Abstract
Plant virus coat proteins (CPs) play a fundamental role in protection of genomic RNAs, virion assembly, and viral movement. Although phosphorylation of several CPs during virus infection have been reported, little information is available about CP phosphorylation of the spherical RNA plant viruses. Here, we demonstrate that the CP of Beet black scorch virus (BBSV), a member of the genus Necrovirus, can be phosphorylated at threonine-41 (T41) by cAMP-dependent protein kinase (PKA)-like kinase in vivo and in vitro. Mutant viruses containing a T41A non-phosphorylatable alanine substitution, and a T41E glutamic acid substitution to mimic threonine phosphorylation were able to replicate but were unable to move systemically in Nicotiana benthamiana. Interestingly, the T41A and T41E mutants generated unstable 17 nm virus-like particles that failed to package viral genomic (g) RNA, compared with wild-type BBSV with 30 nm virions during viral infection in N. benthamiana. Further analyses showed that the T41 mutations had little effect on the gRNA-binding activity of the CP. Therefore, we propose a model whereby CP phosphorylation plays an essential role in long-distance movement of BBSV that involves formation of stable virions.
Highlights
The coat proteins (CPs) of the Tombusviridae are composed of an RNA binding domain (R-domain), a shell domain (S-domain), a hinge domain (H-domain) and a protruding domain (P-domain)[14,15]
A number of studies have shown that CP stability and functions of diverse plant viruses, such as Cauliflower mosaic virus (CaMV), Potato virus A (PVA), Plum pox virus (PPV), Bamboo mosaic virus (BaMV), Potato virus X (PVX), Groundnut bud necrosis virus (GBNV), and Brome mosaic virus (BMV), are regulated by phosphorylation[24,25,26,27,28,29,30,31,32]
Our results indicate that CP threonine 41 (T41) mutants form incompletely-assembled virus-like particles (VLPs) that are unable to package gRNA and are less stable than wild-type virions, and that these mutants compromised in systemic movement during infection of N. benthamiana
Summary
The CPs of the Tombusviridae are composed of an RNA binding domain (R-domain), a shell domain (S-domain), a hinge domain (H-domain) and a protruding domain (P-domain)[14,15]. PVX encapsidated gRNA is completely non-translatable in vitro, but when the N-terminal CP residues are phosphorylated by protein kinase C (PKC) or by a casein kinase mixture (CKI and CKII), the RNA is converted to a translatable form[26]. Another case involves the PVA and BaMV CP, in which phosphorylation at the RNA binding domain reduces RNA binding affinity. Both Hung et al (2014) and Ivanov et al (2001 and 2003) hypothesize that CP phosphorylation promotes virion or ribonucleoprotein (RNP) disassembly to release gRNA for replication or translation[27,28,33]. Our results reveal a correlation between CP phosphorylation, virion assembly and long-distance movement, which has been largely unexplored for spherical RNA plant viruses
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