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Identification of fragments from Autographa californica polyhedrin protein essential for self-aggregation and exogenous protein incorporation.

ABSTRACT: Baculoviruses are widely used for the production of recombinant proteins, biopesticides and as gene delivery systems. One of the viral forms called polyhedra has been recently exploited as a scaffold system to incorporate or encapsulate foreign proteins or peptide fragments. However, an efficient strategy for foreign protein incorporation has not been thoroughly studied.Based on the crystal structure of polyhedrin, we conducted an in silico analysis of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) polyhedrin protein to select the minimum fragments of polyhedrin that could be incorporated into polyhedra. Using confocal and transmission electron microscopy we analyzed the expression and cellular localization of the different polyhedrin fragments fused to the green fluorescent protein (EGFP) used as reporter. The amino fragment 1-110 contains two repeats formed each of two ? sheets followed by a ? helix (amino acids 1-58 and 58-110) that are important for the formation and stability of polyhedra. These fragments 1-58, 58-110 and 1-110 could be incorporated into polyhedra. However, only fragments 1-110 and 58-110 can self-aggregate.These results demonstrate that 58-110 is the minimum fragment that contributes to the assembly of the recombinant polyhedra via self-aggregation. This is the minimum sequence that can be used to efficiently incorporate foreign proteins into polyhedra.

SUBMITTER: Sampieri A

PROVIDER: S-EPMC4320575 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Identification of fragments from Autographa californica polyhedrin protein essential for self-aggregation and exogenous protein incorporation.

Sampieri Alicia A Luz-Madrigal Agustín A Zepeda Jesus J Vaca Luis L

BMC biochemistry 20150204

<h4>Background</h4>Baculoviruses are widely used for the production of recombinant proteins, biopesticides and as gene delivery systems. One of the viral forms called polyhedra has been recently exploited as a scaffold system to incorporate or encapsulate foreign proteins or peptide fragments. However, an efficient strategy for foreign protein incorporation has not been thoroughly studied.<h4>Results</h4>Based on the crystal structure of polyhedrin, we conducted an in silico analysis of the bacu ...[more]

PMID: 25648249

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Project description:Baculoviruses encode a variety of auxiliary proteins that are not essential for viral replication but provide them with a selective advantage in nature. P10 is a 10-kDa auxiliary protein produced in the very late phase of gene transcription by Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The P10 protein forms cytoskeleton-like structures in the host cell that associate with microtubules varying from filamentous forms in the cytoplasm to aggregated perinuclear tubules that form a cage-like structure around the nucleus. These P10 structures may have a role in the release of occlusion bodies (OBs) and thus mediate the horizontal transmission of the virus between insect hosts. Here, using mass spectrometric analysis, it is demonstrated that the C terminus of P10 is phosphorylated during virus infection of cells in culture. Analysis of P10 mutants encoded by recombinant baculoviruses in which putative phosphorylation residues were mutated to alanine showed that serine 93 is a site of phosphorylation. Confocal microscopy examination of the serine 93 mutant structures revealed aberrant formation of the perinuclear tubules. Thus, the phosphorylation of serine 93 may induce the aggregation of filaments to form tubules. Together, these data suggest that the phosphorylation of serine 93 affects the structural conformation of P10.IMPORTANCE The baculovirus P10 protein has been researched intensively since it was first observed in 1969, but its role during viral infection remains unclear. It is conserved in the alphabaculoviruses and expressed at high levels during virus infection. Producing large amounts of a protein is wasteful for the virus unless it is advantageous for the survival of its progeny, and therefore, P10 presents an enigma. As P10 polymerizes to form organized cytoskeletal structures that colocalize with host cell microtubules, the structural relationship of the protein with the host cell may present a key to help understand the function and importance of this protein. This study addresses the importance of the structural changes in P10 during infection and how they may be governed by phosphorylation. The P10 structures affected by phosphorylation are closely associated with the viral progeny and thus may potentially be responsible for its dissemination and survival.

Dataset Information

Identification of fragments from Autographa californica polyhedrin protein essential for self-aggregation and exogenous protein incorporation.

Publications

Identification of fragments from Autographa californica polyhedrin protein essential for self-aggregation and exogenous protein incorporation.

Similar Datasets

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