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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

Functional mapping of the critical residues for VP4 assembly.(a) Schematic diagram of recombinant plasmids with full-length VP4 (wtVP4), truncated VP4 mutants and serial deletion mutants at the C-terminus of IBDV VP4. The amino acids of the hydrophobic stretch at the C-terminus of VP4 are shown. Full-length and mutant VP4 sequences were amplified and subcloned into pEGFP-C2 and pCI-neo vectors to generate the indicated recombinant plasmids. (b) Subcellular expression of truncated VP4 mutants in DF-1 cells at 24 h after transfection. The larger mass-like VP4 protein was present in pEGFP-VP4-C78-243- and pEGFP-VP4-C181-243-transfected cells, but not in pEGFP-VP4-N1-77- and pEGFP-VP4-N1-180-transfected cells. (c) EGPF-VP4 expression in DF-1 cells 24 h after transfection with C-terminal amino acid deletion mutants. The rod-shaped and mass-like VP4 structures appeared in pEGFP-VP4-C∆1- and pEGFP-VP4-C∆1′-transfected DF-1 cells, but not in pEGFP-VP4-C∆2- and pEGFP-VP4-C∆3-transfected DF-1 cells. (d) VP4 expression in DF-1 cells 24 h after transfection with the mutant pCI-VP4-C∆1, pCI-VP4-C∆1′, pCI-VP4-C∆2 and pCI-VP4-C∆3. The filamentous, rod-shaped and mass-like structures of VP4 appeared in pCI-VP4-C∆1- and pCI-VP4-C∆1′-transfected DF-1cells, but not in cells transfected with pCI-VP4-C∆2 or pCI-VP4-C∆3. All cells were labeled with mouse anti-VP4 mAb. Nuclei were stained with DAPI.
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f3: Functional mapping of the critical residues for VP4 assembly.(a) Schematic diagram of recombinant plasmids with full-length VP4 (wtVP4), truncated VP4 mutants and serial deletion mutants at the C-terminus of IBDV VP4. The amino acids of the hydrophobic stretch at the C-terminus of VP4 are shown. Full-length and mutant VP4 sequences were amplified and subcloned into pEGFP-C2 and pCI-neo vectors to generate the indicated recombinant plasmids. (b) Subcellular expression of truncated VP4 mutants in DF-1 cells at 24 h after transfection. The larger mass-like VP4 protein was present in pEGFP-VP4-C78-243- and pEGFP-VP4-C181-243-transfected cells, but not in pEGFP-VP4-N1-77- and pEGFP-VP4-N1-180-transfected cells. (c) EGPF-VP4 expression in DF-1 cells 24 h after transfection with C-terminal amino acid deletion mutants. The rod-shaped and mass-like VP4 structures appeared in pEGFP-VP4-C∆1- and pEGFP-VP4-C∆1′-transfected DF-1 cells, but not in pEGFP-VP4-C∆2- and pEGFP-VP4-C∆3-transfected DF-1 cells. (d) VP4 expression in DF-1 cells 24 h after transfection with the mutant pCI-VP4-C∆1, pCI-VP4-C∆1′, pCI-VP4-C∆2 and pCI-VP4-C∆3. The filamentous, rod-shaped and mass-like structures of VP4 appeared in pCI-VP4-C∆1- and pCI-VP4-C∆1′-transfected DF-1cells, but not in cells transfected with pCI-VP4-C∆2 or pCI-VP4-C∆3. All cells were labeled with mouse anti-VP4 mAb. Nuclei were stained with DAPI.

Mentions: To identify the critical regions for intracellular VP4 self-assembly, four VP4 truncated mutants (pEGFP-VP4-N1–77, pEGFP-VP4-N1–180, pEGFP-VP4-C181–243 and pEGFP-VP4-C78–243) were constructed (Fig. 3a) and transfected into DF-1 cells. EGFP-VP4-N1–77 and EGFP-VP4-N1–180 were diffusely distributed within the cytoplasm and nucleus, while the majority of EGFP-VP4-C181–243 and EGFP-VP4-C78–243 proteins aggregated into larger mass-like structures within the cytoplasm and nucleus (Fig. 3b), indicating that the C-terminus of VP4 contributes to its intracellular assembly. Our results using intracellular system were consistent with recent results using E. Coli-expressed VP4 showing that C-terminal 28mer of VP4 is important for its extracellular self-assembly19. Intriguingly, sequence analysis revealed that the last C-terminal residues 238YHLAMA243 of VP4 form a highly hydrophobic stretch, and sequence alignment indicated that the C-terminal hydrophobic residues 238Y and 240LAMA243 are completely conserved among all isolated IBDV strains, and only 239H are mutated into 239D in very-virulent strains. All these information suggested that the C-terminal hydrophobicity potentially plays key role in the VP4 assembly. To further confirm this hypothesis, a series of C-terminal deletion mutants were subcloned into both pEGFP-C2 and pCI-neo expression systems, respectively, the resulting mutants (pEGFP-VP4-C∆1, ∆1′, ∆2, ∆3 and pCI-VP4-C∆1, ∆1′, ∆2, ∆3) and transfected into DF-1 cells (Fig. 3a). In both expression systems (Fig. 3c,d), deletion of the single C-terminal residue 242M (∆1′) or 243A (∆1) did not disturb VP4 assembly, but C-terminal deletion of two (242MA243), three (241AMA243) or more than three residues caused failure of VP4 assembly. These results suggest that the last hydrophobic residues at the C-terminus are critical for VP4 assembly.


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)

Functional mapping of the critical residues for VP4 assembly.(a) Schematic diagram of recombinant plasmids with full-length VP4 (wtVP4), truncated VP4 mutants and serial deletion mutants at the C-terminus of IBDV VP4. The amino acids of the hydrophobic stretch at the C-terminus of VP4 are shown. Full-length and mutant VP4 sequences were amplified and subcloned into pEGFP-C2 and pCI-neo vectors to generate the indicated recombinant plasmids. (b) Subcellular expression of truncated VP4 mutants in DF-1 cells at 24 h after transfection. The larger mass-like VP4 protein was present in pEGFP-VP4-C78-243- and pEGFP-VP4-C181-243-transfected cells, but not in pEGFP-VP4-N1-77- and pEGFP-VP4-N1-180-transfected cells. (c) EGPF-VP4 expression in DF-1 cells 24 h after transfection with C-terminal amino acid deletion mutants. The rod-shaped and mass-like VP4 structures appeared in pEGFP-VP4-C∆1- and pEGFP-VP4-C∆1′-transfected DF-1 cells, but not in pEGFP-VP4-C∆2- and pEGFP-VP4-C∆3-transfected DF-1 cells. (d) VP4 expression in DF-1 cells 24 h after transfection with the mutant pCI-VP4-C∆1, pCI-VP4-C∆1′, pCI-VP4-C∆2 and pCI-VP4-C∆3. The filamentous, rod-shaped and mass-like structures of VP4 appeared in pCI-VP4-C∆1- and pCI-VP4-C∆1′-transfected DF-1cells, but not in cells transfected with pCI-VP4-C∆2 or pCI-VP4-C∆3. All cells were labeled with mouse anti-VP4 mAb. Nuclei were stained with DAPI.
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f3: Functional mapping of the critical residues for VP4 assembly.(a) Schematic diagram of recombinant plasmids with full-length VP4 (wtVP4), truncated VP4 mutants and serial deletion mutants at the C-terminus of IBDV VP4. The amino acids of the hydrophobic stretch at the C-terminus of VP4 are shown. Full-length and mutant VP4 sequences were amplified and subcloned into pEGFP-C2 and pCI-neo vectors to generate the indicated recombinant plasmids. (b) Subcellular expression of truncated VP4 mutants in DF-1 cells at 24 h after transfection. The larger mass-like VP4 protein was present in pEGFP-VP4-C78-243- and pEGFP-VP4-C181-243-transfected cells, but not in pEGFP-VP4-N1-77- and pEGFP-VP4-N1-180-transfected cells. (c) EGPF-VP4 expression in DF-1 cells 24 h after transfection with C-terminal amino acid deletion mutants. The rod-shaped and mass-like VP4 structures appeared in pEGFP-VP4-C∆1- and pEGFP-VP4-C∆1′-transfected DF-1 cells, but not in pEGFP-VP4-C∆2- and pEGFP-VP4-C∆3-transfected DF-1 cells. (d) VP4 expression in DF-1 cells 24 h after transfection with the mutant pCI-VP4-C∆1, pCI-VP4-C∆1′, pCI-VP4-C∆2 and pCI-VP4-C∆3. The filamentous, rod-shaped and mass-like structures of VP4 appeared in pCI-VP4-C∆1- and pCI-VP4-C∆1′-transfected DF-1cells, but not in cells transfected with pCI-VP4-C∆2 or pCI-VP4-C∆3. All cells were labeled with mouse anti-VP4 mAb. Nuclei were stained with DAPI.
Mentions: To identify the critical regions for intracellular VP4 self-assembly, four VP4 truncated mutants (pEGFP-VP4-N1–77, pEGFP-VP4-N1–180, pEGFP-VP4-C181–243 and pEGFP-VP4-C78–243) were constructed (Fig. 3a) and transfected into DF-1 cells. EGFP-VP4-N1–77 and EGFP-VP4-N1–180 were diffusely distributed within the cytoplasm and nucleus, while the majority of EGFP-VP4-C181–243 and EGFP-VP4-C78–243 proteins aggregated into larger mass-like structures within the cytoplasm and nucleus (Fig. 3b), indicating that the C-terminus of VP4 contributes to its intracellular assembly. Our results using intracellular system were consistent with recent results using E. Coli-expressed VP4 showing that C-terminal 28mer of VP4 is important for its extracellular self-assembly19. Intriguingly, sequence analysis revealed that the last C-terminal residues 238YHLAMA243 of VP4 form a highly hydrophobic stretch, and sequence alignment indicated that the C-terminal hydrophobic residues 238Y and 240LAMA243 are completely conserved among all isolated IBDV strains, and only 239H are mutated into 239D in very-virulent strains. All these information suggested that the C-terminal hydrophobicity potentially plays key role in the VP4 assembly. To further confirm this hypothesis, a series of C-terminal deletion mutants were subcloned into both pEGFP-C2 and pCI-neo expression systems, respectively, the resulting mutants (pEGFP-VP4-C∆1, ∆1′, ∆2, ∆3 and pCI-VP4-C∆1, ∆1′, ∆2, ∆3) and transfected into DF-1 cells (Fig. 3a). In both expression systems (Fig. 3c,d), deletion of the single C-terminal residue 242M (∆1′) or 243A (∆1) did not disturb VP4 assembly, but C-terminal deletion of two (242MA243), three (241AMA243) or more than three residues caused failure of VP4 assembly. These results suggest that the last hydrophobic residues at the C-terminus are critical for VP4 assembly.

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