Tritium planigraphy study of structural alterations in the coat protein of <i>Potato virus X</i> induced by binding of its triple gene block 1 protein to virions

Lukashina, E. V.; Badun, G. A.; Федорова, Н. В.; Ksenofontov, Alexander L.; Nemykh, Maria A.; Serebryakova, Marina V.; Mukhamedzhanova, Anna; Карпова, О. В.; Родионова, Н. В.; Baratova, L. A.; Добров, Е. Н.

doi:10.1111/j.1742-4658.2009.07408.x

Cited by 26 publications

(37 citation statements)

References 30 publications

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“…Indeed, PepMV-NE CP is 100% identical to the LP and EU isolates, and differs from the US1, US2, and CH2 isolates at only 7, 18, and 18 aa positions out of a total of 237 aa. Furthermore, all but four of these variations occur within the Nterminal 26 aa, which are thought to be partially exposed on the surface of the virions (18). The high level of sequence conservation among CPs of diverse PepMV isolates further justifies CP as an ideal target for engineered attenuation.…”

Section: Characterization Of a New Pepmv Isolate From Nebraskamentioning

confidence: 99%

Generation of an Attenuated, Cross-Protective Pepino mosaic virus Variant Through Alignment-Guided Mutagenesis of the Viral Capsid Protein

et al. 2015

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Mild variants of many viruses are able to protect infected plants from subsequent invasion by more severe variants of the same viruses through a process known as cross-protection. In the past, the cross-protective viral variants were commonly derived from mild field isolates that were sometimes genetically heterogeneous, providing variable levels of cross-protection. Here, we report a novel approach to rapidly generate cross-protective variants of the tomato-infecting Pepino mosaic virus (PepMV) independently of the availability of mild field isolates. Our approach sought to attenuate PepMV by mutating less conserved amino acid residues of the abundantly produced capsid protein (CP). These less-conserved amino acid residues were identified through multiple alignments of CPs of six potexviruses including PepMV, and were altered systematically to yield six PepMV mutants. These mutants were subsequently inoculated onto the model plant Nicotiana benthamiana, as well as tomato, to evaluate their accumulation levels, symptom severities, and cross-protection potentials. The mutant KD, in which the threonine (T) and alanine (A) residues at CP positions 66 and 67 were replaced with lysine (K) and aspartic acid (D), respectively, were found to accumulate to low levels in infected plants, cause very mild symptoms, and effectively protect both N. benthamiana and tomato against secondary infections by wild-type PepMV. These data suggest that our approach represents a simple, fast, and reliable way of generating attenuated viral variants capable of cross-protection.

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Section: Characterization Of a New Pepmv Isolate From Nebraskamentioning

confidence: 99%

Generation of an Attenuated, Cross-Protective Pepino mosaic virus Variant Through Alignment-Guided Mutagenesis of the Viral Capsid Protein

et al. 2015

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“…The tags were used for both detection of antigen production and evaluation of antigen presentation on the surface of PVX particles, as no antibodies recognizing the E7 peptide were available. The threedimensional structure of PVX CP in a virion was recently studied by tritium planigraphy (Nemykh et al 2008;Lukashina et al 2009). Although the model provides only limited accuracy, it is possible to use it as a template for designing new insertion sites in PVX CP for antigen presentation.…”

Section: Resultsmentioning

confidence: 99%

“…To test Fig. 1 PVX CP amino acid sequence with secondary structure element prediction (Lukashina et al 2009). Fusion sites are indicated by arrows.…”

Section: Resultsmentioning

confidence: 99%

Potato virus X displaying the E7 peptide derived from human papillomavirus type 16: a novel position for epitope presentation

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Moravec

et al. 2014

Plant Cell Tiss Organ Cult

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Potato virus X (PVX) is widely used as a peptide presentation system in plant biotechnology, mostly by fusion of desired peptides to the N-terminus of its capsid protein (CP). Considering that some epitopes can interfere with the stability and/or self-assembly of PVX CP when fused to its N-terminus, we evaluated four other possible sites for fusion using the E7 epitope derived from human papillomavirus type 16 with different tags. We prepared eight different PVX CP constructs modified with the E7 epitope fused with the 6xHis tag in both orientations (6xHis-E7, E7-6xHis), cloned them into the PVX-based vector pGR106 and expressed them transiently in Nicotiana benthamina plants. Only the fusion site located after amino acid 23 led to systemic infection of plants and the production of recombinant proteins, but no viral particles were detected. When we replaced the 6xHis with StrepII tag, the modified virus infected plants systemically, expressed proteins assembled into viral particles and the epitopes were located on the particle surface. The results of this study indicate that the new position within the PVX CP can be used for peptide presentation on the surface of PVX particles and is promising for PVX based production of therapeutic compounds in plants.

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“…2A, 2B; Table 1) [20]. The nucleotide sequences of short peptides comprising 6xHis tag fused either to the 5'or 3' terminus of the E7 epitope (6xHisE7 and E76xHis, respectively) were inserted by splicing the overlap extension (SOE) PCR [21] into seven putative PVX CP surface exposed loops: 66/67 nt (region of predicted loop A: 67-72 nt), 459/460 nt (region of predicted loop B: 457-468 nt), 576/577 nt (region of predicted loop C: 574-582 nt), 669/670 nt (region of predicted loop D: 661-681 nt), 159/160 (region of predicted loop E: 154-162 nt), 213/214 (region of predicted loop F: 211-213 nt) and 534/535 (region of predicted loop G: 526-552 nt) [19].…”

Section: Dna Constructsmentioning

confidence: 99%

“…1) [19]. These putative surface loops could be promising candidates for new positions to present proteins or peptides.…”

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confidence: 99%

New positions for peptide presentation in Potato virus X capsid protein

et al. 2015

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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...

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Tritium planigraphy study of structural alterations in the coat protein of Potato virus X induced by binding of its triple gene block 1 protein to virions

Cited by 26 publications

References 30 publications

Generation of an Attenuated, Cross-Protective Pepino mosaic virus Variant Through Alignment-Guided Mutagenesis of the Viral Capsid Protein

Generation of an Attenuated, Cross-Protective Pepino mosaic virus Variant Through Alignment-Guided Mutagenesis of the Viral Capsid Protein

Potato virus X displaying the E7 peptide derived from human papillomavirus type 16: a novel position for epitope presentation

New positions for peptide presentation in Potato virus X capsid protein

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