Abstract
BackgroundPseudoviruses (PsVs) that encapsidate a reporter plasmid DNA have been used as surrogates for native human papillomavirus (HPV), whose continuous production is technically difficult. HPV PsVs have been designed to form capsids made up of the major capsid protein L1 and the minor capsid proteins L2. HPV PsVs have been produced in 293TT cells transfected with plasmid expressing L1 and L2 protein and plasmid containing the reporter gene. Several studies have suggested that naturally occurring HPV virions contain cellular histones, and histones have also been identified in mature HPV PsVs. However, the effect of the histones on the properties of the PsVs has not been investigated. Using heparin chromatography, we separated mature HPV type 16 PsVs into three fractions (I, II, and III) according to their heparin-binding affinities.ResultsThe amounts of cellular histone and cellular nucleotides per PsV were found to increase in the order fraction I, II and III. It appeared that PsVs in fraction I contains just small amount of cellular histone in Western blot analysis. The proportions of the three fractions in PsV preparations were 83.4, 7.5, and 9.1 % for fraction I, II, and III PsVs, respectively. In the electron microscope PsVs in fraction I appeared to have a more condensed structure than those in fractions II and III. Under the electron microscope fraction II and III PsVs appeared to be covered by substantial amounts of cellular histone while there was no visible histone covering PsVs of fraction I. Also the levels of reporter gene expression in infections of fraction II and III PsVs to 293TT cells were significantly lower than those in infections of fraction I PsV, and fraction II and III particles had significantly reduced immunogenicity.ConclusionsOur findings suggest that the involvement of large amounts of cellular histones during PsV formation interferes with the structural integrity of the PsVs and affects their immunogenicity. The fraction I particle therefore has the most suitable characteristics for use as an HPV PsV.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0296-3) contains supplementary material, which is available to authorized users.
Highlights
Pseudoviruses (PsVs) that encapsidate a reporter plasmid DNA have been used as surrogates for native human papillomavirus (HPV), whose continuous production is technically difficult
In the present work we identified three types of HPV type 16 (HPV16) PsVs according to their heparin-binding affinities, and compared their structures, infectivity and immunogenicity, in order to identify the specific characteristics of histone-carrying PsVs
Separation of HPV16 PsVs by heparin chromatography Mature HPV16 PsVs were produced in 293TT cells by co-transfection of p16sheLL and pYSEAP and purified by size-exclusion chromatography (SEC, Fig. 1), and the PsVs were subdivided into fractions I, II, and III according to their heparin-binding affinities (Additional file 1: Figure S1 and Additional file 2: Figure S2): the heparin chromatography condition facilitates separation of PsVs containing small amounts of cellular histone from those with large amounts of histone
Summary
Pseudoviruses (PsVs) that encapsidate a reporter plasmid DNA have been used as surrogates for native human papillomavirus (HPV), whose continuous production is technically difficult. HPV PsVs have been produced in 293TT cells transfected with plasmid expressing L1 and L2 protein and plasmid containing the reporter gene. Kim et al BMC Biotechnology (2016) 16:63 and VP2 genes of MCV strain 339 and a Green fluorescent protein (GFP) reporter plasmid of 293TT cells engineered to stably express the cDNA of Simian virus 40 (SV40) T antigen [3]. Similar systems have been used for the PsVs of polyoma virus JCPyV [4], enterovirus 71 [5] and human papillomavirus (HPV) [6]. Some of the known roles of the L2 protein include facilitating capsid assembly, enhancing infectivity, and encapsidation of the HPV genome [14, 15]
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