SV40 VP1 major capsid protein in its self-assembled form allows VP1 pentamers to coat various types of artificial beads in vitro regardless of their sizes and shapes.

The icosahedral capsid structure of simian virus 40 (diameter, 45 nm) consists of 72 pentameric subunits, with each subunit formed by five VP1 molecules. Electron microscopy, immuno-gold labeling, and ζ-potential analysis showed that purified recombinant VP1 pentamers covered polystyrene beads measuring 100, 200, and 500 nm in diameter, as well as silica beads. In addition to covering spherical beads, VP1 pentamers covered cubic magnetite beads, as well as the distorted surface structures of liposomes. These findings indicate that VP1 pentamers could coat artificial beads of various shapes and sizes larger than the natural capsid. Technology based on VP1 pentamers may be useful in providing a capsid-like surface for enclosed materials, enhancing their stability and cellular uptake for drug delivery systems.

Viral protein-coating of magnetic nanoparticles using simian virus 40 VP1.

Artificial beads including magnetite and fluorescence particles are useful to visualize pathologic tissue, such as cancers, from harmless types by magnetic resonance imaging (MRI) or fluorescence imaging. Desirable properties of diagnostic materials include high dispersion in body fluids, and the ability to target specific tissues. Here we report on the development of novel magnetic nanoparticles (MNPs) intended for use as diagnosis and therapy that are coated with viral capsid protein VP1-pentamers of simian virus 40, which are monodispersive in body fluid by conjugating epidermal growth factor (EGF) to VP1. Critically, the coating of MNPs with VP1 facilitated stable dispersion of the MNPs in body fluids. In addition, EGF was conjugated to VP1 coating on MNPs (VP1-MNPs). EGF-conjugated VP1-MNPs were successfully used to target EGF receptor-expressing tumor cells in vitro. Thus, using viral capsid protein VP1 as a coating material would be useful for medical diagnosis and therapy.

Identification of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) as a novel target of bisphenol A.

Bisphenol A (BPA) forms the backbone of plastics and epoxy resins used to produce packaging for various foods and beverages. BPA is also an estrogenic disruptor, interacting with human estrogen receptors (ER) and other related nuclear receptors. Nevertheless, the effects of BPA on human health remain unclear. The present study identified DNA-dependent protein kinase catalytic subunit (DNA-PKcs) as a novel BPA-binding protein. DNA-PKcs, in association with the Ku heterodimer (Ku70/80), is a critical enzyme involved in the repair of DNA double-strand breaks. Low levels of DNA-PK activity are previously reported to be associated with an increased risk of certain types of cancer. Although the Kd for the interaction between BPA and a drug-binding mutant of DNA-PKcs was comparatively low (137 nM), high doses of BPA were required before cellular effects were observed (100-300 µM). The results of an in vitro kinase assay showed that BPA inhibited DNA-PK kinase activity in a concentration-dependent manner. In M059K cells, BPA inhibited the phosphorylation of DNA-PKcs at Ser2056 and H2AX at Ser139 in response to ionizing radiation (IR)-irradiation. BPA also disrupted DNA-PKcs binding to Ku70/80 and increased the radiosensitivity of M059K cells, but not M059J cells (which are DNA-PKcs-deficient). Taken together, these results provide new evidence of the effects of BPA on DNA repair in mammalian cells, which are mediated via inhibition of DNA-PK activity. This study may warrant the consideration of the possible carcinogenic effects of high doses of BPA, which are mediated through its action on DNA-PK.

In vitro reconstitution of SV40 particles that are composed of VP1/2/3 capsid proteins and nucleosomal DNA and direct efficient gene transfer.

SV40 is comprised of the viral minichromosome and the capsid proteins VP1, VP2, and VP3. Complete reconstitution of SV40 virions in vitro remains a challenge. Here we describe in vitro reconstitution of SV40 particles that contain ~5-kb circular nucleosomal DNA with hyperacetylated histones and are encapsidated in a coat composed of VP1, VP2, and VP3, closely mimicking the characteristics of authentic SV40 virions. When inoculated into mammalian cells, VP1/2/3 particles containing nucleosomal DNA carrying a reporter gene yielded a significantly higher level of gene expression than VP1-only particles containing the corresponding naked DNA. The elevated gene expression resulted mainly from enhanced association of the particles with the cell surface and from facilitation of subsequent uptake into cells. Thus, the in vitro reconstitution system reported here should be useful for the elucidation of Polyomaviridae assembly mechanisms and for the development of novel carriers for gene delivery.

Cell selective targeting of a simian virus 40 virus-like particle conjugated to epidermal growth factor.

Simian virus 40 (SV40) virus-like particles (VLPs) are efficient nanocarriers for gene delivery. VLPs conjugated to human epidermal growth factor (hEGF) were prepared and the cell selectivity of the VLP was examined using human epithelial carcinoma A431 cells, which overexpress the EGF receptor. The endocytic efficiency was determined by the level of Gaussia luciferase activity from the encapsulated protein in hEGF-conjugated VLPs. EGF receptor-mediated endocytosis of hEGF-conjugated VLPs was significantly increased and was confirmed by fluorescence imaging using mCherry encapsulated in hEGF-conjugated VLPs. These results suggest that VLPs of SV40 conjugated to a specific ligand could be used for cell selective gene delivery.

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.

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.

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 2,000 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.

Mutational analysis of the carboxyl-terminal region of the SV40 major capsid protein VP1.

Virus-like particles (VLPs), a promising next-generation drug delivery vehicle, can be formed in vitro using a recombinant viral capsid protein VP1 from SV40. Seventy-two VP1 pentamers interconnect to form the T = 7d lattice of SV40 capsids, through three types of C-terminal interactions, alpha-alpha'-alpha'', beta-beta' and gamma-gamma. These appear to require VP1 conformational switch, which involve in particular the region from amino acids 301-312 (herein Region I). Here we show that progressive deletions from the C-terminus of VP1, up to 34 amino acids, cause size and shape variations in the resulting VLPs, including tubular formation, whereas deletions beyond 34 amino acids simply blocked VP1 self-assembly. Mutants carrying in Region I point mutations predicted to disrupt alpha-alpha'-alpha''-type and/or beta-beta'-type interactions formed small VLPs resembling T = 1 symmetry. Chimeric VP1, in which Region I of SV40 VP1 was substituted with the homologous region from VP1 of other polyomaviruses, assembled only into small VLPs. Together, our results show the importance of the integrity of VP1 C-terminal region and the specific amino acid sequences within Region I in the assembly of normal VLPs. By understanding how to alter VLP sizes and shapes contributes to the development of drug delivery systems using VLPs.

Adeno-associated virus site-specific integration is regulated by TRP-185.

Adeno-associated virus (AAV) integrates site specifically into the AAVS1 locus on human chromosome 19. Although recruitment of the AAV nonstructural protein Rep78/68 to the Rep binding site (RBS) on AAVS1 is thought to be an essential step, the mechanism of the site-specific integration, particularly, how the site of integration is determined, remains largely unknown. Here we describe the identification and characterization of a new cellular regulator of AAV site-specific integration. TAR RNA loop binding protein 185 (TRP-185), previously reported to associate with human immunodeficiency virus type 1 TAR RNA, binds to AAVS1 DNA. Our data suggest that TRP-185 suppresses AAV integration at the AAVS1 RBS and enhances AAV integration into a region downstream of the RBS. TRP-185 bound to Rep68 directly, changing the Rep68 DNA binding property and stimulating Rep68 helicase activity. We present a model in which TRP-185 changes the specificity of the AAV integration site from the RBS to a downstream region by acting as a molecular chaperone that promotes Rep68 complex formation competent for 3'-->5' DNA helicase activity.

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.