title: Comparative analysis of recombinant Human Papillomavirus 8 L1 production in plants by a variety of expression systems and purification methods.
abstract: Human papillomavirus 8 (HPV-8), one of the high-risk cutaneous papillomaviruses (cHPVs), is associated with epidermodysplasia verruciformis and nonmelanoma skin cancer in immuno-compromised individuals. Currently, no vaccines against cHPVs have been reported; however, recent studies on cross-neutralizing properties of their capsid proteins (CP) have fostered an interest in vaccine production against these viruses. We examined the potential of producing HPV-8 major CP L1 in Nicotiana benthamiana by agroinfiltration of different transient expression vectors: (i) the binary vector pBIN19 with or without silencing suppressor constructs, (ii) the nonreplicating Cowpea mosaic virus-derived expression vector pEAQ-HT and (iii) a replicating Tobacco mosaic virus (TMV)-based vector alone or with signal peptides. Although HPV-8 L1 was successfully expressed using pEAQ-HT and TMV, a 15-fold increase was obtained with pEAQ-HT. In contrast, no L1 protein could be immune detected using pBIN19 irrespective of whether silencing suppressors were coexpressed, although such constructs were required for identifying L1-specific transcripts. A fourfold yield increase in L1 expression was obtained when 22 C-terminal amino acids were deleted (L1ΔC22), possibly eliminating a nuclear localization signal. Electron microscopy showed that plant-made HPV-8 L1 proteins assembled in appropriate virus-like particles (VLPs) of T = 1 or T = 7 symmetry. Ultrathin sections of L1ΔC22-expressing cells revealed their accumulation in the cytoplasm in the form of VLPs or paracrystalline arrays. These results show for the first time the production and localization of HPV-8 L1 protein in planta and its assembly into VLPs representing promising candidate for potential vaccine production.
authors.affiliation: Istituto di Virologia Vegetale, Consiglio Nazionale delle Ricerche, Strada delle Cacce, Turin, Italy.
authors.forename: Slavica
authors.initials: S
authors.lastname: Matić
authors.forename: Vera
authors.initials: V
authors.lastname: Masenga
authors.forename: Alice
authors.initials: A
authors.lastname: Poli
authors.forename: Riccardo
authors.initials: R
authors.lastname: Rinaldi
authors.forename: Robert G
authors.initials: RG
authors.lastname: Milne
authors.forename: Manuela
authors.initials: M
authors.lastname: Vecchiati
authors.forename: Emanuela
authors.initials: E
authors.lastname: Noris
mesh_terms: D015152:Blotting, Northern; D036022:Capsid Proteins / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000648:ultrastructure; D004269:DNA, Bacterial / Q000235:genetics; D015870:Gene Expression; D005821:Genetic Techniques; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D042541:Intracellular Space / Q000378:metabolism / Q000821:virology; D050505:Mutant Proteins / Q000378:metabolism; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000648:ultrastructure; D053147:Plant Exudates / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000648:ultrastructure; D013347:Subcellular Fractions / Q000821:virology; D014026:Nicotiana / Q000378:metabolism*; D014771:Virion / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D036022:Capsid Proteins; D004269:DNA, Bacterial; C114580:HPV L1 protein, Human papillomavirus; D050505:Mutant Proteins; D009856:Oncogene Proteins, Viral; D053147:Plant Exudates; D011994:Recombinant Proteins; C036483:T-DNA