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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

Intracellular assembly of VP4 does not involve other IBDV-encoded proteins or subcellular organelles.(a) No co-localization of VP4 and IBDV-encoded VP1, VP2 VP3 or VP5 was detected in IBDV-infected cells at 24 hpi. VP1, VP2, VP3 and VP5 in IBDV-infected DF-1 cells were detected with the corresponding mouse mAbs followed by TRITC-conjugated anti-mouse IgG (Red). VP4 was detected by rabbit anti-VP4 antibody followed by FITC-conjugated anti-rabbit IgG (Green). (b) Dot-like VP4 signal partially (white arrow) co-localize with VP2 at 24 hpi in some of IBDV-infected cells. VP2 was detected with mouse anti-VP2 mAb followed by FITC-conjugated anti-mouse IgG (Green). VP4 was detected by rabbit anti-VP4 antibody followed by TRITC-conjugated anti-rabbit IgG (Red). (c) Subcellular relationship of VP4 with subcellular organelles in IBDV-infected cells (MOI = 1) which were pre-transfected with living colors subcellular localization vectors pDsRed2-ER, pDsRed2-Mito, pDsRed2-Peroxi, pEGFP-actin, pEYFP-Golgi or pEYFP-Mem. Cells were fixed and stained with mouse anti-VP4 mAb 24 hpi and FITC/TRITC-conjugated goat anti-mouse IgG. (d) Expression kinetics of VP4 in pCI-wtVP4-transfected DF-1 cells. VP4 was stained at 6, 12 and 24 hpi with anti-VP4 mAb followed by FITC-conjugated anti-mouse IgG. (e) Expression kinetics of EGFP-VP4 protein in pEGFP-wtVP4-transfected DF-1 cells. Rod-shaped and mass-like structures of EGFP-VP4 protein were present in the cytoplasm and nucleus at 6, 12 and 24 h after transfection. Nuclei were stained with DAPI.
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f2: Intracellular assembly of VP4 does not involve other IBDV-encoded proteins or subcellular organelles.(a) No co-localization of VP4 and IBDV-encoded VP1, VP2 VP3 or VP5 was detected in IBDV-infected cells at 24 hpi. VP1, VP2, VP3 and VP5 in IBDV-infected DF-1 cells were detected with the corresponding mouse mAbs followed by TRITC-conjugated anti-mouse IgG (Red). VP4 was detected by rabbit anti-VP4 antibody followed by FITC-conjugated anti-rabbit IgG (Green). (b) Dot-like VP4 signal partially (white arrow) co-localize with VP2 at 24 hpi in some of IBDV-infected cells. VP2 was detected with mouse anti-VP2 mAb followed by FITC-conjugated anti-mouse IgG (Green). VP4 was detected by rabbit anti-VP4 antibody followed by TRITC-conjugated anti-rabbit IgG (Red). (c) Subcellular relationship of VP4 with subcellular organelles in IBDV-infected cells (MOI = 1) which were pre-transfected with living colors subcellular localization vectors pDsRed2-ER, pDsRed2-Mito, pDsRed2-Peroxi, pEGFP-actin, pEYFP-Golgi or pEYFP-Mem. Cells were fixed and stained with mouse anti-VP4 mAb 24 hpi and FITC/TRITC-conjugated goat anti-mouse IgG. (d) Expression kinetics of VP4 in pCI-wtVP4-transfected DF-1 cells. VP4 was stained at 6, 12 and 24 hpi with anti-VP4 mAb followed by FITC-conjugated anti-mouse IgG. (e) Expression kinetics of EGFP-VP4 protein in pEGFP-wtVP4-transfected DF-1 cells. Rod-shaped and mass-like structures of EGFP-VP4 protein were present in the cytoplasm and nucleus at 6, 12 and 24 h after transfection. Nuclei were stained with DAPI.

Mentions: His-tagged VP4 proteins expressed in E. coli can self-assemble into tubule-like structures18 which are very similar to tubules found in IBDV-infected cells17. However, it is unclear if other viral proteins or intracellular factors are involved in the VP4 assembly during IBDV replication. Firstly, we examined the subcellular relationship of tubule-like VP4 structures with other IBDV-encoded viral proteins in infected DF-1 cells by dual-staining IFAs. Co-localization analysis showed that the needle-like VP4 structures did not overlap with viral proteins VP1, VP2, VP3 or VP5 (Fig. 2a), only dot-like VP4 structures co-localize with the VP2 at 24 hpi (Fig. 2b). To further analyze the relationship of VP4 structures with subcellular organelles, DF-1 cells were infected with IBDV after individually transfected with the living colors subcellular localization vectors pDsRed2-ER, pDsRed2-Mito, pEGFP-actin, pDsRed2-Peroxi, pEYFP-Golgi or pEYFP-Mem. Confocal microscopy of VP4-stained cells revealed no co-localization of VP4 with host endoplasmic reticulum (ER), mitochondria (Mito), actin, peroxisomes (Peroxi), Golgi apparatus (Golgi) or membrane (Mem) (Fig. 2c), indicating that VP4 assembly is not directly associated with other viral proteins or host organelles. To further investigate whether intracellular VP4 protein alone can form these unique structures, VP4 expression in DF-1 cells transfected with pEGFP-VP4 or pCI-VP4 was detected at 6, 12 and 24 hours post transfection (hpt). Confocal microscopy showed that wild-type VP4 (wtVP4) proteins with or without an enhanced green fluorescent protein (EGFP) tag at the N terminus can assemble within the cytoplasm and nucleus of transfected cells. Specially, VP4 assembly was initiated before 6 h, both wtVP4 (Fig. 2d) and EGFP-wtVP4 (Fig. 2e) gradually assembled from small granules into filamentous or mass-like structures. Under a living-cell confocal microscope, the intracellular EGFP-wtVP4 fusion protein was dynamically observed to gradually aggregate within the cytoplasm and nucleus, and coalesce into larger mass-like structures (Supplementary Video S1). The dynamic expression of EGFP-VP4 or wtVP4 in transfected cells (Fig. 2d,e, Supplementary Video S1) was similar to those in IBDV-infected cells (Fig. 1b). These results indicate that intracellular VP4 can self-assemble into unique structures within the cytoplasm and nucleus which is independent of IBDV-encoded viral proteins and host organelles.


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)

Intracellular assembly of VP4 does not involve other IBDV-encoded proteins or subcellular organelles.(a) No co-localization of VP4 and IBDV-encoded VP1, VP2 VP3 or VP5 was detected in IBDV-infected cells at 24 hpi. VP1, VP2, VP3 and VP5 in IBDV-infected DF-1 cells were detected with the corresponding mouse mAbs followed by TRITC-conjugated anti-mouse IgG (Red). VP4 was detected by rabbit anti-VP4 antibody followed by FITC-conjugated anti-rabbit IgG (Green). (b) Dot-like VP4 signal partially (white arrow) co-localize with VP2 at 24 hpi in some of IBDV-infected cells. VP2 was detected with mouse anti-VP2 mAb followed by FITC-conjugated anti-mouse IgG (Green). VP4 was detected by rabbit anti-VP4 antibody followed by TRITC-conjugated anti-rabbit IgG (Red). (c) Subcellular relationship of VP4 with subcellular organelles in IBDV-infected cells (MOI = 1) which were pre-transfected with living colors subcellular localization vectors pDsRed2-ER, pDsRed2-Mito, pDsRed2-Peroxi, pEGFP-actin, pEYFP-Golgi or pEYFP-Mem. Cells were fixed and stained with mouse anti-VP4 mAb 24 hpi and FITC/TRITC-conjugated goat anti-mouse IgG. (d) Expression kinetics of VP4 in pCI-wtVP4-transfected DF-1 cells. VP4 was stained at 6, 12 and 24 hpi with anti-VP4 mAb followed by FITC-conjugated anti-mouse IgG. (e) Expression kinetics of EGFP-VP4 protein in pEGFP-wtVP4-transfected DF-1 cells. Rod-shaped and mass-like structures of EGFP-VP4 protein were present in the cytoplasm and nucleus at 6, 12 and 24 h after transfection. Nuclei were stained with DAPI.
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f2: Intracellular assembly of VP4 does not involve other IBDV-encoded proteins or subcellular organelles.(a) No co-localization of VP4 and IBDV-encoded VP1, VP2 VP3 or VP5 was detected in IBDV-infected cells at 24 hpi. VP1, VP2, VP3 and VP5 in IBDV-infected DF-1 cells were detected with the corresponding mouse mAbs followed by TRITC-conjugated anti-mouse IgG (Red). VP4 was detected by rabbit anti-VP4 antibody followed by FITC-conjugated anti-rabbit IgG (Green). (b) Dot-like VP4 signal partially (white arrow) co-localize with VP2 at 24 hpi in some of IBDV-infected cells. VP2 was detected with mouse anti-VP2 mAb followed by FITC-conjugated anti-mouse IgG (Green). VP4 was detected by rabbit anti-VP4 antibody followed by TRITC-conjugated anti-rabbit IgG (Red). (c) Subcellular relationship of VP4 with subcellular organelles in IBDV-infected cells (MOI = 1) which were pre-transfected with living colors subcellular localization vectors pDsRed2-ER, pDsRed2-Mito, pDsRed2-Peroxi, pEGFP-actin, pEYFP-Golgi or pEYFP-Mem. Cells were fixed and stained with mouse anti-VP4 mAb 24 hpi and FITC/TRITC-conjugated goat anti-mouse IgG. (d) Expression kinetics of VP4 in pCI-wtVP4-transfected DF-1 cells. VP4 was stained at 6, 12 and 24 hpi with anti-VP4 mAb followed by FITC-conjugated anti-mouse IgG. (e) Expression kinetics of EGFP-VP4 protein in pEGFP-wtVP4-transfected DF-1 cells. Rod-shaped and mass-like structures of EGFP-VP4 protein were present in the cytoplasm and nucleus at 6, 12 and 24 h after transfection. Nuclei were stained with DAPI.
Mentions: His-tagged VP4 proteins expressed in E. coli can self-assemble into tubule-like structures18 which are very similar to tubules found in IBDV-infected cells17. However, it is unclear if other viral proteins or intracellular factors are involved in the VP4 assembly during IBDV replication. Firstly, we examined the subcellular relationship of tubule-like VP4 structures with other IBDV-encoded viral proteins in infected DF-1 cells by dual-staining IFAs. Co-localization analysis showed that the needle-like VP4 structures did not overlap with viral proteins VP1, VP2, VP3 or VP5 (Fig. 2a), only dot-like VP4 structures co-localize with the VP2 at 24 hpi (Fig. 2b). To further analyze the relationship of VP4 structures with subcellular organelles, DF-1 cells were infected with IBDV after individually transfected with the living colors subcellular localization vectors pDsRed2-ER, pDsRed2-Mito, pEGFP-actin, pDsRed2-Peroxi, pEYFP-Golgi or pEYFP-Mem. Confocal microscopy of VP4-stained cells revealed no co-localization of VP4 with host endoplasmic reticulum (ER), mitochondria (Mito), actin, peroxisomes (Peroxi), Golgi apparatus (Golgi) or membrane (Mem) (Fig. 2c), indicating that VP4 assembly is not directly associated with other viral proteins or host organelles. To further investigate whether intracellular VP4 protein alone can form these unique structures, VP4 expression in DF-1 cells transfected with pEGFP-VP4 or pCI-VP4 was detected at 6, 12 and 24 hours post transfection (hpt). Confocal microscopy showed that wild-type VP4 (wtVP4) proteins with or without an enhanced green fluorescent protein (EGFP) tag at the N terminus can assemble within the cytoplasm and nucleus of transfected cells. Specially, VP4 assembly was initiated before 6 h, both wtVP4 (Fig. 2d) and EGFP-wtVP4 (Fig. 2e) gradually assembled from small granules into filamentous or mass-like structures. Under a living-cell confocal microscope, the intracellular EGFP-wtVP4 fusion protein was dynamically observed to gradually aggregate within the cytoplasm and nucleus, and coalesce into larger mass-like structures (Supplementary Video S1). The dynamic expression of EGFP-VP4 or wtVP4 in transfected cells (Fig. 2d,e, Supplementary Video S1) was similar to those in IBDV-infected cells (Fig. 1b). These results indicate that intracellular VP4 can self-assemble into unique structures within the cytoplasm and nucleus which is independent of IBDV-encoded viral proteins and host organelles.

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