Hamster polyomavirus-derived virus-like particles are able to transfer in vitro encapsidated plasmid DNA to mammalian cells.
The authentic major capsid protein 1 (VP1) of hamster polyomavirus (HaPyV) consists of 384 amino acid (aa) residues (42 kDa). Expression from an additional in-frame initiation codon located upstream from the authentic VP1 open reading frame (at position -4) might result in the synthesis of a 388 aa-long, amino-terminally extended VP1 (aa -4 to aa 384; VP1(ext)). In a plasmid-mediated Drosophila Schneider (S2) cell expression system, both VP1 derivatives as well as a VP1(ext) variant with an amino acid exchange of the authentic Met1Gly (VP1(ext-M1)) were expressed to a similar high level. Although all three proteins were detected in nuclear as well as cytoplasmic fractions, formation of virus-like particles (VLPs) was observed exclusively in the nucleus as confirmed by negative staining electron microscopy. The use of a tryptophan promoter-driven Escherichia coli expression system resulted in the efficient synthesis of VP1 and VP1(ext) and formation of VLPs. In addition, establishment of an in vitro disassembly/reassembly system allowed the encapsidation of plasmid DNA into VLPs. Encapsidated DNA was found to be protected against the action of DNase I. Mammalian COS-7 and CHO cells were transfected with HaPyV-VP1-VLPs carrying a plasmid encoding enhanced green fluorescent protein (eGFP). In both cell lines eGFP expression was detected indicating successful transfer of the plasmid into the cells, though at a still low level. Cesium chloride gradient centrifugation allowed the separation of VLPs with encapsidated DNA from "empty" VLPs, which might be useful for further optimization of transfection. Therefore, heterologously expressed HaPyV-VP1 may represent a promising alternative carrier for foreign DNA in gene transfer applications.
Voronkova
Tatyana
T
Biomedical Research and Study Centre, Ratsupites 1, Riga LV-1067, Latvia. tatyanav@biomed.lu.lv
Kazaks
Andris
A
Ose
Velta
V
Ozel
Muhsin
M
Scherneck
Siegfried
S
Pumpens
Paul
P
Ulrich
Rainer
R
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D004330:Drosophila; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010957:Plasmids; D011120:Polyomavirus / Q000235:genetics; D014162:Transfection / Q000379:methods*; D014760:Viral Fusion Proteins / Q000737:chemistry / Q000235:genetics; D014771:Virion
D036022:Capsid Proteins; D014760:Viral Fusion Proteins