Natural insertions within the N-terminal region of the coat protein
of Maize dwarf mosaic potyvirus (MDMV) have an effect
on the RNA stability
Kathrin Petrik • Endre Sebestye´n • Gyo¨ngyve´r Gell •
Ervin Bala´zs
Received: 8 July 2009 / Accepted: 10 November 2009 / Published online: 24 November 2009

Springer Science+Business Media, LLC 2009
Abstract A 13 amino acid residue insertion was found in
the N-terminal region of the coat protein of several Maize
dwarf mosaic virus isolates (MDMV). These insertions seem
to be the result of a direct duplication event, but differ in
some positions. In order to evaluate the influence of the
insertion on the RNA secondary structure and stability, the
RNA secondary structures and minimum free energies
(MFE) of all existing MDMV coat protein sequences were
estimated using three different softwares, the Vienna RNA
Package, NUPACK, and UNAFold, and compared to the
secondary structure and MFE of various random sequence
collections preserving the nucleotide distribution of
MDMV. The bioinformatic analysis showed that the inser-
tion stabilizes the RNA structure of the coat protein gene.
Keywords Potyvirus
Coat protein

RNA secondary structure
RNA stability
Insertion
Potyviruses are single-stranded positive-sense RNA viru-
ses, allowing them to escape from the innate defence
mechanisms and acquired immune surveillance of the host,
due to the inherently error-prone replication, and to rapidly
adapt to new cell types, tissues or species [1]. The N-ter-
minal region of potyvirus coat proteins is one of the most
diverse regions within their genome and is important for the
adaptation of the virus to its host [2]. Maize dwarf mosaic
potyvirus (MDMV), which infects maize, sweet corn, mil-
let, and several sorghum species, is an economically
important virus disease worldwide. The morphological
structure is composed of approximately 2,000 copies of the
coat protein, the main functions of which are the encapsi-
dation of the viral RNA, virus replication, cell-to-cell and
systemic movement, and the regulation of viral RNA
amplification [3, 4]. The coat protein core and C-terminal
regions are mainly involved in the encapsidation and cell-
to-cell movement [5]. The more flexible N-terminal region
plays a role in long distance and systemic movement [6, 7]
and also contains the DAG motif, which is responsible for
the competence of aphid transmission. Differences in the
N-terminal region are important for potyvirus discrimina-
tion [8]. The N- and C-termini of the coat proteins are
surface-located and these termini constitute the most
immunodominant regions in the virus particle. To´bia´s and
Palkovics [9] described a 13 amino acid (39 bp) residue
insertion within the N-terminal region of the MDMV coat
protein, which appears to be the result of a direct duplica-
tion event, although the duplicated region has since accu-
mulated mutations. Earlier investigations showed that these
insertions are relatively frequent, and four other MDMV
isolates with an insertion were recovered [10].
In addition to a large number of functional studies on the
RNA secondary structure of the 50- and 30-terminal regions
of various virus species [11, 12] studies were also carried
out on genome-scale ordered RNA structures (GORS),
which show significant variability between genera [1, 13]. It
was shown that GORS are absent from potyviruses, sug-
gesting that the association with nucleocapsid proteins
limits or restricts the need for this type of viral structure [1].
In the present study, a minimum free energy (MFE)
analysis of the viral RNA is described to estimate the
influence of the 39-bp insertions within the N-terminal
region of MDMV on the RNA secondary structure and
stability.
K. Petrik (&)
E. Sebestye´n
G. Gell
E. Bala´zs
Department of Applied Genomics, Agricultural Research
Institute of the Hungarian Academy of Sciences, Brunszvik u. 2,
2462 Martonva´sa´r, Hungary
e-mail: kathrin.petrik@gmail.com
123
Virus Genes (2010) 40:135–139
DOI 10.1007/s11262-009-0425-3

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T
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av
er
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e
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es
,
th
e
st
an
da
rd
de
vi
at
io
n,
m
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um
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um
va
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es
w
er
e
ba
se
d
on
th
e
or
ig
in
al
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qu
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ce
or
a
gi
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n
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ta
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t,
co
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a
1,
00
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qu
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s
(s
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te
xt
)
N
A
:
no
t
av
ai
la
bl
e
Virus Genes (2010) 40:135–139 137
123

The influence of the insertion on RNA stability was
inspected by comparing the MFE of the RNA of 93
MDMV coat protein sequences to various types of random
and shuffled sequences. The dataset used in the compari-
sons contained a collection of 1,000 random sequences
with the same trinucleotide distribution as the MDMV coat
protein sequence RNA. Sequences were generated for three
different lengths: random1000867 bp for Spanish MDMV
isolates (AJ416633-AJ416645, AJ416648) missing the first
three amino acids, random1000915 bp for MDMV isolates
with an insertion (AJ563725, FM883164, FM883177,
FM883200, and DQ973169) and random1000876 bp for all
other MDMV isolates (FM883165-FM883176, FM883178-
FM883199, FM883201-FM883228, AJ563726, A34974,
AF395135, AJ001691, AJ542536, AJ563724, AM490848,
AM490849, AM110758, AM491607, MDU07216, and
NC003377). The average MFE values and the standard
deviation of a given random sequence dataset, containing
1,000 sequences, were calculated and compared with the
MFE values of the respective MDMV isolates.
The comparison of the random sequence datasets with the
MDMV sequence MFE values generated by the Vienna RNA
Package showed that the MFE of MDMV coat protein RNA
without the 39-bp insertion was 5.4% lower on average than
the MFE of the corresponding random sequence collection
(random1000867 bp and random1000876 bp). Isolates with
insertions had 7.7% lower MFE values on average than the
appropriate random sequences (random1000915 bp), while
the MFE average of all MDMV sequences with an insertion
was 7.2 % lower than the MFE average of all MDMV
sequences without an insertion (Fig. 1a). In the following
step, three sequences (FM883164: Mv0801, FM883177:
Mv0814, and FM883200: Mv0702) containing the insertion
were compared to four different types of shuffled or random
sequences (Fig. 1b). Every sequence type was generated for
a 1,000 variations. The first sequence type was identical to
the previously mentioned 915 bp random sequences. The
second sequence type contained the original bases of a given
sequence but in shuffled positions. The third sequence type
consisted of sequences generated with the original insertion
sequence and a random surrounding sequence with the same
trinucleotide distribution as the MDMV isolate. The fourth
sequence type consisted of a 39-bp length random insert
sequences surrounded by the original sequence of the isolate
(Fig. 1c).
The results of UNAFold and NUPACK showed similar
trends, although the exact minimum free energy values are
varying, due to the differences in the algorithms. The
average MFE, the standard deviation, and minimum and
maximum values predicted by the various programs of
these random, shuffled and original sequences are descri-
bed in Table 1.
After comparing the MFE values of the MDMV
sequences and the different generated sequences, a one-way
ANOVA test was carried out on the results of the Vienna
RNA Package to determine the statistically significant dif-
ferences as follows. The MFE values of the above-men-
tioned insert-containing sequences and the average MFE
values of the 915-bp length random sequences, shuffled
isolates, 876 bp original insert containing random sequen-
ces and original isolates with random inserts were selected
(Table 1). The result of the test (F = 8.024722019,
Fcrit = 3.478049691, LSD = 9.327616604) showed that
most of the differences between the groups were statisti-
cally significant at the 95% level. The only exception was
the difference between the original sequences and the
sequences with the sequence type 4 (random insertion and
original surrounding sequence). Significant differences
between the isolate and the various types of shuffled or
random sequences are marked with an asterisk (*).
Insertions within the N-terminal region of the coat protein
are a frequent feature of potyviruses and were associated
with virus adaptation to outside influences and immuno-
dominant features. In general, it can be assumed that pot-
yviruses have a mutational hotspot in the N-terminal region
of the coat protein resulting in insertions and deletions. In
this study, the influences of the insertion of different MDMV
isolates on the basis of the different MFE datasets were
investigated. The positive effect of the insertion on the RNA
stability of MDMV isolates was shown. However, this sta-
bilizing effect does not depend on the composition of the
sequence, but only on the presence of it. The increased RNA
stability could be a further reason for the location specific
integration of short RNA segments in the MDMV genome.
Since the RNA secondary structure is known to play a fun-
damental role in RNA viral replication [1], the decrease in
the MFE values of the RNA sequence with the insertions
raises also the possibility that the insertion may have an
influence of the virus replication. This is the first bioinfor-
matic report on the possibly positive influence of an insertion
within the RNA sequence of the coat protein of MDMV.
Acknowledgments This study was supported by grants from the
German Academic Exchange Service (DAAD), the Hungarian Sci-
entific Research Fund (OTKA NI 61023) and the EU-FP7-REGPOT
2007-1 (AGRISAFE 203288) project. The authors are grateful to
B. Harasztos for the English revision of the manuscript.
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