Limits...
The C-terminal amyloidogenic peptide contributes to self-assembly of Avibirnavirus viral protease.

Zheng X, Jia L, Hu B, Sun Y, Zhang Y, Gao X, Deng T, Bao S, Xu L, Zhou J - Sci Rep (2015)

Bottom Line: Interestingly, the last C-terminal hydrophobic and amyloidogenic stretch (238)YHLAMA(243) with two "aggregation-prone" alanine residues was found to be essential for its intracellular self-assembly.Importantly, the assembly of VP4 significantly reduced the cytotoxicity of protease activity in host cells which potentially prevent the premature cell death and facilitate viral replication.This study provides novel insights into the formation mechanism and biological functions of the Avibirnavirus protease-related fibrils.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Animal Virology of Ministry of Agriculture, Zhejiang University, Hangzhou 310058, PR China.

ABSTRACT
Unlike other viral protease, Avibirnavirus infectious bursal disease virus (IBDV)-encoded viral protease VP4 forms unusual intracellular tubule-like structures during viral infection. However, the formation mechanism and potential biological functions of intracellular VP4 tubules remain largely elusive. Here, we show that VP4 can assemble into tubules in diverse IBDV-infected cells. Dynamic analysis show that VP4 initiates the assembly at early stage of IBDV infection, and gradually assembles into larger size of fibrils within the cytoplasm and nucleus. Intracellular assembly of VP4 doesn't involve the host cytoskeleton, other IBDV-encoded viral proteins or vital subcellular organelles. Interestingly, the last C-terminal hydrophobic and amyloidogenic stretch (238)YHLAMA(243) with two "aggregation-prone" alanine residues was found to be essential for its intracellular self-assembly. The assembled VP4 fibrils show significantly low solubility, subsequently, the deposition of highly assembled VP4 structures ultimately deformed the host cytoskeleton and nucleus, which was potentially associated with IBDV lytic infection. Importantly, the assembly of VP4 significantly reduced the cytotoxicity of protease activity in host cells which potentially prevent the premature cell death and facilitate viral replication. This study provides novel insights into the formation mechanism and biological functions of the Avibirnavirus protease-related fibrils.

No MeSH data available.


Related in: MedlinePlus

Assembled VP4 structures mechanically alter cytoskeleton and nucleus.IBDV-infected DF-1 cells (a), pEGFP-wtVP4-transfected DF-1 cells (b) and mock-infected DF-1 cells (c) were fixed for confocal analysis at 24 h and 36 h following infection or transfection. VP4 was detected with mouse anti-VP4 mAb or rabbit anti-VP4 polyclonal antibody followed by FITC-conjugated goat anti-mouse or anti-rabbit IgG. Host F-actin was probed with TRITC-phalloidin. Anti-β-tubulin mAb and TRITC-conjugated anti-mouse IgG were used to label host microtubules. Rabbit anti-vimentin mAb and TRITC-conjugated anti-rabbit IgG were used to stain intermediate filaments. Nuclear DNA was stained with DAPI. Scale bar is 7.5 μm for microtubules at 24 h and F-actin and vimentin at 36 h; scale bars are 10 μm for other panels. Ultrastructure of assembled VP4 in IBDV-infected DF-1 cells (d) and pEGFP-wtVP4-transfected DF-1 cells (e). The ultrathin section was stained with mouse anti-VP4 mAb followed by goat anti-mouse immunogold-labeled secondary antibody. Black arrowheads indicate bundles of VP4 tubules, N is nucleus and CP is cytoplasm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4594098&req=5

f6: Assembled VP4 structures mechanically alter cytoskeleton and nucleus.IBDV-infected DF-1 cells (a), pEGFP-wtVP4-transfected DF-1 cells (b) and mock-infected DF-1 cells (c) were fixed for confocal analysis at 24 h and 36 h following infection or transfection. VP4 was detected with mouse anti-VP4 mAb or rabbit anti-VP4 polyclonal antibody followed by FITC-conjugated goat anti-mouse or anti-rabbit IgG. Host F-actin was probed with TRITC-phalloidin. Anti-β-tubulin mAb and TRITC-conjugated anti-mouse IgG were used to label host microtubules. Rabbit anti-vimentin mAb and TRITC-conjugated anti-rabbit IgG were used to stain intermediate filaments. Nuclear DNA was stained with DAPI. Scale bar is 7.5 μm for microtubules at 24 h and F-actin and vimentin at 36 h; scale bars are 10 μm for other panels. Ultrastructure of assembled VP4 in IBDV-infected DF-1 cells (d) and pEGFP-wtVP4-transfected DF-1 cells (e). The ultrathin section was stained with mouse anti-VP4 mAb followed by goat anti-mouse immunogold-labeled secondary antibody. Black arrowheads indicate bundles of VP4 tubules, N is nucleus and CP is cytoplasm.

Mentions: Morphologically, the assembled VP4 structures are apparently similar to amyloid fibrils which are thought to be insoluble and resistant to degradation28. As VP4 was largely found to assemble within IBDV-infected cells, and maintained in the cell debris after lytic IBDV infection around 36 hpi (Fig. 6a,b), it is possibly to speculate that the assembly of VP4 affects its intracellular solubility. To test this hypothesis, DF-1 cells infected with IBDV or transfected with plasmids pCI-wtVP4, pCI-VP4-C∆3, pCI-VP4-C∆2, pCI-VP4-C∆1 and pCI-VP4-C∆1′ were lysed with RAPI lysis buffer containing 1% Triton X-100 (TX-100). VP4 protein in the TX-100-insoluble and soluble fractions were then detected by Western blotting using anti-VP4 mAb. As shown in Fig. 5, assembled VP4 proteins in IBDV-infected cells or wtVP4, VP4-C∆1 and VP4-C∆1′ in transfected cells were mainly (~80%) detected in the TX-100-insoluble fractions. In contrast, diffusely distributed VP4-C∆2 and VP4-C∆3 in transfected cells were rarely (~20%) detected in the TX-100-insoluble fraction. These results demonstrate that assembly of VP4 greatly reduced the solubility, and assembled VP4 tubules mainly exist as an insoluble component within the cells.


The C-terminal amyloidogenic peptide contributes to self-assembly of Avibirnavirus viral protease.

Zheng X, Jia L, Hu B, Sun Y, Zhang Y, Gao X, Deng T, Bao S, Xu L, Zhou J - Sci Rep (2015)

Assembled VP4 structures mechanically alter cytoskeleton and nucleus.IBDV-infected DF-1 cells (a), pEGFP-wtVP4-transfected DF-1 cells (b) and mock-infected DF-1 cells (c) were fixed for confocal analysis at 24 h and 36 h following infection or transfection. VP4 was detected with mouse anti-VP4 mAb or rabbit anti-VP4 polyclonal antibody followed by FITC-conjugated goat anti-mouse or anti-rabbit IgG. Host F-actin was probed with TRITC-phalloidin. Anti-β-tubulin mAb and TRITC-conjugated anti-mouse IgG were used to label host microtubules. Rabbit anti-vimentin mAb and TRITC-conjugated anti-rabbit IgG were used to stain intermediate filaments. Nuclear DNA was stained with DAPI. Scale bar is 7.5 μm for microtubules at 24 h and F-actin and vimentin at 36 h; scale bars are 10 μm for other panels. Ultrastructure of assembled VP4 in IBDV-infected DF-1 cells (d) and pEGFP-wtVP4-transfected DF-1 cells (e). The ultrathin section was stained with mouse anti-VP4 mAb followed by goat anti-mouse immunogold-labeled secondary antibody. Black arrowheads indicate bundles of VP4 tubules, N is nucleus and CP is cytoplasm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4594098&req=5

f6: Assembled VP4 structures mechanically alter cytoskeleton and nucleus.IBDV-infected DF-1 cells (a), pEGFP-wtVP4-transfected DF-1 cells (b) and mock-infected DF-1 cells (c) were fixed for confocal analysis at 24 h and 36 h following infection or transfection. VP4 was detected with mouse anti-VP4 mAb or rabbit anti-VP4 polyclonal antibody followed by FITC-conjugated goat anti-mouse or anti-rabbit IgG. Host F-actin was probed with TRITC-phalloidin. Anti-β-tubulin mAb and TRITC-conjugated anti-mouse IgG were used to label host microtubules. Rabbit anti-vimentin mAb and TRITC-conjugated anti-rabbit IgG were used to stain intermediate filaments. Nuclear DNA was stained with DAPI. Scale bar is 7.5 μm for microtubules at 24 h and F-actin and vimentin at 36 h; scale bars are 10 μm for other panels. Ultrastructure of assembled VP4 in IBDV-infected DF-1 cells (d) and pEGFP-wtVP4-transfected DF-1 cells (e). The ultrathin section was stained with mouse anti-VP4 mAb followed by goat anti-mouse immunogold-labeled secondary antibody. Black arrowheads indicate bundles of VP4 tubules, N is nucleus and CP is cytoplasm.
Mentions: Morphologically, the assembled VP4 structures are apparently similar to amyloid fibrils which are thought to be insoluble and resistant to degradation28. As VP4 was largely found to assemble within IBDV-infected cells, and maintained in the cell debris after lytic IBDV infection around 36 hpi (Fig. 6a,b), it is possibly to speculate that the assembly of VP4 affects its intracellular solubility. To test this hypothesis, DF-1 cells infected with IBDV or transfected with plasmids pCI-wtVP4, pCI-VP4-C∆3, pCI-VP4-C∆2, pCI-VP4-C∆1 and pCI-VP4-C∆1′ were lysed with RAPI lysis buffer containing 1% Triton X-100 (TX-100). VP4 protein in the TX-100-insoluble and soluble fractions were then detected by Western blotting using anti-VP4 mAb. As shown in Fig. 5, assembled VP4 proteins in IBDV-infected cells or wtVP4, VP4-C∆1 and VP4-C∆1′ in transfected cells were mainly (~80%) detected in the TX-100-insoluble fractions. In contrast, diffusely distributed VP4-C∆2 and VP4-C∆3 in transfected cells were rarely (~20%) detected in the TX-100-insoluble fraction. These results demonstrate that assembly of VP4 greatly reduced the solubility, and assembled VP4 tubules mainly exist as an insoluble component within the cells.

Bottom Line: Interestingly, the last C-terminal hydrophobic and amyloidogenic stretch (238)YHLAMA(243) with two "aggregation-prone" alanine residues was found to be essential for its intracellular self-assembly.Importantly, the assembly of VP4 significantly reduced the cytotoxicity of protease activity in host cells which potentially prevent the premature cell death and facilitate viral replication.This study provides novel insights into the formation mechanism and biological functions of the Avibirnavirus protease-related fibrils.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Animal Virology of Ministry of Agriculture, Zhejiang University, Hangzhou 310058, PR China.

ABSTRACT
Unlike other viral protease, Avibirnavirus infectious bursal disease virus (IBDV)-encoded viral protease VP4 forms unusual intracellular tubule-like structures during viral infection. However, the formation mechanism and potential biological functions of intracellular VP4 tubules remain largely elusive. Here, we show that VP4 can assemble into tubules in diverse IBDV-infected cells. Dynamic analysis show that VP4 initiates the assembly at early stage of IBDV infection, and gradually assembles into larger size of fibrils within the cytoplasm and nucleus. Intracellular assembly of VP4 doesn't involve the host cytoskeleton, other IBDV-encoded viral proteins or vital subcellular organelles. Interestingly, the last C-terminal hydrophobic and amyloidogenic stretch (238)YHLAMA(243) with two "aggregation-prone" alanine residues was found to be essential for its intracellular self-assembly. The assembled VP4 fibrils show significantly low solubility, subsequently, the deposition of highly assembled VP4 structures ultimately deformed the host cytoskeleton and nucleus, which was potentially associated with IBDV lytic infection. Importantly, the assembly of VP4 significantly reduced the cytotoxicity of protease activity in host cells which potentially prevent the premature cell death and facilitate viral replication. This study provides novel insights into the formation mechanism and biological functions of the Avibirnavirus protease-related fibrils.

No MeSH data available.


Related in: MedlinePlus