CryoEM and stability analysis of virus-like particles of potyvirus and ipomovirus infecting a common host

Abstract

Sweet potato feathery mottle virus (SPFMV) and Sweet potato mild mottle virus (SPMMV) are members of the genera Potyvirus and Ipomovirus, family Potyviridae, sharing Ipomoea batatas as common host, but transmitted, respectively, by aphids and whiteflies. Virions of family members consist of flexuous rods with multiple copies of a single coat protein (CP) surrounding the RNA genome. Here we report the generation of virus-like particles (VLPs) by transient expression of the CPs of SPFMV and SPMMV in the presence of a replicating RNA in Nicotiana benthamiana. Analysis of the purified VLPs by cryo-electron microscopy, gave structures with resolutions of 2.6 and 3.0 Å, respectively, showing a similar left-handed helical arrangement of 8.8 CP subunits per turn with the C-terminus at the inner surface and a binding pocket for the encapsidated ssRNA. Despite their similar architecture, thermal stability studies reveal that SPMMV VLPs are more stable than those of SPFMV.

From structural polymorphism to structural metamorphosis of the coat protein of flexuous filamentous potato virus Y

“…The remaining 8% of picked particles also exhibited left-handed helical symmetry and encapsidated RNA (VLP h+RNA ), closely resembling PVY virion 6 (Fig. 1c ; Supplementary Table 1 ) and SPFMV VLPs 27 . An improved data analysis procedure seemed to play a crucial role here, as such a distribution of polymorphic filaments was also obtained by reprocessing our previous data 6 (Supplementary Fig.…”

“…23 ) or potato virus A 36 . Previous studies already suggested that recombinant potyviral CP encapsidates its own mRNA 27 , 37 . To verify this, we performed analysis of RNA extracted from VLP T43C+D136C filaments.…”

“…Although the structures of several flexuous filamentous potexviruses 22 – 26 and potyviruses 5 – 7 have been recently determined, information on the structural diversity of these viruses and their VLPs is lacking 6 , 7 , 23 , 26 , 27 . In our work, we have investigated the structural landscape of self-assemblies formed by potyviral CP.…”

“…On the other hand, the structure of TuMV VLPs produced by transient expression in tobacco at 8.0 Å resolution indicated an RNA-free filamentous arrangement of CP units in left-handed helical symmetry 7 . Interestingly, the structure of VLPs determined at 2.6 Å resolution based on CP of another potyvirus, sweet potato feathery mottle virus (SPFMV), and produced in tobacco by transient expression in the presence of a replicating RNA, showed a virus-like architecture, with ssRNA directing the helical arrangement of CPs along the filament 27 . These studies showed that a specific potyviral CP self-assembles into filaments of a single architectural type under selected experimental conditions, but this differed among the three experimental arrangements, suggesting that polymorphism may also exist within a species of CP under certain conditions.…”

Structural Insights into Plant Viruses Revealed by Small-Angle X-ray Scattering and Atomic Force Microscopy

“…The IHRSR algorithm developed by Egelman [ 31 ] has been used for further helical reconstructions. Recently, cryo-EM and stability analyses have been carried out on virus-like particles (VLP) from representatives of the genera Potyvirus and Ipomovirus of the family Potyviridae, again lacking the N-terminal 91 or 66 amino acid residues, respectively, and a sequence-based prediction of the N-terminal domain has been made using AlphaFold to obtain the full-length structure [ 32 ].…”