Turnip Crinkle Virus Coat Protein Research Articles

Symptom Attenuation by a Satellite RNA in Vivo Is Dependent on Reduced Levels of Virus Coat Protein

Many plant RNA viruses provide replication and encapsidation functions for one or more satellite RNAs (sat-RNAs) that can modulate the symptoms of the associated helper virus. Sat-RNA C, a virulent sat-RNA associated with turnip crinkle virus (TCV), normally intensifies symptoms but can attenuate symptoms if the TCV coat protein (CP) is replaced with that of cardamine chlorotic fleck carmovirus [Kong et al. (1995) Plant Cell 7, 1625–1634] or if TCV contains an alteration in the CP initiation codon (TCV-CPm) [Kong et al. (1997b) Plant Cell 9, 2051–2063]. To further elucidate the mechanism of symptom attenuation by sat-RNA C, the composition of the CP produced by TCV-CPm (CPCPm) was determined. Our results reveal that CPCPm likely has two additional amino acids at its N-terminus compared with wild-type TCV CP. TCV-CPm produces reduced levels of CP, and this reduction, not the two additional residues at the CP N-terminus, is responsible for symptom attenuation by sat-RNA C.

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)≈0.5μ 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 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.

Asp → Asn Substitutions in the Putative Calcium-Binding Site of the Turnip Crinkle Virus Coat Protein Affect Virus Movement in Plants

Many plant virus coat proteins have binding sites for divalent cations, particularly calcium. It has been speculated that the purpose of such sites is to ensure that plant viruses release their RNA only in the host cytoplasm which has a low calcium concentration. By comparison to tomato bushy stunt virus, two of the amino acids that probably interact directly with calcium ions in turnip crinkle virus (TCV) capsids, Asp155 and Asp157, were substituted with Asn residues by oligonucleotide-directed mutagenesis to create the mutant TCV-M18. TCV-M18 coat protein, and virions accumulated to wild-type levels in isolated protoplasts. Mutant virions were able to uncoat and to initiate de novo replication in protoplasts although no symptoms were observed in plants inoculated with TCV-M18. Mutant RNA accumulated to much lower levels than wild-type RNA in inoculated leaves and was not detected in upper, uninoculated leaves. The lower infectivity of TCV-M18 in plants may be due to a decreased capacity to move efficiently from cell to cell, and we suggest the possibility that TCV coat protein plays an active, pivotal role in cell-to-cell movement interactions.

Interactions between viral coat protein and a specific binding region on turnip crinkle virus RNA

The turnip crinkle virus coat protein binding sites in the ribonucleoprotein complex resulting from virion dissociation have been identified previously. In this study, RNA binding characteristics of viral coat protein to a region encompassing the protected RNA fragments Fa, Ff, and Fc (Fafc) have been investigated further using an RNA transcript (the Fafc fragment). These experiments have shown that coat protein requires no additional viral RNA elements to bind to this region. Such binding was shown to be specific for turnip crinkle virus coat protein using an ultra-violet light cross-linking assay. Gel mobility shift analyses demonstrated that the protein-RNA interactions produced two complexes: a homogeneous small ribonucleoprotein complex, and larger complexes which failed to migrate into gels. High salt and limiting protein concentrations favored the formation of the small ribonucleoprotein complex, whereas low salt and excess protein concentrations favored the larger complexes. RNA competition experiments demonstrated that small ribonucleoprotein complex formation coincided with specific RNA binding of the coat protein to the Fafc fragment. In addition, the coat protein possessed a poly(U)-binding site(s), which enabled it to interact with single-stranded RNA in a sequence non-specific manner to form large complexes. The results suggest that the coat protein contains both specific and non-specific RNA binding activities located at physically distinct sites. These results are consistent with the proposed assembly model for turnip crinkle virus.

Structure and assembly of turnip crinkle virus: IV. Analysis of the coat protein gene and implications of the subunit primary structure

The structure of the turnip crinkle virus (TCV) coat protein and coat protein gene has been examined by cDNA cloning, nucleotide sequencing and high-resolution mRNA mapping. We have cloned a 1450-nucleotide cDNA fragment, representing the 3′ end of the TCV genome, using genomic RNA polyadenylated in vitro as the reverse transcriptional template. Nucleic acid sequence analysis reveals the presence of a 1053 nucleotide open reading frame capable of encoding a protein of 38,131 M r, identified as the coat protein subunit. The 1446 base subgenomic mRNA for the coat protein, mapped using high-resolution primer extension techniques, contains a 137 nucleotide leader sequence upstream from the initiation codon. We have characterized a second subgenomic RNA of approximately 1700 bases, roughly 250 nucleotides longer than the 1446 base species in the 5′ direction. No TCV-related RNAs are polyadenylated in vivo. The derived amino acid sequence of the TCV coat protein has been built into the 3.2 Å resolution electron density map of TCV reported in paper I of this series. We describe here some of the important features of the structure. Alignment of the three-dimensional structures of tomato bushy stunt virus and southern bean mosaic virus shows significant sequence relationships in the arms and S domains, although the conserved residues do not appear to have any special role in stabilizing the β-barrel fold or in mediating subunit interactions. The sequences of TCV and carnation mottle virus can be aligned. Comparisons among the four are discussed in terms of the organization of the S domain.