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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

Mutational effects of C-terminal amyloidogenic peptide on VP4 assembly.Each residue of the last C-terminal stretch 239HLAMA243 is randomly mutated into NNK degenerate codon, the mutation effects of individual mutants on self-assembly were examined at 12 h after transfecting DF-1 cells with indicated pEGFP plasmids.
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f4: Mutational effects of C-terminal amyloidogenic peptide on VP4 assembly.Each residue of the last C-terminal stretch 239HLAMA243 is randomly mutated into NNK degenerate codon, the mutation effects of individual mutants on self-assembly were examined at 12 h after transfecting DF-1 cells with indicated pEGFP plasmids.

Mentions: We attempted to predict the possibility of amyloidogenic peptides for VP4 protein using the online prediction tool which utilizes five independently published methods20. Consensus prediction of VP4 protein primary sequence revealed 8 potentially amyloidogenic peptides (aa 7–13, 36–40, 45–54, 67–71, 113–117, 143–151, 206–210 and 238–242). Importantly, C-terminal residues 238YHLAM242 of VP4 are highly predicted as a top hit for amyloid propensity based on two independent methods, including the average packing density value21 and conformational energy values22, and A243 is predicted as hit by conformational energy values. The consensus prediction of amyloidogenic regions 238YHLAMA243 is also consistent with the results of deletion mutation (Fig. 3d). To further define the property of C-terminal amino acids for VP4 assembly, each of C-terminal residues 239HLAMA243 was randomly substituted with degenerate codon NNK (N = A/C/T/G, K = G/T) and respectively subcloned into pEGFP-C2 vector. The resulting clones of VP4 random mutants were individually transfected into DF-1 cells to investigate how the mutations affect VP4 self-assembly (Fig. 4). Aromatic, hydrophobic and electrostatic interaction were known to play essential roles in the formation of amyloid fibrils2324. Our mutagenic analysis extensively confirmed that substitutions of the C-terminal residues with aromatic residues (M242Y, A241Y, L240Y and H239Y) or electrostatic residues (M242N, L240N, H239N, M242Q, L240Q and H239Q) maintain the capability of fibril assembly (Fig. 4). Proline (P) residue was generally known to have the helical-breaking and β-sheet-disrupting effects which will destabilize amyloid fibrils2526. As expected, substitutions of H239, A241, M242 or A243 with proline residue result in destruction of VP4 self-assembly (Fig. 4). Small amino acids, including alanine and glycine, were reported to be important for self-assembly, and thus commonly used as aggregation-prone system to study self-assembly27. As expected, substitution of 240LAMA243 with glycine (L240G, A241G, M242G and A243G) maintained the VP4 self-assembly (Fig. 4). Additionally, the substitutions of A241 and A243 with charged amino acids (A243H, A243R, A241H, A241K and A241D), some hydrophobic residues (A243I and A241L), or electrostatic residue (A241N) mostly abolish the VP4 assembly, while M242, L240 and H239 tolerate various substitutions except proline (Fig. 4). Although H239D mainly exists in very virulent IBDV strains, the VP4-H239D aggregates like wt-VP4, thus suggesting position 239 of VP4 may be associated with virus virulence via aggregation-independent mechanism (Fig. 4). In summary, these results indicate that C-terminal stretch of VP4 has the amyloidogenic properties which are dominated by the two aggregation-prone alanine residues, 241A and 243A.


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)

Mutational effects of C-terminal amyloidogenic peptide on VP4 assembly.Each residue of the last C-terminal stretch 239HLAMA243 is randomly mutated into NNK degenerate codon, the mutation effects of individual mutants on self-assembly were examined at 12 h after transfecting DF-1 cells with indicated pEGFP plasmids.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Mutational effects of C-terminal amyloidogenic peptide on VP4 assembly.Each residue of the last C-terminal stretch 239HLAMA243 is randomly mutated into NNK degenerate codon, the mutation effects of individual mutants on self-assembly were examined at 12 h after transfecting DF-1 cells with indicated pEGFP plasmids.
Mentions: We attempted to predict the possibility of amyloidogenic peptides for VP4 protein using the online prediction tool which utilizes five independently published methods20. Consensus prediction of VP4 protein primary sequence revealed 8 potentially amyloidogenic peptides (aa 7–13, 36–40, 45–54, 67–71, 113–117, 143–151, 206–210 and 238–242). Importantly, C-terminal residues 238YHLAM242 of VP4 are highly predicted as a top hit for amyloid propensity based on two independent methods, including the average packing density value21 and conformational energy values22, and A243 is predicted as hit by conformational energy values. The consensus prediction of amyloidogenic regions 238YHLAMA243 is also consistent with the results of deletion mutation (Fig. 3d). To further define the property of C-terminal amino acids for VP4 assembly, each of C-terminal residues 239HLAMA243 was randomly substituted with degenerate codon NNK (N = A/C/T/G, K = G/T) and respectively subcloned into pEGFP-C2 vector. The resulting clones of VP4 random mutants were individually transfected into DF-1 cells to investigate how the mutations affect VP4 self-assembly (Fig. 4). Aromatic, hydrophobic and electrostatic interaction were known to play essential roles in the formation of amyloid fibrils2324. Our mutagenic analysis extensively confirmed that substitutions of the C-terminal residues with aromatic residues (M242Y, A241Y, L240Y and H239Y) or electrostatic residues (M242N, L240N, H239N, M242Q, L240Q and H239Q) maintain the capability of fibril assembly (Fig. 4). Proline (P) residue was generally known to have the helical-breaking and β-sheet-disrupting effects which will destabilize amyloid fibrils2526. As expected, substitutions of H239, A241, M242 or A243 with proline residue result in destruction of VP4 self-assembly (Fig. 4). Small amino acids, including alanine and glycine, were reported to be important for self-assembly, and thus commonly used as aggregation-prone system to study self-assembly27. As expected, substitution of 240LAMA243 with glycine (L240G, A241G, M242G and A243G) maintained the VP4 self-assembly (Fig. 4). Additionally, the substitutions of A241 and A243 with charged amino acids (A243H, A243R, A241H, A241K and A241D), some hydrophobic residues (A243I and A241L), or electrostatic residue (A241N) mostly abolish the VP4 assembly, while M242, L240 and H239 tolerate various substitutions except proline (Fig. 4). Although H239D mainly exists in very virulent IBDV strains, the VP4-H239D aggregates like wt-VP4, thus suggesting position 239 of VP4 may be associated with virus virulence via aggregation-independent mechanism (Fig. 4). In summary, these results indicate that C-terminal stretch of VP4 has the amyloidogenic properties which are dominated by the two aggregation-prone alanine residues, 241A and 243A.

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