COPI and COPII transport systems. (A) COPI and COPII transport systems. COPII vesicles transport cargo from the ER to the Golgi compartment (anterograde transport). COPI vesicles transport cargo from the Golgi compartment to the ER (retrograde transport), but also play a role in anterograde cargo transport from the ERGIC to the Golgi compartment. (B) COPI vesicle formation. Rab1b activates GBF1, which then activates ARF1 (step 1). Activated ARF1 recruits preassembled COPI complexes to the Golgi/ERGIC membrane (step 2). Assembled COPI complexes recruit cargo, followed by membrane curvature (step 3).
FIG. 2.
FIG. 2.
Dominant-negative mutants of Rab1b reduce Ebolavirus VLP production. (A) Inhibition of secretory pathways by dominant-negative mutants of Rab1a or Rab1b. Secreted alkaline phosphatase (SEAP) was coexpressed with Rab1a, Rab1b, or their dominant-negative mutants in 293 cells. Twenty-four hours posttransfection, SEAP activities were measured. For cells expressing wild-type Rab1 proteins, the secretion index (i.e., the ratio of SEAP activity detected in the culture supernatant to the cell-associated SEAP activity) was defined as 100%. Experiments were carried out in triplicate. (B) Ebolavirus VP40-induced VLP production is reduced by dominant-negative Rab1b mutants. VP40 was coexpressed in 293T cells in the absence (“Empty”) or presence of FLAG-Rab1a, FLAG-Rab1b, or their dominant-negative mutants (“N124I” or “S22N” and “N121I” or “S22N,” respectively). Twenty-four hours posttransfection, the released VLPs and total cell lysates were analyzed by Western blot analysis with an anti-FLAG antibody or an anti-VP40 antibody. The intensities of the VP40 double bands were quantified, and VLP release efficiencies were calculated based on the ratio of VP40 in VLPs and cell lysates. The values obtained with wild-type Rab1a and Rab1b proteins were set to 100%. The results shown are representative of three independent experiments. (C) Morphological changes in cells expressing Venus-VP40 fusion protein. The reporter protein Venus or Venus-VP40 fusion protein was expressed in 293 cells. Twenty-four hours posttransfection, the cells were imaged by using confocal microscopy. Arrowheads indicate filamentous cell protrusions. (D) Dominant-negative Rab1b mutants reduce the frequency of VP40-induced formation of cell protrusions. Venus-VP40 was coexpressed with wild-type or dominant-negative Rab1 proteins in 293 cells. The cells were imaged 24 h posttransfection by using confocal microscopy.
FIG. 3.
FIG. 3.
Role of GBF1 in Ebolavirus VP40-mediated VLP formation. (A) Functionality of a dominant-negative GBF1 mutant. 293 cells were transfected with a plasmid encoding FLAG-GBF1 or its dominant-negative mutant. After 24 h, cells were fixed and stained with an anti-GM130 antibody (green; GE Health Care, England) and an anti-FLAG antibody (red). Hoechst 33342 (Invitrogen, CA) was used to stain the nuclei (blue). Arrows indicate the GM130 redistribution in cells expressing dominant-negative GBF1_E794K, confirming the functionality of this mutant. (B) Reduction of Ebolavirus VP40-mediated VLP production by a dominant-negative GBF1 mutant. VP40 was expressed in the absence (lane 1) or presence of wild-type (lane 2) or dominant-negative (lane 3) GBF1 in 293T cells. VLP production efficiency was calculated as described in the legend to Fig. 2B. The results shown are representative of two independent experiments. (C) A dominant-negative GBF1 mutant reduces the frequency of VP40-induced formation of cell protrusions. Venus-VP40 was coexpressed with FLAG-GBF1 or its mutant in 293 cells. The cells were imaged 24 h posttransfection by confocal microscopy.
FIG. 4.
FIG. 4.
Role of ARF1 Ebolavirus VP40-mediated VLP formation. (A) Inhibition of secretory pathways by ARF1 mutants. SEAP was coexpressed with FLAG-tagged wild-type ARF1 or with dominant-negative (FLAG-ARF1_T31N) or constitutively active (FLAG-ARF1_Q71L) mutants. The secretion index was calculated as described in the legend to Fig. 2A. (B) Ebolavirus VP40-mediated VLP production is reduced by overexpression of ARF1 or its mutants. VP40 was coexpressed in the absence (lane 1) or presence of FLAG-ARF1 (lane 2), FLAG-ARF1_T31N (dominant negative; lane 3), or FLAG-ARF1_Q71L (constitutively active; lane 4) in 293T cells. VLP production efficiency was calculated as described in the legend to Fig. 2B. The results shown are representative of three independent experiments. (C) ARF1 or its mutants reduce the frequency of VP40-induced formation of cell protrusions. Venus-VP40 was coexpressed with ARF1 or its mutants in 293 cells. The cells were imaged 24 h posttransfection by confocal microscopy.
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