Characteristics of Artificial Virus-like Particles Assembled in vitro from Potato Virus X Coat Protein and Foreign Viral RNAs

Abstract

Potato virus X (PVX) and some other potexviruses can be reconstitutedin vitrofrom viral coat protein (CP) and RNA. PVX CP is capable of forming viral ribonucleoprotein complexes (vRNP) not only with homologous, but also with foreign RNAs. This paper presents the structure and properties of vRNP assembledin vitroupon incubation of PVX CP and RNAs of various plant and animal viruses belonging to different taxonomic groups. We have shown that the morphology and translational properties of vRNPs containing foreign (heterologous) RNA are identical to those of homological vRNP (PVX RNA – PVX CP). Our data suggest that the assembly of the “mixed” vRNPin vitrocould be started at the 5’-proximal region of the RNA, producing a helical structure of vRNPs with foreign nucleic acids. The formation of heterologous vRNPin vitrowith PVX CP appears not to require a specific 5’ end RNA nucleotide sequence, and the PVX CP seems to be able to pack foreign genetic material of various sizes and compositions into artificial virus-like particles.

Design of virus-based nanomaterials for medicine, biotechnology, and energy

“…This was demonstrated recently with the plant virus cowpea chlorotic mosaic virus (CCMV) through modulating several factors: an E. coli strain resistant to chloramphenicol was utilized, which helps inhibit protein transition to an insoluble state, and to give time for coat proteins to fold and maintain solubility, lower temperature, lower concentration of isopropyl-β-D-1-thiogalactopyranoside (IPTG) for induction of expression, and E. coli with a slower rate of protein synthesis were used. 37 Returning to in vitro assembly, this method has been demonstrated for a wide range of VLPs, including those based on bacteriophages P22 38 and PP7, 39 plant viruses potato virus X (PVX) 40 and CCMV, 37 and mammalian viruses human papilloma virus (HPV) 41 and human immunodeficiency virus (HIV). 42 Some particles require a nucleic acid template in order to self-assemble, 30, 39, 40 but others can be assembled to form empty capsids simply by altering conditions such as temperature, pH, and molarity.…”

“…37 Returning to in vitro assembly, this method has been demonstrated for a wide range of VLPs, including those based on bacteriophages P22 38 and PP7, 39 plant viruses potato virus X (PVX) 40 and CCMV, 37 and mammalian viruses human papilloma virus (HPV) 41 and human immunodeficiency virus (HIV). 42 Some particles require a nucleic acid template in order to self-assemble, 30, 39, 40 but others can be assembled to form empty capsids simply by altering conditions such as temperature, pH, and molarity. 37, 38, 41, 42 For templated self-assembly, TMV for example has an origin of assembly that is thought to be required to drive its assembly, 30 but other filamentous particles may not have such sequence specificity.…”

“…37, 38, 41, 42 For templated self-assembly, TMV for example has an origin of assembly that is thought to be required to drive its assembly, 30 but other filamentous particles may not have such sequence specificity. 40, 43 Knowledge of the self-assembly process of viruses can be important for determining the types of payloads that can be encapsulated as well as inform the design of novel architectures. 15 Assembly of pure, empty particles in vivo in high yields is unique and has so far only been accomplished for the plant virus cowpea mosaic virus (CPMV), which will be discussed later with plant-based production systems.…”

Translational Cross-Activation of the Encapsidated RNA of Potexviruses

“…The translation was performed in a wheat germ extract cell-free system (Promega), following a modified manufacturer protocol as described [ 12 ]. The RNA input was 40 µg/µl.…”

Plant Viruses as Nanoparticle-Based Vaccines and Adjuvants

“…The less practical and more costly baculovirus expression system seems to avoid these problems [ 94 , 95 , 96 ]. When capsid protein production does not induce particle formation, it is also possible to perform the assembly process in vitro , with or without the addition of nucleic acids [ 78 , 97 , 98 , 99 ]. Although this process is more laborious, it allows for better control of the RNA present inside the particle since diverse and uncontrolled sources of RNA can be incorporated in self-assembled viral particles in vivo [ 93 , 94 , 96 ].…”