Masaaki Kawano

Engineering of SV40-based nano-capsules for delivery of heterologous proteins as fusions with the minor capsid proteins VP2/3.

The capsid of SV40 is regarded as a potential nano-capsule for delivery of biologically active materials. The SV40 capsid is composed of 72 pentamers of the VP1 major capsid protein and 72 copies of the minor coat proteins VP2/3. We have previously demonstrated that, when expressed in insect Sf9 cells by the baculovirus system, VP1 self-assembles into virus-like particles (VP1-VLPs), which are morphologically indistinguishable from the SV40 virion and can be easily purified. Here, we show that heterologous proteins fused to VP2/3 can be efficiently incorporated into the VP1-VLPs. Using EGFP as a model protein, we have optimized this encapsulation system and found that fusion to the C-terminus of VP2/3 is preferable and that the C-terminal VP1-interaction domain of VP2/3 is sufficient for incorporation into VLPs. The VLPs encapsulating EGFP retain the ability to attach to the cell surface and enter the cells. Using this system, we have encapsulated yeast cytosine deaminase (yCD), a prodrug-modifying enzyme that converts 5-fluorocytosine to 5-fluorouracil, into VLPs. When CV-1 cells are challenged by the yCD-encapsulating VLPs, they become sensitive to 5-fluorocytosine-induced cell death. Therefore, proteins of interest can be encapsulated in VP1-VLPs by fusion to VP2/3 and successfully delivered to cells.

Importance of Vp1 Calcium-Binding Residues in Assembly, Cell Entry, and Nuclear Entry of Simian Virus 40

ABSTRACT For polyomaviruses, calcium ions are known to be essential for virion integrity and for the assembly of capsid structures. To define the role of calcium ions in the life cycle of the virus, we analyzed simian virus 40 (SV40) mutants in which structurally deduced calcium-binding amino acids of Vp1 were mutated singly and in combination. Our study provides evidence that calcium ions mediate not only virion assembly but also the initial infection processes of cell entry and nuclear entry. Mutations at Glu48, Glu157, Glu160, Glu216, and/or Glu330 are correlated with different extents of packaging defects. The low packaging ability of mutant E216R suggests the need to position the Glu216 side chain for proper virion formation. All other mutants selected for further analysis produced virus-like particles (VLPs) but were poorly infectious. The VLPs of mutant E330K could not attach to or enter the cell, and mutant E157A-E160A and E216K VLPs entered the cell but failed to enter the nucleus, apparently as a result of premature VLP dissociation. Our results show that five of the seven acidic side chains at the two calcium-binding sites—Glu48 and Glu330 (site 1), Glu157 and Glu160 (site 2), and Glu216 (both sites)—are important for SV40 infection. We propose that calcium coordination imparts not only stability but also structural flexibility to the virion, allowing the acquisition or loss of the ion at the two sites to control virion formation in the nucleus, as well as virion structural alterations at the cell surface and in the cytoplasm early during infection.

Structural Evidence of Glycoprotein Assembly in Cellular Membrane Compartments prior to Alphavirus Budding

ABSTRACT Membrane glycoproteins of alphavirus play a critical role in the assembly and budding of progeny virions. However, knowledge regarding transport of viral glycoproteins to the plasma membrane is obscure. In this study, we investigated the role of cytopathic vacuole type II (CPV-II) through in situ electron tomography of alphavirus-infected cells. The results revealed that CPV-II contains viral glycoproteins arranged in helical tubular arrays resembling the basic organization of glycoprotein trimers on the envelope of the mature virions. The location of CPV-II adjacent to the site of viral budding suggests a model for the transport of structural components to the site of budding. Thus, the structural characteristics of CPV-II can be used in evaluating the design of a packaging cell line for replicon production.

Presentation of functional foreign peptides on the surface of SV40 virus-like particles.

Viral capsids of simian virus 40 (SV40) are highly efficient gene delivery vehicles that infect a broad range of cells and tissues. To develop a controlled, cell type-specific delivery system, we sought to display foreign peptides on the capsid surface by genetically manipulating the major capsid protein Vp1. Here we report the identification of two sites within the surface loops of Vp1 that can accommodate foreign peptides in such a way that the foreign peptides are displayed on the surface of the virus-like particles (VLPs) without interfering with VLP assembly or the packaging of viral DNA. Insertion of Flag-tags but not RGD integrin-binding motifs at these sites strongly inhibited cell attachment of VLPs, which normally associate with host cells through cell surface molecules such as major histocompatibility complex (MHC) class I and ganglioside GM1. Instead, VLPs carrying the RGD motifs bound to integrin in vitro and to the cell surface in an RGD-dependent manner. Thus, insertion of foreign sequences into the surface loops of Vp1 can reduce natural virus-cell interactions and even confer an ability to bind to a new target receptor. This study demonstrates the potential usefulness of this strategy for the development of novel delivery vehicles with different cell tropisms.

The VP2/VP3 Minor Capsid Protein of Simian Virus 40 Promotes the in Vitro Assembly of the Major Capsid Protein VP1 into Particles *

The SV40 capsid is composed primarily of 72 pentamers of the VP1 major capsid protein. Although the capsid also contains the minor capsid protein VP2 and its amino-terminally truncated form VP3, their roles in capsid assembly remain unknown. An in vitro assembly system was used to investigate the role of VP2 in the assembly of recombinant VP1 pentamers. Under physiological salt and pH conditions, VP1 alone remained dissociated, and at pH 5.0, it assembled into tubular structures. A stoichiometric amount of VP2 allowed the assembly of VP1 pentamers into spherical particles in a pH range of 7.0 to 4.0. Electron microscopy observation, sucrose gradient sedimentation analysis, and antibody accessibility tests showed that VP2 is incorporated into VP1 particles. The functional domains of VP2 important for VP1 binding and for enhancing VP1 assembly were further explored with a series of VP2 deletion mutants. VP3 also enhanced VP1 assembly, and a region common to VP2 and VP3 (amino acids 119-272) was required to promote VP1 pentamer assembly. These results are relevant for controlling recombinant capsid formation in vitro, which is potentially useful for the in vitro development of SV40 virus vectors.

Calcium Bridge Triggers Capsid Disassembly in the Cell Entry Process of Simian Virus 40

The calcium bridge between the pentamers of polyoma viruses maintains capsid metastability. It has been shown that viral infection is profoundly inhibited by the substitution of lysine for glutamate in one calcium-binding residue of the SV40 capsid protein, VP1. However, it is unclear how the calcium bridge affects SV40 infectivity. In this in vitro study, we analyzed the influence of host cell components on SV40 capsid stability. We used an SV40 mutant capsid (E330K) in which lysine had been substituted for glutamate 330 in protein VP1. The mutant capsid retained the ability to interact with the SV40 cellular receptor GM1, and the internalized mutant capsid accumulated in caveolin-1-mediated endocytic vesicles and was then translocated to the endoplasmic reticulum (ER) region. However, when placed in ER-rich microsome, the mutant capsid retained its spherical structure in contrast to the wild type, which disassembled. Structural analysis of the mutant capsid with cryo-electron microscopy and image reconstruction revealed altered pentamer coordination, possibly as a result of electrostatic interaction, although its overall structure resembled that of the wild type. These results indicate that the calcium ion serves as a trigger at the pentamer interface, which switches on capsid disassembly, and that the failure of the E330K mutant capsid to disassemble is attributable to an inadequate triggering system. Our data also indicate that calcium depletion-induced SV40 capsid disassembly may occur in the ER region and that this is essential for successful SV40 infection.

The structure of avian polyomavirus reveals variably sized capsids, non-conserved inter-capsomere interactions, and a possible location of the minor capsid protein VP4.

Avian polyomavirus (APV) causes a fatal, multi-organ disease among several bird species. Using cryogenic electron microscopy and other biochemical techniques, we investigated the structure of APV and compared it to that of mammalian polyomaviruses, particularly JC polyomavirus and simian virus 40. The structure of the pentameric major capsid protein (VP1) is mostly conserved; however, APV VP1 has a unique, truncated C-terminus that eliminates an intercapsomere-connecting β-hairpin observed in other polyomaviruses. We postulate that the terminal β-hairpin locks other polyomavirus capsids in a stable conformation and that absence of the hairpin leads to the observed capsid size variation in APV. Plug-like density features were observed at the base of the VP1 pentamers, consistent with the known location of minor capsid proteins VP2 and VP3. However, the plug density is more prominent in APV and may include VP4, a minor capsid protein unique to bird polyomaviruses.

Evidence that SV40 VP1-DNA interactions contribute to the assembly of 40-nm spherical viral particles.

The simian virus 40 (SV40) particle is mainly composed of the major capsid protein termed VP1. VP1 self‐assembles into virus‐like particles (VLPs) of approximately 40 nm in diameter when over‐expressed in bacteria or in insect cells, but purified VP1 does not form such a structure under physiological conditions, and thus, the mechanism of VP1 assembly is not well understood. Using a highly purified VP1 assembly/disassembly system in vitro, here we provide evidence that DNA is a factor that contributes to VP1 assembly into 40‐nm spherical particles. At pH 5, for example, VP1 preferentially assembles into 40‐nm particles in the presence of DNA, whereas VP1 assembles into tubular structures in the absence of DNA. Electron microscopic observations revealed that the concentration of DNA and its length are important for the formation of 40‐nm particles. In addition, sucrose gradient sedimentation analysis and DNase I‐sensitivity assays indicated that DNA of up to 2000 bp is packaged into the 40‐nm particles under the conditions examined. We propose that DNA may facilitate the formation of 40‐nm spherical particles by acting as a scaffold that increases the local concentration of VP1 and/or by acting as an allosteric effector that alters the structure of VP1.

Chimeric SV40 virus-like particles induce specific cytotoxicity and protective immunity against influenza A virus without the need of adjuvants

Virus-like particles (VLPs) are a promising vaccine platform due to the safety and efficiency. However, it is still unclear whether polyomavirus-based VLPs are useful for this purpose. Here, we attempted to evaluate the potential of polyomavirus VLPs for the antiviral vaccine using simian virus 40 (SV40). We constructed chimeric SV40-VLPs carrying an HLA-A*02:01-restricted, cytotoxic T lymphocyte (CTL) epitope derived from influenza A virus. HLA-A*02:01-transgenic mice were then immunized with the chimeric SV40-VLPs. The chimeric SV40-VLPs effectively induced influenza-specific CTLs and heterosubtypic protection against influenza A viruses without the need of adjuvants. Because DNase I treatment of the chimeric SV40-VLPs did not disrupt CTL induction, the intrinsic adjuvant property may not result from DNA contaminants in the VLP preparation. In addition, immunization with the chimeric SV40-VLPs generated long-lasting memory CTLs. We here propose that the chimeric SV40-VLPs harboring an epitope may be a promising CTL-based vaccine platform with self-adjuvant properties.

The route of immunization with adenoviral vaccine influences the recruitment of cytotoxic T lymphocytes in the lung that provide potent protection from influenza A virus

Virus-specific cytotoxic T lymphocytes (CTLs) in the lung are considered to confer protection from respiratory viruses. Several groups demonstrated that the route of priming was likely to have an implication for the trafficking of antigen-specific CTLs. Therefore, we investigated whether the route of immunization with adenoviral vaccine influenced the recruitment of virus-specific CTLs in the lung that should provide potent protection from influenza A virus. Mice were immunized with recombinant adenovirus expressing the matrix (M1) protein of influenza A virus via various immunization routes involving intraperitoneal, intranasal, intramuscular, or intravenous administration as well as subcutaneous administration in the hind hock. We found that the immunization route dramatically impacted the recruitment of M1-specific IFN-γ(+) CD8(+) T cells both in the lung and the spleen. Surprisingly, hock immunization was most effective for the accumulation in the lung of IFN-γ-producing CD8(+) T cells that possessed M1-specific cytolytic activity. Further, antigen-driven IFN-γ(+) CD8(+) T cells in the lung, but not in the spleen, were likely to be correlated with the resistance to challenge with influenza A virus. These results may improve our ability to design vaccines that target virus-specific CTL responses to respiratory viruses such as influenza A virus.