Multivalent Ligand Displayed on Plant Virus Induces Rapid Onset of Bone Differentiation

Viruses are monodispersed biomacromolecules with well-defined 3-D structures at the nanometer level. The relative ease to manipulate viral coat protein gene to display numerous functional groups affords an attractive feature for these nanomaterials, and the inability of plant viruses to infect mammalian hosts poses little or no cytotoxic concerns. As such, these nanosized molecular tools serve as powerful templates for many pharmacological applications ranging as multifunctional theranostic agents with tissue targeting motifs and imaging agents, potent vaccine scaffolds to induce cellular immunity and for probing cellular functions as synthetic biomaterials. The results herein show that combination of serum-free, chemically defined media with genetically modified plant virus induces rapid onset of key bone differentiation markers for bone marrow derived mesenchymal stem cells within two days. The xeno-free culture is often a key step toward development of ex vivo implants, and the early onset of osteocalcin, BMP-2 and calcium sequestration are some of the key molecular markers in the progression toward bone formation. The results herein will provide some key insights to engineering functional materials for rapid bone repair

Mutant Plant Viruses with Cell Binding Motifs Provide Differential Adhesion Strengths and Morphologies

The ability of Tobacco mosaic virus (TMV) to tolerate various amino acid insertions near its carboxy terminus is well-known. Typically these inserts are based on antigenic sequences for vaccine development with plant viruses as carriers. However, we determined that the structural symmetries and the size range of the viruses could also be modeled to mimic the extracellular matrix proteins by inserting cell-binding sequences to the virus coat protein. The extracellular matrix proteins play important roles in guiding cell adhesion, migration, proliferation, and stem cell differentiation. Previous studies with TMV demonstrated that the native and phosphate-modified virus particles enhanced stem cell differentiation toward bone-like tissues. Based on these studies, we sought to design and screen multiple genetically modified TMV mutants with reported cell adhesion sequences to expand the virus-based tools for cell studies. Here, we report the design of these mutants with cell binding amino acid motifs derived from several proteins, the stabilities of the mutants against proteases during purification and storage, and a simple and rapid functional assay to quantitatively determine adhesion strengths by centrifugal adhesion assay. Among the mutants, we found that cells on TMV expressing RGD motifs formed filopodial extensions with weaker attachment profiles, whereas the cells on TMV expressing collagen I mimetic sequence displayed little spreading but higher attachment strengths