Quantitative Analysis of the Binding of Turnip Crinkle Virus Coat Protein to RNA Fails to Demonstrate Binding Specificity but Reveals a Highly Cooperative Assembly Interaction

An element(s) within a 386-nucleotide segment (TCV-386 RNA) of the turnip crinkle virus (TCV) genome was previously implicated in virus assembly nucleation. To localize the proposed high-affinity binding determinants, we analyzed the ability of the coat protein to bind the full-length and truncated derivatives of the TCV-386 RNA using gel retardation and nitrocellulose filter retention assays. Quantitation of the binding data indicated that the coat protein did not preferentially recognize a particular region of the RNA Moreover, the affinity of the coat protein for the TCV-386 RNA [apparent dissociation constant (K_d)≈0.5μM) did not appreciably differ from its affinity for other comparably sized RNAs tested, including nonviral RNAs. However, the quantitative studies also suggested that the coat protein binds RNA in a cooperative manner and this was supported by evidence that all of the RNAs examined were bound by multiple copies of the coat protein Based on the number of binding intermediates which could be detected in titrations involving RNAs of different chain length, it appeared that each coat protein binding unit occupies 35-40 nucleotides, Our results demonstrate that encapsidation of TCV RNA results from highly cooperative binding of the coat protein on the large viral genome. However, we were not able to confirm that assembly is mediated by initiation at a high-affinity binding site on the viral RNA.