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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

Protease activity of VP4 with C-terminal MA deletion induces cell death.(a) Western blotting of in vitro proteolytic assay of polyprotein VP243 synthesized by the TNT system. Plasmids pCI-wtVP243 and pCI-VP243-∆MA were used for in vitro translation to produce polyprotein VP243, and empty plasmid pCI-neo was used as negative control (CK). TNT products were subjected to Western blotting using anti-VP4 and anti-VP3 mAbs. No VP3 or VP4 protein bands were detected in the product of template pCI-VP243-∆MA. (b) Confocal analysis of DF-1 cells transfected with pCI-wtVP243 and pCI-VP243-∆MA. The transfected cells were fixed after 24 h, and incubated with a mixture of rabbit anti-VP3 antiserum and mouse anti-VP4 mAb followed by FITC-conjugated goat anti-mouse IgG and TRITC-conjugated goat anti-rabbit IgG. Nuclear DNA was stained with DAPI. (c) Substitution of protease activity site S140 or K180 of VP4 or VP4-C∆2 with alanine (A) failed to induce cell death in transfected DF-1 cells. DF-1 cells transfected with indicated mutants were directly observed under microscopy at 24 h following transfection. Empty plasmid pEGFP-C2 was used as control.
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f8: Protease activity of VP4 with C-terminal MA deletion induces cell death.(a) Western blotting of in vitro proteolytic assay of polyprotein VP243 synthesized by the TNT system. Plasmids pCI-wtVP243 and pCI-VP243-∆MA were used for in vitro translation to produce polyprotein VP243, and empty plasmid pCI-neo was used as negative control (CK). TNT products were subjected to Western blotting using anti-VP4 and anti-VP3 mAbs. No VP3 or VP4 protein bands were detected in the product of template pCI-VP243-∆MA. (b) Confocal analysis of DF-1 cells transfected with pCI-wtVP243 and pCI-VP243-∆MA. The transfected cells were fixed after 24 h, and incubated with a mixture of rabbit anti-VP3 antiserum and mouse anti-VP4 mAb followed by FITC-conjugated goat anti-mouse IgG and TRITC-conjugated goat anti-rabbit IgG. Nuclear DNA was stained with DAPI. (c) Substitution of protease activity site S140 or K180 of VP4 or VP4-C∆2 with alanine (A) failed to induce cell death in transfected DF-1 cells. DF-1 cells transfected with indicated mutants were directly observed under microscopy at 24 h following transfection. Empty plasmid pEGFP-C2 was used as control.

Mentions: A recent study using in vitro assay of E. Coli-derived VP4 showed that VP4 tubules have significantly lower endopeptidase than monomeric or dimeric VP418, which provides extracellular evidence that VP4 self-assembly potentially blocks VP4 protease activity. The proteolytic activity of proteases is important for inducing apoptotic cell death34, it raised our hypothesis that the apoptosis induced by intracellular VP4-C∆2 may relate to its protease activity. Firstly, we determine the proteolytic capacity of VP4-C∆2 in processing polyprotein VP243 using the TNT system. Although our anti-VP2 mAb unfortunately failed to react with VP2 protein in Western blots, protein bands with the expected sizes of VP4 (approximately 28 kDa) and VP3 (approximately 32 kDa) were detected in the product of template pCI-wtVP243 (Fig. 8a), and no more bands with larger size were detected, indicating that VP4 and VP3 were successfully and completely released from wtVP243. Conversely, approximately 60 kDa of VP4-VP3 polyprotein bands were detected by both anti-VP4 and anti-VP3 mAbs in the product of the pCI-VP243-∆MA template, but no single VP4 or VP3 bands were individually detected (Fig. 8a), indicating that VP4-VP3 junction released from VP243-∆MA precursor was not successfully cleaved into VP4 and VP3. Consistent results were obtained in cells transfected with pCI-A and pCI-A-∆MA which contain the full-length A segment or A segment with MA deletion from the C-terminus of VP4. The results further revealed that VP4 in pCI-A-transfected cells was successfully released and assembled into structures (Fig. 8b), which were similar to the VP4 structures in pCI-wtVP4-transfected cells (Fig. 2d). However, VP4 in pCI-A-∆MA-transfected cells failed to assemble into specific structures (Fig. 8b), indicating MA deletion from VP4 C-terminus affects the VP4-VP3 cleavage, which was consistent with the destruction of cleavage motif (Thr/Ala)–X–Ala↓Ala motifs11. Overall, these data suggested that deletion of the C-terminal residues 242MA243 of VP4 maintains the cleavage activity which can efficiently cleave VP2–VP4 junctions but not VP4–VP3 junctions with destructive cleavage motif.


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)

Protease activity of VP4 with C-terminal MA deletion induces cell death.(a) Western blotting of in vitro proteolytic assay of polyprotein VP243 synthesized by the TNT system. Plasmids pCI-wtVP243 and pCI-VP243-∆MA were used for in vitro translation to produce polyprotein VP243, and empty plasmid pCI-neo was used as negative control (CK). TNT products were subjected to Western blotting using anti-VP4 and anti-VP3 mAbs. No VP3 or VP4 protein bands were detected in the product of template pCI-VP243-∆MA. (b) Confocal analysis of DF-1 cells transfected with pCI-wtVP243 and pCI-VP243-∆MA. The transfected cells were fixed after 24 h, and incubated with a mixture of rabbit anti-VP3 antiserum and mouse anti-VP4 mAb followed by FITC-conjugated goat anti-mouse IgG and TRITC-conjugated goat anti-rabbit IgG. Nuclear DNA was stained with DAPI. (c) Substitution of protease activity site S140 or K180 of VP4 or VP4-C∆2 with alanine (A) failed to induce cell death in transfected DF-1 cells. DF-1 cells transfected with indicated mutants were directly observed under microscopy at 24 h following transfection. Empty plasmid pEGFP-C2 was used as control.
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f8: Protease activity of VP4 with C-terminal MA deletion induces cell death.(a) Western blotting of in vitro proteolytic assay of polyprotein VP243 synthesized by the TNT system. Plasmids pCI-wtVP243 and pCI-VP243-∆MA were used for in vitro translation to produce polyprotein VP243, and empty plasmid pCI-neo was used as negative control (CK). TNT products were subjected to Western blotting using anti-VP4 and anti-VP3 mAbs. No VP3 or VP4 protein bands were detected in the product of template pCI-VP243-∆MA. (b) Confocal analysis of DF-1 cells transfected with pCI-wtVP243 and pCI-VP243-∆MA. The transfected cells were fixed after 24 h, and incubated with a mixture of rabbit anti-VP3 antiserum and mouse anti-VP4 mAb followed by FITC-conjugated goat anti-mouse IgG and TRITC-conjugated goat anti-rabbit IgG. Nuclear DNA was stained with DAPI. (c) Substitution of protease activity site S140 or K180 of VP4 or VP4-C∆2 with alanine (A) failed to induce cell death in transfected DF-1 cells. DF-1 cells transfected with indicated mutants were directly observed under microscopy at 24 h following transfection. Empty plasmid pEGFP-C2 was used as control.
Mentions: A recent study using in vitro assay of E. Coli-derived VP4 showed that VP4 tubules have significantly lower endopeptidase than monomeric or dimeric VP418, which provides extracellular evidence that VP4 self-assembly potentially blocks VP4 protease activity. The proteolytic activity of proteases is important for inducing apoptotic cell death34, it raised our hypothesis that the apoptosis induced by intracellular VP4-C∆2 may relate to its protease activity. Firstly, we determine the proteolytic capacity of VP4-C∆2 in processing polyprotein VP243 using the TNT system. Although our anti-VP2 mAb unfortunately failed to react with VP2 protein in Western blots, protein bands with the expected sizes of VP4 (approximately 28 kDa) and VP3 (approximately 32 kDa) were detected in the product of template pCI-wtVP243 (Fig. 8a), and no more bands with larger size were detected, indicating that VP4 and VP3 were successfully and completely released from wtVP243. Conversely, approximately 60 kDa of VP4-VP3 polyprotein bands were detected by both anti-VP4 and anti-VP3 mAbs in the product of the pCI-VP243-∆MA template, but no single VP4 or VP3 bands were individually detected (Fig. 8a), indicating that VP4-VP3 junction released from VP243-∆MA precursor was not successfully cleaved into VP4 and VP3. Consistent results were obtained in cells transfected with pCI-A and pCI-A-∆MA which contain the full-length A segment or A segment with MA deletion from the C-terminus of VP4. The results further revealed that VP4 in pCI-A-transfected cells was successfully released and assembled into structures (Fig. 8b), which were similar to the VP4 structures in pCI-wtVP4-transfected cells (Fig. 2d). However, VP4 in pCI-A-∆MA-transfected cells failed to assemble into specific structures (Fig. 8b), indicating MA deletion from VP4 C-terminus affects the VP4-VP3 cleavage, which was consistent with the destruction of cleavage motif (Thr/Ala)–X–Ala↓Ala motifs11. Overall, these data suggested that deletion of the C-terminal residues 242MA243 of VP4 maintains the cleavage activity which can efficiently cleave VP2–VP4 junctions but not VP4–VP3 junctions with destructive cleavage motif.

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