Restoration of potato virus X coat protein capacity for assembly
with RNA after His-tag removal
Olga Zayakina Æ Marina Arkhipenko Æ
Alexander Smirnov Æ Nina Rodionova Æ
Olga Karpova Æ Joseph Atabekov
Received: 15 September 2008 / Accepted: 4 December 2008 / Published online: 8 January 2009
Ó Springer-Verlag 2009
Abstract We found that the fusion of hexahistidine (H)
tag to the potato virus X (PVX) coat protein (CP) abolished
its ability to be assembled with viral RNA into helical
nucleoprotein virus-like particles (VLPs). Instead, irregular
agglomerates were produced upon incubation of PVX RNA
-tagged PVX CP. A factor Xa recognition site,
IEGR, was inserted upstream of the CP coding sequence.
Removal of the (H)
tag from PVX CP by Xa protease
restored its ability to bind RNA and to assemble VLPs. In
addition to the canonical IEGR site, the factor Xa protease
was found to cleave PVX CP at a second (non-consensus)
site, AVTRGR, located close to the C-terminus of PVX
CP. The latter cleavage did not affect reassembly of the
PVX RNA and CP into VLPs.
Fusion of a polyhistidine tag (H)
with a protein molecule
is a commonly used method for puriﬁcation of recombinant
proteins. In most cases the (H)
tag does not affect protein
properties [2, 7, 12], but sometimes it can alter the bio-
logical activities of the protein [4, 11, 14, 16–19].
Potato virus X (PVX) is a ﬁlamentous positive-strand
RNA plant virus, the type member of the genus Potexvirus.
PVX coat protein (CP) binds PVX RNA to form helical
virions. CP plays an important role in cell-to-cell PVX
movement  and translational regulation of PVX genome
RNA in the course of the infection cycle [1, 9, 10, 15].
Previously, we examined the structure of the particles
reassembled in vitro from native PVX CP and RNA .
The major product of assembly was represented by single-
tailed virus-like particles with the 5
-proximal region of
PVX RNA encapsidated in a helical, head-like structure.
However, we have shown recently that N-terminally (H)
tagged preparations of the PVX CP were not functional in
reassembly of virus-like particles (VLPs) in vitro with PVX
RNA . In contrast Kwon et al.  assembled PVX-
like particles using a bacterially expressed PVX CP with an
Activation of (H)
-tagged PVX CP reassembly
-tag removal with Xa protease
Full-length recombinant (H)
-CP and native CP isolated
from PVX were used in experiments on in vitro PVX RNA
and CP assembly. PVX (Russian strain) was isolated from
systemically infected Datura stramonium L. plants by
differential centrifugation. Virus preparations were used
for CP and RNA isolation. PVX CP was puriﬁed by the
method of salt deproteinization . RNA was isolated with
phenol and analyzed by 1% agarose gel electrophoresis .
Expression and puriﬁcation of recombinant CP have been
described previously . The (H)
tag was added to the
N-terminus of the CP molecules because none of the
N-terminal modiﬁcations (including removal of 19 residues
by trypsin, phosphorylation and amino acid substitutions)
affected the PVX CP RNA-binding properties .
Assembly of VLPs was performed as described [9, 10].
PVX RNA (1 lg) and CP were incubated for 20 min at
O. Zayakina Á M. Arkhipenko Á A. Smirnov Á N. Rodionova Á
O. Karpova Á J. Atabekov (&)
Department of Virology, Moscow State University,
119991 Moscow, Russia
A. N. Belozersky Institute of Physico-Chemical Biology,
Moscow State University, 119991 Moscow, Russia
Arch Virol (2009) 154:337–341