Intracellular trafficking and endocytic uptake pathway of Pepper vein banding virus-like particles in epithelial cells.

Aim: Plant virus-like particles (VLPs) have emerged as a novel platform for delivery of drugs/antibodies. The aim of the present investigation is to establish the entry mechanism of flexuous rod-shaped virus particles into mammalian cells. Methods: Far-Western blot analysis, pull-down and ELISA were used to characterize vimentin and Hsp60 interaction with VLPs. The mode/kinetics of internalization of VLPs was deciphered using pharmacological inhibitors/endosomal markers. Results & discussion: The flexuous rod-shaped VLPs of Pepper vein banding virus (PVBV) enter HeLa and HepG2 cells via cell-surface proteins: vimentin and Hsp60, respectively. VLPs internalize via different modes of endocytosis in HeLa, HepG2 cells and are biodegradable. Vimentin and Hsp60 could be potential epithelial ligands that facilitate targeting of nanoparticles to tumor cells.

Development of sesbania mosaic virus nanoparticles for imaging.

The capsids of viruses have a high degree of symmetry. Therefore, virus nanoparticles (VNPs) can be programmed to display many imaging agents precisely. Plant VNPs are biocompatible, biodegradable and non-infectious to mammals. We have carried out bioconjugation of sesbania mosaic virus (SeMV), a well characterized plant virus, with fluorophores using reactive lysine-N-hydroxysuccinimide ester and cysteine-maleimide chemistries. Monitoring of cellular internalization of labelled SeMV nanoparticles (NPs) by confocal microscopy and flow cytometry showed that the particles have a natural preference for entry into MDA-MB-231 (breast cancer) cells, although they could also enter various other cell lines. The fluorescence of SeMV NPs labelled via the cysteines with Cy5.5 dye was found to be more stable and was detectable with greater sensitivity than that of particles labelled via the lysines with Alexa Fluor. Live-cell imaging using SeMV internally labelled with Cy5.5 showed that it could bind to MDA-MB-231 cells in less than 5 minutes and enter the cells within 15 minutes. The particles undergo endolysosomal degradation by 6 h as evidenced by their co-localization with LAMP-1. Far-western blot analysis with a HeLa cell membrane protein fraction showed that SeMV interacts with 54-, 35- and 33-kDa proteins, which were identified by mass spectrometry as vimentin, voltage-dependent anion-selective channel protein (VDAC1), and annexin A2 isoform 2 (ANXA2), respectively, suggesting that the particles may bind and enter the cell through these proteins. The results presented here demonstrate that the SeMV NPs provide a new platform technology that could be used to develop in vivo imaging and targeted drug delivery agents for cancer diagnosis and therapy.

Intracellular delivery of antibodies by chimeric Sesbania mosaic virus (SeMV) virus like particles.

The therapeutic potential of antibodies has not been fully exploited as they fail to cross cell membrane. In this article, we have tested the possibility of using plant virus based nanoparticles for intracellular delivery of antibodies. For this purpose, Sesbania mosaic virus coat protein (CP) was genetically engineered with the B domain of Staphylococcus aureus protein A (SpA) at the ßH-ßI loop, to generate SeMV loop B (SLB), which self-assembled to virus like particles (VLPs) with 43 times higher affinity towards antibodies. CP and SLB could internalize into various types of mammalian cells and SLB could efficiently deliver three different monoclonal antibodies-D6F10 (targeting abrin), anti-α-tubulin (targeting intracellular tubulin) and Herclon (against HER2 receptor) inside the cells. Such a mode of delivery was much more effective than antibodies alone treatment. These results highlight the potential of SLB as a universal nanocarrier for intracellular delivery of antibodies.

Structural studies on chimeric Sesbania mosaic virus coat protein: Revisiting SeMV assembly.

The capsid protein (CP) of Sesbania mosaic virus (SeMV, a T=3 plant virus) consists of a disordered N-terminal R-domain and an ordered S-domain. Removal of the R-domain results in the formation of T=1 particles. In the current study, the R-domain was replaced with unrelated polypeptides of similar lengths: the B-domain of Staphylococcus aureus SpA, and SeMV encoded polypeptides P8 and P10. The chimeric proteins contained T=3 or larger virus-like particles (VLPs) and could not be crystallized. The presence of metal ions during purification resulted in a large number of heterogeneous nucleoprotein complexes. N∆65-B (R domain replaced with B domain) could also be purified in a dimeric form. Its crystal structure revealed T=1 particles devoid of metal ions and the B-domain was disordered. However, the B-domain was functional in N∆65-B VLPs, suggesting possible biotechnological applications. These studies illustrate the importance of N-terminal residues, metal ions and robustness of the assembly process.

Identification of cellular isoform of oviduct-specific glycoprotein: role in oviduct tissue remodeling?

The oviduct is known to secrete mucins (MUC1 and MUC9) under the influence of ovarian steroids. The secreted form of MUC1 binds gametes in the oviduct, whereas the cellular form seen in breast cancers has been implicated in cell adhesion and morphogenesis. The secreted MUC9 or oviduct-specific glycoprotein (OGP), in addition to being a mucin, belongs to family 18 glycosylhydrolases and is known to bind gametes and embryos in the oviduct. Studies in our laboratory have identified non-muscle myosin IIA (involved in cell shape, polarity, and morphogenesis) as the protein partner to OGP in gametes. In view of the crucial role of the cortical cytoskeleton in the selective internalization of tight junctions (TJs) /adherent junctions (AJs) or apical junctional complex (AJC) in simple epithelial cells during tissue remodeling, the present study has been undertaken to evaluate the existence of a cellular form of OGP in oviductal tissue, which itself undergoes cyclic tissue remodeling. In silico analysis of the deduced amino-acid sequence of OGP has revealed the presence of several conserved motifs; these imply that OGP is a component of multi-protein complexes such as TJs. Corroborative immunoelectron-microscopic analysis in peri-ovulatory oviduct epithelia in the bonnet monkey has revealed the presence of OGP at the TJ. Co-localization studies of OGP and cadherin demonstrate that, whereas OGP is localized at the tonofilaments of the TJs, cadherin is localized at the intercellular space of the AJ. The possible role of OGP in oviductal tissue remodeling is discussed in light of the present findings and those reported in the literature.

Fertilization, embryonic development and oviductal environment: role of estrogen induced oviductal glycoprotein.

Mammalian oviduct is the physiological site for sperm capacitation, gamete fertilization and early embryonic development. The secretory cells lining the lumen of the mammalian oviduct synthesize and secrete high molecular weight glycoprotein (OGP) in response to estrogen. The protein has been shown to interact with gametes and early embryo. Several key functions have been postulated particularly its role in pre-implantation events which would have far reaching implications in assisted reproductive technology and in the development of non-hormonal contraceptive vaccine. The intention of this article is to discuss the current status of the protein and analyze how far the postulated function of OGP has been borne out by the available data.