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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

VP4-C∆2 protein without intracellular assembly induces apoptotic cell death.DF-1 cells were infected with IBDV strain NB or transfected with pEGFP-wtVP4 or pEGFP-VP4-C∆2. After 24 h, cells were fixed with 4% formaldehyde and incubated with TUNEL reaction solution to stain apoptotic cells. DAPI staining was used to demonstrate the nuclear morphology of apoptotic and normal cells. The white arrowheads indicate TUNEL-positive cells. (a) TUNEL assay of IBDV-infected cells. VP4 was detected using mouse anti-VP4 mAb followed by FITC-conjugated goat anti-mouse IgG. (b) TUNEL assay of pEGFP-wtVP4-transfected cells. The inset shows the magnification of one VP4-positive cell. (c) TUNEL assay of pEGFP-wtVP4-C∆2-transfected cells. The cells were TUNEL-positive and showed typical apoptotic bodies. (d) Magnification of TUNEL-positive cells in top left corner of panel c.
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f7: VP4-C∆2 protein without intracellular assembly induces apoptotic cell death.DF-1 cells were infected with IBDV strain NB or transfected with pEGFP-wtVP4 or pEGFP-VP4-C∆2. After 24 h, cells were fixed with 4% formaldehyde and incubated with TUNEL reaction solution to stain apoptotic cells. DAPI staining was used to demonstrate the nuclear morphology of apoptotic and normal cells. The white arrowheads indicate TUNEL-positive cells. (a) TUNEL assay of IBDV-infected cells. VP4 was detected using mouse anti-VP4 mAb followed by FITC-conjugated goat anti-mouse IgG. (b) TUNEL assay of pEGFP-wtVP4-transfected cells. The inset shows the magnification of one VP4-positive cell. (c) TUNEL assay of pEGFP-wtVP4-C∆2-transfected cells. The cells were TUNEL-positive and showed typical apoptotic bodies. (d) Magnification of TUNEL-positive cells in top left corner of panel c.

Mentions: Previous report showed that IBDV-encoded serine protease VP4 did not induce apoptosis31. Intriguingly, VP4 with various C-terminal deletions (deletion of two or more residues) failing to assemble induced extensive cell death, including cell shrinkage and chromatin condensation (Supplementary Video S2). To further confirm the apoptotic effects, terminal deoxynucleotidyl transferase (TdT)-mediated nick end-labeling (TUNEL) assay and confocal microscopy were applied to examine apoptosis in DF-1 cells transfected with pEGFP-wtVP4 and pEGFP-VP4-C∆2, IBDV-infected cells were used as apoptosis-positive control, as IBDV infection was known to induce host apoptosis by multiple pathways3233. As expected, IBDV-infected cells were TUNEL-positive (Fig. 7a), pEGFP-wtVP4-transfected cells showed VP4 assembly, presented a normal cellular morphology and were TUNEL-negative (Fig. 7b), whereas pEGFP-VP4-C∆2-transfected cells without VP4 assembly were TUNEL-positive, and exhibited morphological features typical of apoptosis, including cell shrinkage, chromatin condensation and apoptotic body formation (Fig. 7c,d and Supplementary Video S2). These results further confirmed that assembled VP4 fails to induce apoptosis while diffusely distributed EGFP-VP4-C∆2 was capable of eliciting apoptotic cell death.


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)

VP4-C∆2 protein without intracellular assembly induces apoptotic cell death.DF-1 cells were infected with IBDV strain NB or transfected with pEGFP-wtVP4 or pEGFP-VP4-C∆2. After 24 h, cells were fixed with 4% formaldehyde and incubated with TUNEL reaction solution to stain apoptotic cells. DAPI staining was used to demonstrate the nuclear morphology of apoptotic and normal cells. The white arrowheads indicate TUNEL-positive cells. (a) TUNEL assay of IBDV-infected cells. VP4 was detected using mouse anti-VP4 mAb followed by FITC-conjugated goat anti-mouse IgG. (b) TUNEL assay of pEGFP-wtVP4-transfected cells. The inset shows the magnification of one VP4-positive cell. (c) TUNEL assay of pEGFP-wtVP4-C∆2-transfected cells. The cells were TUNEL-positive and showed typical apoptotic bodies. (d) Magnification of TUNEL-positive cells in top left corner of panel c.
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Related In: Results  -  Collection

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f7: VP4-C∆2 protein without intracellular assembly induces apoptotic cell death.DF-1 cells were infected with IBDV strain NB or transfected with pEGFP-wtVP4 or pEGFP-VP4-C∆2. After 24 h, cells were fixed with 4% formaldehyde and incubated with TUNEL reaction solution to stain apoptotic cells. DAPI staining was used to demonstrate the nuclear morphology of apoptotic and normal cells. The white arrowheads indicate TUNEL-positive cells. (a) TUNEL assay of IBDV-infected cells. VP4 was detected using mouse anti-VP4 mAb followed by FITC-conjugated goat anti-mouse IgG. (b) TUNEL assay of pEGFP-wtVP4-transfected cells. The inset shows the magnification of one VP4-positive cell. (c) TUNEL assay of pEGFP-wtVP4-C∆2-transfected cells. The cells were TUNEL-positive and showed typical apoptotic bodies. (d) Magnification of TUNEL-positive cells in top left corner of panel c.
Mentions: Previous report showed that IBDV-encoded serine protease VP4 did not induce apoptosis31. Intriguingly, VP4 with various C-terminal deletions (deletion of two or more residues) failing to assemble induced extensive cell death, including cell shrinkage and chromatin condensation (Supplementary Video S2). To further confirm the apoptotic effects, terminal deoxynucleotidyl transferase (TdT)-mediated nick end-labeling (TUNEL) assay and confocal microscopy were applied to examine apoptosis in DF-1 cells transfected with pEGFP-wtVP4 and pEGFP-VP4-C∆2, IBDV-infected cells were used as apoptosis-positive control, as IBDV infection was known to induce host apoptosis by multiple pathways3233. As expected, IBDV-infected cells were TUNEL-positive (Fig. 7a), pEGFP-wtVP4-transfected cells showed VP4 assembly, presented a normal cellular morphology and were TUNEL-negative (Fig. 7b), whereas pEGFP-VP4-C∆2-transfected cells without VP4 assembly were TUNEL-positive, and exhibited morphological features typical of apoptosis, including cell shrinkage, chromatin condensation and apoptotic body formation (Fig. 7c,d and Supplementary Video S2). These results further confirmed that assembled VP4 fails to induce apoptosis while diffusely distributed EGFP-VP4-C∆2 was capable of eliciting apoptotic cell death.

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