New positions for peptide presentation in Potato virus X capsid protein

Abstract

viruses represents potentially a promising new delivery and adjuvant system mainly for the following reasons: (i) the capsid of plant virus represents a scaffold exposing desired epitopes to the immune system in an abundant and organized manner and can serve as an adjuvant by itself, (ii) plant viruses are not infectious to animals, lowering the safety risk associated with their deployment, and (iii) scalability [3,4].Human papillomavirus (HPV) infection is one of the most common sexually transmitted infections worldwide. Infection by high risk HPVs is causally associated with cervical cancer, which represents the second most common cancer among women (after breast cancer) [5]. The most common oncogenic HPV genotypes are 16 and 18, together causing approximately 70 % of all cervical cancers, while HPV 16 alone causing 50 % of all cervical cancers [6]. The E7 oncoprotein plays an important role in both malignant transformation and in the maintenance of the transformed state of HPV infected cells. This represents a promising target for immunotherapy because the E7 oncoprotein is expressed in all cervical tumors and in precancerous lesions [7]. The main known role of E7 oncoprotein in virus cycle is to prevent cell death by binding tumor-suppressor protein Rb (pRb), resulting in the dissociation of pRB from E2F-family transcription factors and the premature cell progression into the S phase of the cell cycle which leads to uncontrollable cell division [8].Potato virus X (PVX) belongs to the order Tymovirales, family Alphaflexiviridae and genus Potexvirus. It is a filamentous, positive single stranded RNA (+ssRNA) virus. Virus particles are about 515 nm long and have about 13 nm in diameter. The PVX genome is capped, has a poly(A) tail and encodes five open reading frames (viral replicase, three movement proteins belonging to the triple-gene block family and capsid protein -approximately 1 300 CP subunits per virion) [9]. The PVX CP can be used for presentation by direct or indirect fusion of a desired protein or peptide to the N-terminus [10][11][12]. Recently, it has been also shown that the C-terminus of PVX CP can be used for protein presentation [13]. However, the Abstract: Potato virus X (PVX) is widely used as a peptide presentation system in plant biotechnology, mostly for transient expression of desired peptides fused to the N-terminus of its capsid protein (CP). Here we describe testing/investigation of new positions for peptide presentation based on seven putative surface loops of PVX CP. We performed bacterial expression of fourteen different PVX CPs modified by the insertion of the epitope derived from the E7 oncoprotein (E7 epitope) of Human papillomavirus type 16 fused with His tag. The expression from vector pMPM-A4Ω in Escherichia coli MC1061 was performed to evaluate the capacity of the PVX CP platform to tolerate the insertion of E7 epitope fused with His tag in the seven putative surface loops. The immunological characteristics of expressed epitopes were assessed by Western blot using both anti-P...

Small, Smaller, Nano: New Applications for Potato Virus X in Nanotechnology

“…Vaculik et al (2015a) identified four promising internal transgene insertion positions within the surface loops of the PVX CP. The tested epitope insertion mutant was infectious and produced particles only after amino acid 23 (Vaculik et al, 2015b).…”