Department of Chemistry and Biochemistry, University of South Carolina, 631 Sumter Street, Columbia, South Carolina 29208, United States.
Biomacromolecules. 2012 Feb 13;13(2):422-31. doi: 10.1021/bm2014558. Epub 2012 Jan 12.
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.
烟草花叶病毒（TMV）在其羧基末端附近耐受各种氨基酸插入的能力是众所周知的。这些插入物通常基于植物病毒作为载体的抗原序列，用于疫苗开发。然而，我们确定病毒的结构对称性和大小范围也可以通过将细胞结合序列插入病毒外壳蛋白来模拟细胞外基质蛋白。细胞外基质蛋白在指导细胞黏附、迁移、增殖和干细胞分化方面发挥着重要作用。以前的 TMV 研究表明，天然和磷酸化修饰的病毒颗粒增强了干细胞向骨样组织的分化。基于这些研究，我们试图设计和筛选具有报道的细胞黏附序列的多种遗传修饰的 TMV 突变体，以扩展基于病毒的细胞研究工具。在这里，我们报告了这些突变体的设计，其细胞结合氨基酸基序源自几种蛋白质，突变体在纯化和储存过程中对蛋白酶的稳定性，以及一种简单快速的功能测定法，通过离心黏附测定法定量测定黏附强度。在这些突变体中，我们发现表达 RGD 基序的 TMV 上的细胞形成了具有较弱附着特征的丝状伪足延伸，而表达胶原 I 模拟序列的 TMV 上的细胞则很少扩展，但附着强度更高。
