Virus Research 155 (2011) 381–388
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Virus Research
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The anti-apoptotic protein L
*
of Theiler’s murine encephalomyelitis virus
(TMEV) contains a mitochondrial targeting signal
Toshiki Himeda, Takako Okuwa, Masafumi Nojiri, Yasushi Muraki, Yoshiro Ohara
∗
Department of Microbiology, Kanazawa Medical University School of Medicine, 1-1 Uchinada, Ishikawa 920-0293, Japan
article info
Article history:
Received 1 September 2010
Received in revised form
16 November 2010
Accepted 19 November 2010
Available online 2 December 2010
Keywords:
TMEV
L
*
protein
Mitochondria
abstract
L
*
protein of TMEV is out-of-frame with the viral polyprotein from an alternative initiation codon AUG, 13
nucleotides downstream from the authentic polyprotein AUG. Anti-apoptotic activity of L
*
was demon-
strated by both ‘loss of function’ and ‘gain of function’ experiments. However, the precise mechanism(s)
of anti-apoptotic activity of L
*
remains to be clarified. In this study, L
*
was demonstrated to be localized
to mitochondria. It was also shown by the GFP fusion protein that N-terminal sequence of L
*
may con-
tain a mitochondrial targeting signal (MTS). Surprisingly, L
*
(5–70)
–GFP and L
*
(41–70)
–GFP were localized
to mitochondria although L
*
(1–70)
–GFP was distributed in the cytosol, suggesting L
*
has an MTS between
amino acid (AA) positions 41 and 70, and that L
*
(1–4)
inhibits its mitochondrial targeting. Furthermore,
L
*
(1–70)
–GFP was localized to the mitochondria by co-expression of L
*
(65–156)
, indicating that L
*
(65–156)
suppresses the inhibition of mitochondrial targeting by L
*
(1–4)
. These results suggest that the intra- or
inter-molecular interaction of L
*
regulates its mitochondrial localization. It is also suggested that L
*
may
inhibit the intrinsic apoptosis through the localization to mitochondria.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
Theiler’s murine encephalomyelitis virus (TMEV) belongs to the
genus Cardiovirus of the family Picornaviridae and is divided into
two subgroups on the basis of their different biological activities
(Oleszak et al., 2004; Roos, 2010; Takano-Maruyama et al., 2006).
GDVII subgroup strains cause acute and fatal encephalomyelitis in
mice without virus persistence or demyelination (Oleszak et al.,
2004; Roos, 2010; Takano-Maruyama et al., 2006). In contrast, TO
subgroup including DA and some (other) viral strains induces an
early, non-fatal polioencephalomyelitis of weanling mice, followed
by virus persistence and chronic demyelination in the spinal cords
(Oleszak et al., 2004; Roos, 2010; Takano-Maruyama et al., 2006).
This late demyelinating disease serves as an experimental model for
the human demyelinating disease, multiple sclerosis (MS) (Oleszak
et al., 2004; Roos, 2010; Takano-Maruyama et al., 2006). Viral per-
sistence is essential for bystander demyelination (Drescher et al.,
1997; Lipton et al., 2005; Monteyne et al., 1997). However, the pre-
cise mechanism(s) of virus persistence and demyelination remains
to be elucidated.
A small 17–18 kDa protein designated L
*
is out-of-frame with
the viral polyprotein from an alternative initiation codon AUG,
13 nucleotides downstream from the authentic polyprotein AUG
∗
Corresponding author. Tel.: +81 76 286 2211; fax: +81 76 286 3961.
E-mail address: ohara@kanazawa-med.ac.jp (Y. Ohara).
(Kong and Roos, 1991). L
*
protein is synthesized in TO subgroup
strains, but not GDVII subgroup strains (Michiels et al., 1995; Roos,
2010; Takano-Maruyama et al., 2006). It is shown to be essential for
virus growth in J774.1 macrophage cells, the major site of virus per-
sistence, by both ‘loss of function’ and ‘gain of function’ experiments
(Himeda et al., 2005a; Obuchi et al., 1999; Takata et al., 1998).
Anti-apoptotic activity of L
*
protein was also demonstrated by
both ‘loss of function’ and ‘gain of function’ experiments (Ghadge
et al., 1998; Himeda et al., 2005b). However, precise mecha-
nism(s) of inhibition of apoptosis by L
*
protein remains to be
clarified. We previously reported that L
*
is immunocytologically
co-localized with ␣/-tubulin (Obuchi et al., 2001). Thereafter, it
was reported that tubulin is an inherent component of mitochon-
drial membranes that interacts with the voltage-dependent anion
channel (VDAC), which is the major channel of mitochondrial outer
membrane (MOM) (Carre et al., 2002; Rostovtseva et al., 2008). Reg-
ulation of MOM permeability is important in apoptosis by release of
apoptogenic factors into the cytosol. Therefore, it is of great interest
to investigate the relationship between L
*
and mitochondria.
Interestingly, a PSORTII analysis of the amino acid (AA) sequence
of L
*
protein predicted a cleavage site of mitochondrial targeting
signal (MTS) between residues 64 and 65 that matched R-3 motif
(Schneider et al., 1998)(Fig. 1A). In addition, it was reported that
3×FLAGL
*
, N-terminus of L
*
was tagged with 3×FLAG, could not
rescue the growth of DAL
*
-1 in J774 cells (Himeda et al., 2005a),
suggesting that N-terminus of L
*
may play an important role for
its activity. Therefore, we studied the distribution of L
*
in the L
*
-
0168-1702/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.virusres.2010.11.006