Evidence for the role of basic amino acids in the coat protein arm region of Cucumber necrosis virus in particle assembly and selective encapsidation of viral RNA

Cucumber necrosis virus (CNV) is an icosahedral virus particle (protein coat) with a single stranded RNA genome packaged inside. The CNV coat protein contains a short section of amino acids that connects the RNA binding domain (R-domain) and the shell domain that forms the outer casing of the virus particle. This short segment of connecting amino acids is referred to as the arm, and contains a conserved, highly basic amino acid sequence (“KGRKPR”), which we show to be involved in RNA encapsidation during virion assembly. Seven mutants were constructed by altering the CNV “KGRKPR” sequence; the four basic residues (K or R) were mutated to the amino acid alanine individually, in pairs, or in total. It is previously known that the negatively charged RNA molecules can interact with basic amino acid residues, so we postulated that since this sequence “KGRKPR” is located in the interior of the virion, and are highly conserved between other related viruses, that they may be involved in RNA encapsidation. We found that virion accumulation and virion RNA encapsidation were significantly reduced in mutants containing two or four substitutions In addition, virion morphology was also affected, where both small and intermediate-sized particles were produced. Mutants with two or four substitutions encapsidate significantly higher levels of truncated RNA than that of wild type CNV, suggesting that basic residues in the “KGRKPR” sequence are important for encapsidation of full-length CNV RNA. Interestingly, “KGRKPR” mutants also encapsidated relatively higher levels of host RNA, suggesting that the “KGRKPR” sequence also contributes to selective encapsidation of CNV specific RNA. This research brings new knowledge to light regarding how plant viruses work, and contributes to the overall understanding of how plant viruses have evolved.