Generation of recombinant virus-like particles of human and non-human polyomaviruses in yeast Saccharomyces cerevisiae.
OBJECTIVE
Non-viral methods of gene transfer have been preferred in gene therapy approaches for several reasons, particularly for their safety, simplicity and convenience in introducing heterologous DNA into cells. Polyomavirus virus-like particles (VLPs) represent a promising carrier for encapsidation of foreign nucleic acids for gene therapy. For the development of such gene delivery systems as well as for providing reagents for improving virus diagnostics, an efficient yeast expression system for the generation of different polyomavirus VLPs was established.


METHODS
A galactose-inducible Saccharomyces cerevisiae yeast expression system was used. Formation of empty VLPs was confirmed by cesium chloride ultracentrifugation, agarose gel electrophoresis and electron microscopy. Cross-reactivity of the major capsid proteins (VP1) of different polyomaviruses was analyzed by Western blot using rabbit and mice sera raised against the VP1 proteins.


RESULTS
VP1 of polyomaviruses from humans (JC polyomavirus and serotypes AS and SB of BK polyomavirus), rhesus monkeys (simian virus 40), hamsters (hamster polyomavirus), mice (murine polyomavirus) and birds (budgerigar fledgling disease virus) were expressed at high levels in yeast. Empty VLPs formed by all yeast-expressed VP1 proteins were dissociated into pentamers and reassociated into VLPs by defined ion and pH conditions. Different patterns of cross-reactivity of the VP1 proteins with heterologous mice and rabbit sera were observed.


CONCLUSIONS
The developed heterologous yeast expression system is suitable for high-level production of polyomavirus VLPs. Yeast-derived VLPs are generally free of toxins, host cell DNA and proteins. These VLPs might be useful for the generation of new diagnostical tools, gene delivery systems and antiviral vaccines.
Sasnauskas
K
K
Institutes of Biotechnology, Leeds, UK. sasnausk@ibt.lt
Bulavaite
A
A
Hale
A
A
Jin
L
L
Knowles
W A
WA
Gedvilaite
A
A
Dargeviciūte
A
A
Bartkeviciūte
D
D
Zvirbliene
A
A
Staniulis
J
J
Brown
D W G
DW
Ulrich
R
R
D000595:Amino Acid Sequence; D000818:Animals; D036022:Capsid Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology; D006801:Humans; D008969:Molecular Sequence Data; D011120:Polyomavirus / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014771:Virion / Q000502:physiology*
D036022:Capsid Proteins; D011994:Recombinant Proteins; C062481:VP1 protein, polyomavirus