Effect of mutations K97A and E128A on RNA binding and self assembly of papaya mosaic potexvirus coat protein

Abstract

Papaya mosaic potexvirus (PapMV) is a member of the potexvirus family. The virion is a flexible rod 500 nm in length and 15 nm in diameter composed of 1400 subunits of viral coat protein (CP) [1] assembled around a plus-strand genomic RNA of 6656 nucleotides (nt) [2]. In vitro reconstitution of PapMV nucleocapsid-like particles (NLPs) was previously studied using CP prepared from the acetic acid degradation of purified virus [3]. Using this method, PapMV CP can be isolated from the genomic RNA and used for in vitro assembly assays [3]. Extracted CP has been found as a variety of aggregates ranging from 14S to 25S [4] that include a disk-like structure (14 S) made of 18-20 subunits. These disks are helical structures (two turns of the helix) similar in architecture to the native virus particle [3,5]. The addition of RNA to the isolated disks triggers the assembly of long rod-shaped particles that are very similar to the WT virus [3]. The in vitro assembly of PapMV was shown to be specific and triggered by 47 nucleotides of the 5¢ noncoding region of the virus [6]. This region is free of any discernable secondary structure, an important feature for initiation of the assembly process [6]. In vitro assembly is specific when performed at a pH of 8.0-8.5 in a buffer of low ionic strength [4] and elongation proceeds in the 5¢fi3¢ direction [7]. One atom of Ca 2+ is attached to each subunit, which is probably important for the structure of the protein [8]. Alignment of PapMV CP with the CP of other potexviruses reveals that these proteins share 35% identity [9]. The N terminus of the proteins is the most divergent and their length is also variable. It can reach more than 50 amino acids in the case of potato aucuba mosaic virus. Phosphorylation of the N terminus of potato virus X CP by host Papaya mosaic potexvirus (PapMV) coat protein (CP) was expressed (CPDN5) in Escherichia coli and showed to self assemble into nucleocapsid like particles (NLPs). Twenty per cent of the purified protein was found as NLPs of 50 nm in length and 80% was found as a multimer of 450 kDa (20 subunits) arranged in a disk. Two mutants in the RNA binding domain of the PapMV CP, K97A and E128A showed interesting properties. The proteins of both mutants could be easily purified and CD spectra of these proteins showed secondary and tertiary structures similar to the WT protein. The mutant K97A was unable to self assemble and bind RNA. On the contrary, the mutant E128A showed an improved affinity for RNA and self assembled more efficiently in NLPs. E128A NLPs were longer (150 nm) than the recombinant CPDN5 and 100% percent of the protein was found as NLPs in bacteria. E128A NLPs were more resistant to digestion by trypsin than the CPDN5 but were more sensitive to denaturation by heat. We discuss the possible role of K97 and E128 in the assembly of PapMV.Abbreviations CP, coat protein; NLP, nucleocapsid like particles; nt, nucleotide; PapMV, Papaya mosaic potexvirus.

Novel Plant Virus-Based Vaccine Induces Protective Cytotoxic T-Lymphocyte-Mediated Antiviral Immunity through Dendritic Cell Maturation

“…We recently described the generation of a VLP system based on the papaya mosaic virus (PapMV) (39). PapMV, a member of the potexvirus family, displays a flexible rod-like structure composed of 1,400 subunits of the viral CP assembled around a positive-strand RNA (8,34,39,46).…”

“…PapMV, a member of the potexvirus family, displays a flexible rod-like structure composed of 1,400 subunits of the viral CP assembled around a positive-strand RNA (8,34,39,46). PapMV VLPs, formed by the self-assembly of the CP after overexpression in Escherichia coli (39), can carry foreign epitopes and induce the production of specific antibodies in vaccinated mice (7). Moreover, we have recently shown that a human B-cell line can cross-present CTL epitopes displayed on PapMV VLPs to specific T cells in vitro in a proteasome-independent fashion (14).…”

New positions for peptide presentation in Potato virus X capsid protein

“…Twenty percent of the purified protein was found as NLPs of 50 nm in length and 80% was found as a multimer of 450 kDa (20 subunits) arranged in a disk. [28]. Two new tested insertion sites in CP led to the production of recombinant proteins capable of assembly into PapMV nanoparticles [29].…”

“…1). The engineering of Papaya mosaic virus (PapMV), another member of the family Flexiviridae in genus Potexvirus has been recently described [28]. PapMV CP was expressed in Escherichia coli and showed to self assemble into nucleocapsid like particles (NLPs).…”

Adjuvanted influenza vaccines

“…68 Virus-like particles (also called nanoparticles) derived from the expression of papaya mosaic virus coat protein (PaPMV) in bacteria and consisting of several hundred recombinant coat proteins organized in a repetitive and ordered manner have been tried as TIV adjuvants. 69 They are used as an epitope display system that leads to the production of antibodies against surfaceexposed peptides, thus providing protection. 70,71 Savard et al treated mice and ferrets with PaPMV nanoparticles and observed an increase in the global humoral response to TIV by measuring the increase in total IgG titers, particularly those of the IgG2a subclass.…”