Bink Hugo H J, Roepan Shalendra K, Pleij Cornelis W A
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Leiden, The Netherlands.
Proteins. 2004 May 1;55(2):236-44. doi: 10.1002/prot.10600.
RNA-coat protein interactions in turnip yellow mosaic virus (TYMV) have been shown to involve low pK proton-donating groups. Two different types of interaction have been proposed. In the so-called type I interaction, protonated C-residues interact with acidic amino acids at low pH, thereby providing a rationale for the high C-content (38%) of the genomic RNA. The type II interaction involves charged histidines interacting with phosphates of the RNA backbone. Site-directed mutagenesis of the TYMV coat protein and subsequent in vivo analysis were performed to distinguish between these two types of RNA-protein interaction. The results reveal a prominent role for the histidines H68 and H180, since mutation to an alanine residue inhibits symptom development on secondary leaves, indicating that spreading of the virus in the plant is blocked. Viral RNA and coat protein synthesis are not altered, showing that these two histidines may play a role in the process of RNA encapsidation. Overexpression of the TYMV coat protein in Escherichia coli leads to the formation of bona fide capsids, showing that the two histidines are not critical in capsid assembly. Mutagenesis of the acidic amino acids D11, E135, and D143 to alanine apparently did not interfere with virus viability. The functional role of the histidines during the infection cycle is discussed in terms of the structure of the coat protein, both at the level of amino acid sequence conservation among the members of the Tymoviridae family and as the three-dimensional structure of the coat protein.
芜菁黄花叶病毒（TYMV）中的RNA-衣壳蛋白相互作用已被证明涉及低pK质子供体基团。人们提出了两种不同类型的相互作用。在所谓的I型相互作用中，质子化的C残基在低pH下与酸性氨基酸相互作用，从而为基因组RNA的高C含量（38%）提供了一种解释。II型相互作用涉及带电荷的组氨酸与RNA主链的磷酸基团相互作用。对TYMV衣壳蛋白进行定点诱变并随后进行体内分析，以区分这两种类型的RNA-蛋白相互作用。结果揭示了组氨酸H68和H180的重要作用，因为突变为丙氨酸残基会抑制次生叶上症状的发展，这表明病毒在植物中的传播受阻。病毒RNA和衣壳蛋白的合成没有改变，这表明这两个组氨酸可能在RNA衣壳化过程中起作用。TYMV衣壳蛋白在大肠杆菌中的过表达导致了真正衣壳的形成，这表明这两个组氨酸在衣壳组装中并不关键。将酸性氨基酸D11、E135和D143突变为丙氨酸显然不会干扰病毒的活力。从衣壳蛋白的结构方面，无论是在芜菁病毒科成员之间氨基酸序列保守性的层面，还是作为衣壳蛋白的三维结构，讨论了组氨酸在感染周期中的功能作用。
