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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

Assembly greatly decreases the solubility of VP4 protein.Plasmids pEGFP-wtVP4, pEGFP-VP4-C∆1, pEGFP-VP4-C∆1′, pEGFP-VP4-C∆2 and pEGFP-VP4-C∆3 were transfected into DF-1 cells, and IBDV-infected cells were used as a control. Cells were lysed with TX-100-containing lysis buffer at 24 h after transfection or infection. The TX-100-insoluble and TX-100-soluble fractions were analyzed by 12% SDS-PAGE and Western blotting using anti-VP4 mAb. (a) Volume quantification of VP4 bands was detected using Quantity One software (N = 2). (b) Western blots of bands corresponding to VP4. Soluble VP4 protein was increased in DF-1 cells transfected with pEGFP-VP4-C∆2 and EGFP-VP4-C∆3.
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f5: Assembly greatly decreases the solubility of VP4 protein.Plasmids pEGFP-wtVP4, pEGFP-VP4-C∆1, pEGFP-VP4-C∆1′, pEGFP-VP4-C∆2 and pEGFP-VP4-C∆3 were transfected into DF-1 cells, and IBDV-infected cells were used as a control. Cells were lysed with TX-100-containing lysis buffer at 24 h after transfection or infection. The TX-100-insoluble and TX-100-soluble fractions were analyzed by 12% SDS-PAGE and Western blotting using anti-VP4 mAb. (a) Volume quantification of VP4 bands was detected using Quantity One software (N = 2). (b) Western blots of bands corresponding to VP4. Soluble VP4 protein was increased in DF-1 cells transfected with pEGFP-VP4-C∆2 and EGFP-VP4-C∆3.

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)

Assembly greatly decreases the solubility of VP4 protein.Plasmids pEGFP-wtVP4, pEGFP-VP4-C∆1, pEGFP-VP4-C∆1′, pEGFP-VP4-C∆2 and pEGFP-VP4-C∆3 were transfected into DF-1 cells, and IBDV-infected cells were used as a control. Cells were lysed with TX-100-containing lysis buffer at 24 h after transfection or infection. The TX-100-insoluble and TX-100-soluble fractions were analyzed by 12% SDS-PAGE and Western blotting using anti-VP4 mAb. (a) Volume quantification of VP4 bands was detected using Quantity One software (N = 2). (b) Western blots of bands corresponding to VP4. Soluble VP4 protein was increased in DF-1 cells transfected with pEGFP-VP4-C∆2 and EGFP-VP4-C∆3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Assembly greatly decreases the solubility of VP4 protein.Plasmids pEGFP-wtVP4, pEGFP-VP4-C∆1, pEGFP-VP4-C∆1′, pEGFP-VP4-C∆2 and pEGFP-VP4-C∆3 were transfected into DF-1 cells, and IBDV-infected cells were used as a control. Cells were lysed with TX-100-containing lysis buffer at 24 h after transfection or infection. The TX-100-insoluble and TX-100-soluble fractions were analyzed by 12% SDS-PAGE and Western blotting using anti-VP4 mAb. (a) Volume quantification of VP4 bands was detected using Quantity One software (N = 2). (b) Western blots of bands corresponding to VP4. Soluble VP4 protein was increased in DF-1 cells transfected with pEGFP-VP4-C∆2 and EGFP-VP4-C∆3.
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