Cell-line-dependent crystal morphology and sublocalization of the Thyrinteina arnobia cypovirus polyhedrin expressed from a recombinant baculovirus.

We describe an unexpected feature observed for the heterologous expression of the Thyrinteina arnobia cypovirus polyhedrin from a recombinant baculovirus infection in different insect cell lines. The in cellulo-formed crystals varied in size and shape depending on the cell line. Crystals formed in Trichoplusia ni-derived cells were cubic (0.1-2 µm) and localized in both the nucleus and cytoplasm, whereas those formed in Spodoptera frugiperda-derived cells were ovate and ellipsoidal (0.1-3 µm) and also localized in both the nucleus and cytoplasm. The molecular basis for differences in the morphology, size, and location of cypovirus occlusion bodies is unclear, and cellular proteins might play a role in their formation and location.

Biotechnological applications of occlusion bodies of Baculoviruses.

The ability of Baculoviruses to hyper-express very late genes as polyhedrin, the major component of occlusion bodies (OBs) or polyhedra, has allowed the evolution of a system of great utility for biotechnology. The main function of polyhedra in nature is to protect Baculovirus in the environment. The possibility of incorporating foreign proteins into the crystal by fusing them to polyhedrin (POLH) opened novel potential biotechnological uses. In this review, we summarize different applications of Baculovirus chimeric OBs. Basically, the improvement of protein expression and purification with POLH as a fusion partner; the use of recombinant polyhedra as immunogens and antigens, and the incorporation of proteins into polyhedra to improve Baculoviruses as bioinsecticides. The results obtained in each area and the future trends in these topics are also discussed.

Codon Optimization-based Whole-gene Scanning Identifies Hidden Nucleotides Essential for Bombyx mori Nucleopolyhedrovirus polyhedrin Hyperexpression.

During the late stage of infection, alphabaculoviruses produce many occlusion bodies (OBs) in the nuclei of the insect host's cells through the hyperexpression of polyhedrin (POLH), a major OB component encoded by polh. The strong polh promoter has been used to develop a baculovirus expression vector system for recombinant protein expression in cultured insect cells and larvae. However, the relationship between POLH accumulation and the polh coding sequence remains largely unelucidated. This study aimed to assess the importance of polh codon usage and/or nucleotide sequences in POLH accumulation by generating a baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) expressing mutant polh (co-polh) optimized according to the codon preference of its host insect. Although the deduced amino acid sequence of CO-POLH was the same as that of wild-type POLH, POLH accumulation was significantly lower in cells infected with the co-polh mutant. This reduction was due to decreased polh mRNA levels rather than translational repression. Analysis of mutant viruses with chimeric polh revealed that a 30 base-pair (bp) 5' proximal polh coding region was necessary for maintaining high polh mRNA levels. Sequence comparison of wild-type polh and co-polh identified five nucleotide differences in this region, indicating that these nucleotides were critical for polh hyperexpression. Furthermore, luciferase reporter assays showed that the 30 bp 5' coding region was sufficient for maintaining the polh promoter-driven high level of polh mRNA. Thus, our whole-gene scanning by codon optimization identified important hidden nucleotides for polh hyperexpression in alphabaculoviruses.

Molecular mechanism responsible for the hyperexpression of baculovirus polyhedrin.

One of the amazing phenomena in the baculovirus life cycle is the hyperexpression of the very late gene, polyhedrin (polh), causing the production of the occlusion bodies where progeny virions are embedded. However, to date, the molecular mechanism underlying its hyperexpression is not completely elucidated. Considering that, in this review, the mechanism responsible for its hyperexpression from the previous studies up to now was comprehensively summarized from three aspects, namely, the structure characteristics of the polh promoter and transcription regulation, the structure and translation regulation of the polh mRNA, and especially the regulators that influence the expression of polh gene. Moreover, this review will help us obtain a better understanding about the hyperexpression of polh, and also provide guidance for improving the expression efficiency of the foreign proteins by adopting the baculovirus expression vector system.

Actin Contributes to the Hyperexpression of Baculovirus Polyhedrin (polh) and p10 as a Component of Transcription Initiation Complex (TIC).

Hyperexpression of polh and p10, two very late genes, is one of the remarkable characteristics in the baculovirus life cycle. However, the mechanisms underlying the hyperexpression of these two genes are still incompletely understood. In this study, actin was identified as a highly potential binding partner of polh and p10 promoters by conducting DNA pull-down and LC-MS/MS analyses. Inhibiting actin dynamics delayed and decreased the transcription of polh and p10. Actin interacted with viral RNA polymerase and transcription regulators, and the nuclear import of viral polymerase was inhibited with the disruption of actin dynamics. Simultaneously, the high enrichment of actin in polh and p10 promoters discovered via a chromatin immunoprecipitation (ChIP) assay indicated that actin was a component of the viral polymerase TIC. Moreover, overexpression of actin surprisingly upregulated the expression of luciferase (Luc) under the control of polh and p10 promoters. Taken together, actin participated in the hyperexpression of polh and p10 as a component of TIC. These results facilitate the promotion of the expression efficiency of foreign genes in the baculovirus expression vector system (BEVS).

A simplified strategy to package foreign proteins into baculovirus occlusion bodies without engineering the viral genome.

Polyhedron envelope protein (PEP) is the major component of the calyx that surrounds the baculovirus occlusion body (OB). PEP has been associated with the stabilization and resistance of polyhedra in the environment. Due to the abundant levels of PEP in OBs, we decided to use this protein as a fusion partner to redirect foreign proteins to baculovirus polyhedra. In this study we developed a strategy that involves the generation of a monoclonal transformed insect cell line expressing a protein of interest fused to the the Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) N-terminus of PEP that enables the packaging of foreign proteins into the OBs without generating a recombinant baculovirus. This proved to be an efficient platform that could be exploited to improve wild type baculovirus for their use as bioinsecticides without facing the concerns of releasing genetically modified DNA to the environment and bypassing the associated regulatory issues. We demonstrated, using immunological, proteomic and microscopy techniques, that the envelope of AgMNPV OBs can effectively trap chimeric proteins in an infected insect cell line expressing AgMNPV PEP fused to the enhanced green fluorescent protein (eGFP). Furthermore, packaging of chimeric PEP also took place with heterologous OBs such as those of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), another group I alphabaculovirus.

Major capsid protein of Autographa californica multiple nucleopolyhedrovirus contributes to the promoter activity of the very late viral genes.

The baculovirus expression vector system (BEVS) is one of the most powerful eukaryotic expression systems. Recombinant protein expression is usually controlled by promoters of the baculovirus very late genes (i.e., polyhedrin and p10); therefore, identifying novel regulatory factors for these promoters is key to increasing BEVS productivity. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the viral vector most frequently used in BEVS. VP39 is the major nucleocapsid protein of AcMNPV and plays a pivotal role in nucleocapsid assembly in the nucleus. In this study, we found that knocking out vp39 from the AcMNPV genome resulted in decreased protein abundance of polyhedrin and P10. Further assays revealed that the mRNA transcripts and the promoter activities of polyhedrin and p10 were decreased in the absence of vp39, suggesting that VP39 contributes to the activity of the very late viral gene promoters and may represent a means of optimizing the current BEVS.

Sustained Neurotrophin Release from Protein Nanoparticles Mediated by Matrix Metalloproteinases Induces the Alignment and Differentiation of Nerve Cells.

The spatial and temporal availability of cytokines, and the microenvironments this creates, is critical to tissue development and homeostasis. Creating concentration gradients in vitro using soluble proteins is challenging as they do not provide a self-sustainable source. To mimic the sustained cytokine secretion seen in vivo from the extracellular matrix (ECM), we encapsulated a cargo protein into insect virus-derived proteins to form nanoparticle co-crystals and studied the release of this cargo protein mediated by matrix metalloproteinase-2 (MMP-2) and MMP-8. Specifically, when nerve growth factor (NGF), a neurotrophin, was encapsulated into nanoparticles, its release was promoted by MMPs secreted by a PC12 neuronal cell line. When these NGF nanoparticles were spotted onto a cover slip to create a uniform circular field, movement and alignment of PC12 cells via their extended axons along the periphery of the NGF nanoparticle field was observed. Neural cell differentiation was confirmed by the expression of specific markers of tau, neurofilament, and GAP-43. Connections between the extended axons and the growth cones were also observed, and expression of connexin 43 was consistent with the formation of gap junctions. Extensions and connection of very fine filopodia occurred between growth cones. Our studies indicate that crystalline protein nanoparticles can be utilized to generate a highly stable cytokine gradient microenvironment that regulates the alignment and differentiation of nerve cells. This technique greatly simplifies the creation of protein concentration gradients and may lead to therapies for neuronal injuries and disease.

Protein composition analysis of polyhedra matrix of Bombyx mori nucleopolyhedrovirus (BmNPV) showed powerful capacity of polyhedra to encapsulate foreign proteins.

Polyhedra can encapsulate other proteins and have potential applications as protein stabilizers. The extremely stable polyhedra matrix may provide a platform for future engineered micro-crystal devices. However, the protein composition of the polyhedra matrix remains largely unknown. In this study, the occlusion-derived virus (ODV)-removed BmNPV polyhedra matrix fraction was subjected to SDS-PAGE and then an LC-ESI-MS/MS analysis using a Thermo Scientific Q Exactive mass spectrometer. In total, 28 host and 91 viral proteins were identified. The host components were grouped into one of six categories, i.e., chaperones, ubiquitin and related proteins, host helicases, cytoskeleton-related proteins, RNA-binding proteins and others, according to their predicted Pfam domain(s). Most viral proteins may not be essential for polyhedra assembly, as evidenced by studies in the literature showing that polyhedra formation occurs in the nucleus upon the disruption of individual genes. The structural role of these proteins in baculovirus replication will be of significant interest in future studies. The immobilization of enhanced green fluorescent protein (eGFP) into the polyhedra by fusing with the C-terminus of BM134 that is encoded by open reading frame (ORF) 134 suggested that the polyhedra had a powerful capacity to trap foreign proteins, and BM134 was a potential carrier for incorporating proteins of interest into the polyhedra.