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 tur nip crinkle virus (TCV) genome was previously implicated in virus asse mbly nucleation. To localize the proposed high-affinity binding determ inants, we analyzed the ability of the coat protein to bind the full-l ength and truncated derivatives of the TCV-386 RNA using gel retardati on and nitrocellulose filter retention assays. Quantitation of the bin ding data indicated that the coat protein did not preferentially recog nize a particular region of the RNA Moreover, the affinity of the coat protein for the TCV-386 RNA [apparent dissociation constant (K-d) app roximate to 0.5 mu M] 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 R NA in a cooperative manner and this was supported by evidence that all of the RNAs examined were bound by multiple copies of the coat protei n. Based on the number of binding intermediates which could he detecte d in titrations involving RNAs of different chain length, it appeared that each coat protein binding unit occupies 35-40 nucleotides. Our re sults demonstrate that encapsidation of TCV RNA results from highly co operative binding of the coat protein on the large viral genome. Howev er, we were not able to confirm that assembly is mediated by initiatio n at a high-affinity binding site on the viral RNA. (C) 1995 Academic Press, Inc.