Extreme evolutionary stability of conserved non-protein coding element of baculovirus genome
Aim. To estimate a level of evolutionary stability of the baculovirus conserved non protein-coding element (CNE) playing an essential role in baculovirus pathogenesis. Methods. NCBI-BLAST was applied to identify the orthologous sequences in genomes of 50 alphabaculoviruses. The orthologous sequences...
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Makarenko, V.E. Kikhno, I.M. Kashuba, V.I. 2019-06-13T07:24:52Z 2019-06-13T07:24:52Z 2016 Extreme evolutionary stability of conserved non-protein coding element of baculovirus genome / V.E. Makarenko, I.M. Kikhno, V.I. Kashuba // Вiopolymers and Cell. — 2016. — Т. 32, № 2. — С. 131-139. — Бібліогр.: 37 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000916 https://nasplib.isofts.kiev.ua/handle/123456789/152811 577 Aim. To estimate a level of evolutionary stability of the baculovirus conserved non protein-coding element (CNE) playing an essential role in baculovirus pathogenesis. Methods. NCBI-BLAST was applied to identify the orthologous sequences in genomes of 50 alphabaculoviruses. The orthologous sequences were aligned by using ClustalW software. Alistat tool was applied to obtain the pairwise percent identity ( %ID) matrix data. Results. An average pairwise ID value (73 %) calculated for the 1225-member sample was shown to be comparable with those of coding parts of the most conserved baculovirus genes polh and pif2 as well as with those of the polh and p18 promoter regions which are the most conserved representatives of a small group of the evolutionary stable promoter regions of the alphabaculovirus late genes. Conclusion. CNE is one of the most conserved elements of the alphabaculovirus genome. Мета. Визначити рівень еволюційної стабільності CNE (conserved non protein-coding element) – некодуючого функціонального елементу бакуловірусного геному, що грає виключну роль у патогенезі бакуловірусів. Методи. Для ідентифікації ортологічних послідовностей в геномах 50 альфабакуловірусів застосовували NCBI-BLAST. Ортологічні послідовності вирівнювали за допомогою алгоритму вирівнювання ClustalW. Отримували матрицю значень відсотків ідентичності (percent identity, %ID) для попарних порівняннь за допомогою програми Alistat. Результати. Середній рівень ID (73 %), розрахований для 1225-членної вибірки, виявився порівнюваним з такими показниками кодуючих послідовностей найконсервативніших бакуловірусних генів polh та pif2, а також з показниками промоторних ділянок polh та p18, що є найконсервативнішими представниками невеликої групи еволюційно стабільних промоторних ділянок пізніх генів. Висновки. CNE є одним з найконсервативніших елементів геномів альфабакуловірусів. Цель. Определить уровень эволюционной стабильности CNE (conserved non protein-coding element) – некодирующего функционального элемента бакуловирусного генома, который играет исключительную роль в патогенезе бакуловирусов. Методы. Для идентификации ортологичных последовательностей в геномах 50 альфабакуловирусов применяли NCBI-BLAST. Ортологичные последовательности выравнивали при помощи алгоритма выравнивания ClustalW. Получали матрицу значений процентов идентичности (percent identity, %ID) для попарных сравнений при помощи программы Alistat. Результаты. Средний уровень ID (73 %), рассчитанный для 1225-членной выборки, является сравнимым с таковыми показателями кодирующих последовательностей наиболее консервативных бакуловирусных генов polh и pif2, а также с показателями промоторных участков polh и p18, которые являются самыми консервативними представителями небольшой группы эволюционно стабильных промоторных участков поздних генов. Выводы. CNE является одним из наиболее консервативных элементов геномов альфабакуловирусов. en Інститут молекулярної біології і генетики НАН України Вiopolymers and Cell Viruses and Cell Extreme evolutionary stability of conserved non-protein coding element of baculovirus genome Надзвичайна еволюційна стабільність консервативного некодуючого елементу (CNE) геному бакуловірусів Чрезвычайная эволюционная стабильность консервативного некодирующего элемента (CNE) геномов бакуловирусов Article published earlier |
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| title |
Extreme evolutionary stability of conserved non-protein coding element of baculovirus genome |
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Extreme evolutionary stability of conserved non-protein coding element of baculovirus genome Makarenko, V.E. Kikhno, I.M. Kashuba, V.I. Viruses and Cell |
| title_short |
Extreme evolutionary stability of conserved non-protein coding element of baculovirus genome |
| title_full |
Extreme evolutionary stability of conserved non-protein coding element of baculovirus genome |
| title_fullStr |
Extreme evolutionary stability of conserved non-protein coding element of baculovirus genome |
| title_full_unstemmed |
Extreme evolutionary stability of conserved non-protein coding element of baculovirus genome |
| title_sort |
extreme evolutionary stability of conserved non-protein coding element of baculovirus genome |
| author |
Makarenko, V.E. Kikhno, I.M. Kashuba, V.I. |
| author_facet |
Makarenko, V.E. Kikhno, I.M. Kashuba, V.I. |
| topic |
Viruses and Cell |
| topic_facet |
Viruses and Cell |
| publishDate |
2016 |
| language |
English |
| container_title |
Вiopolymers and Cell |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| format |
Article |
| title_alt |
Надзвичайна еволюційна стабільність консервативного некодуючого елементу (CNE) геному бакуловірусів Чрезвычайная эволюционная стабильность консервативного некодирующего элемента (CNE) геномов бакуловирусов |
| description |
Aim. To estimate a level of evolutionary stability of the baculovirus conserved non protein-coding element (CNE) playing an essential role in baculovirus pathogenesis. Methods. NCBI-BLAST was applied to identify the orthologous sequences in genomes of 50 alphabaculoviruses. The orthologous sequences were aligned by using ClustalW software. Alistat tool was applied to obtain the pairwise percent identity ( %ID) matrix data. Results. An average pairwise ID value (73 %) calculated for the 1225-member sample was shown to be comparable with those of coding parts of the most conserved baculovirus genes polh and pif2 as well as with those of the polh and p18 promoter regions which are the most conserved representatives of a small group of the evolutionary stable promoter regions of the alphabaculovirus late genes. Conclusion. CNE is one of the most conserved elements of the alphabaculovirus genome.
Мета. Визначити рівень еволюційної стабільності CNE (conserved non protein-coding element) – некодуючого функціонального елементу бакуловірусного геному, що грає виключну роль у патогенезі бакуловірусів. Методи. Для ідентифікації ортологічних послідовностей в геномах 50 альфабакуловірусів застосовували NCBI-BLAST. Ортологічні послідовності вирівнювали за допомогою алгоритму вирівнювання ClustalW. Отримували матрицю значень відсотків ідентичності (percent identity, %ID) для попарних порівняннь за допомогою програми Alistat. Результати. Середній рівень ID (73 %), розрахований для 1225-членної вибірки, виявився порівнюваним з такими показниками кодуючих послідовностей найконсервативніших бакуловірусних генів polh та pif2, а також з показниками промоторних ділянок polh та p18, що є найконсервативнішими представниками невеликої групи еволюційно стабільних промоторних ділянок пізніх генів. Висновки. CNE є одним з найконсервативніших елементів геномів альфабакуловірусів.
Цель. Определить уровень эволюционной стабильности CNE (conserved non protein-coding element) – некодирующего функционального элемента бакуловирусного генома, который играет исключительную роль в патогенезе бакуловирусов. Методы. Для идентификации ортологичных последовательностей в геномах 50 альфабакуловирусов применяли NCBI-BLAST. Ортологичные последовательности выравнивали при помощи алгоритма выравнивания ClustalW. Получали матрицу значений процентов идентичности (percent identity, %ID) для попарных сравнений при помощи программы Alistat. Результаты. Средний уровень ID (73 %), рассчитанный для 1225-членной выборки, является сравнимым с таковыми показателями кодирующих последовательностей наиболее консервативных бакуловирусных генов polh и pif2, а также с показателями промоторных участков polh и p18, которые являются самыми консервативними представителями небольшой группы эволюционно стабильных промоторных участков поздних генов. Выводы. CNE является одним из наиболее консервативных элементов геномов альфабакуловирусов.
|
| issn |
0233-7657 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/152811 |
| citation_txt |
Extreme evolutionary stability of conserved non-protein coding element of baculovirus genome / V.E. Makarenko, I.M. Kikhno, V.I. Kashuba // Вiopolymers and Cell. — 2016. — Т. 32, № 2. — С. 131-139. — Бібліогр.: 37 назв. — англ. |
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131
V. E. Makarenko, I. M. Kikhno, V. I. Kashuba
© 2016 V. E. Makarenko et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/),
which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited
UDC 577
Extreme evolutionary stability of conserved non-protein coding element
of baculovirus genome
V. E. Makarenko, I. M. Kikhno, V. I. Kashuba
Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
i.m.kikhno@imbg.org.ua
Aim. To estimate a level of evolutionary stability of the baculovirus conserved non protein-coding element
(CNE) playing an essential role in baculovirus pathogenesis. Methods. NCBI-BLAST was applied to identify
the orthologous sequences in genomes of 50 alphabaculoviruses. The orthologous sequences were aligned by
using ClustalW software. Alistat tool was applied to obtain the pairwise percent identity (%ID) matrix data.
Results. An average pairwise ID value (73 %) calculated for the 1225-member sample was shown to be com-
parable with those of coding parts of the most conserved baculovirus genes polh and pif2 as well as with those
of the polh and p18 promoter regions which are the most conserved representatives of a small group of the
evolutionary stable promoter regions of the alphabaculovirus late genes. Conclusion. CNE is one of the most
conserved elements of the alphabaculovirus genome.
K e y w o r d s. Baculovirus, conserved non-coding element
Introduction
The recent advances in genome-wide studies have
revealed the abundance of extended non-coding re-
gions that are conserved at the sequence level across
eukaryotic genomes and that are believed to be re-
lated to basic processes in a eukaryotic cell [1].
Some of these regions known by several different
names (conserved non-coding element, CNE is one
among them) have been shown to encode the genes
of ncRNA or the 5` and 3` untranslated regions
(UTRs) of mRNAs. Others appear to be true non-
coding sequences, the functional significance of
which is still poorly understandable. Herewith, at
least some of them were shown to be involved in
transcriptional regulation of the key developmental
genes in metazoan genomes [2].
The amenability of RNA-coding CNEs to nega-
tive selection reflected in the CNE sequence conser-
vation can be explained by the necessity of strictly
defined structure of their RNA products for function-
al efficiency and/or molecule stability. In contrast,
none of the available hypotheses explaining amena-
bility of true non-coding CNEs to selective pressure
can be found quite satisfactory [3]. The point is that
the vast majority of known non-coding elements of
the genome (transcription and translation regulators,
origins of replication, sites of recombination, scaf-
fold attachment sites, etc.) recognized as platforms
for the protein trans-factors performing particular
DNA trans-actions have been found to evolve too
fast to be detected on the basis of their overall se-
quence conservation in the genomes of evolution-
arily distant organisms, and only short nucleotide
stretches (5-10bp), targets for the protein trans-fac-
tors, may be expected to constitute their conserved
parts. Herewith, regardless of the function that can
be attributed to CNEs, the length of these sequences
(up to several hundred bps) is much bigger than nec-
essary to specify the binding of a single protein
ISSN 1993-6842 (on-line); ISSN 0233-7657 (print)
Biopolymers and Cell. 2016. Vol. 32. N 2. P 131–139
doi: http://dx.doi.org/10.7124/bc.000916
132
V. E. Makarenko, I. M. Kikhno, V. I. Kashuba
trans-factor or even several cooperatively acting
proteins.
Pairwise comparison of eukaryotic genomes re-
sults in the identification of a variety of CNEs char-
acterized by vastly different levels of the sequence
conservation. While the conservation of some of
these CNEs appears to be a result of negative selec-
tion, the conservation of others may simply reflect
the short evolutionary distance or slow rate of neu-
tral divergence among the species compared [4].
Therefore, only the sequences showing an extreme
level of conservation in comparison with other ge-
nome elements can be reliably identified as CNEs
which are likely under evolutionary constraint. Some
of the most conserved CNEs may exhibit up to 100
% sequence identity (for instance, ultraconserved
CNEs in mammals [5]), and some of those may be
shared by so distantly related lineages as inverte-
brates and vertebrates [6].
Non protein-coding element essential for produc-
tive infection has been revealed recently in the ge-
nomes of insect pathogens, large dsDNA viruses of
family Baculoviridae [7]. The newly discovered ele-
ment was shown to express extended homology
(152–156 bp) across the representatives of genus
Alphabaculovirus and, accordingly it was called
CNE. The alphabaculovirus CNE is composed of
7 blocks of absolutely conserved nucleotides inter-
spaced by relatively variable nucleotide stretches of
conserved length. CNE of Autographa californica
nucleopolyhedrovirus (AcMNPV) was found to
overlap with short ORF and three ncRNA genes [8],
it includes three previously described protein-bind-
ing motifs [9] and sequence that activates minimal
promoter of adjacent gene ie-2 [10]. The transfec-
tion-infection assays using CNE knock out AcMNPV
demonstrated that an additional not-yet-defined es-
sential function is also attributed to СNE [7].
The present research was aimed at estimation of
the degree of CNE sequence conservation measured
as percentage of pairwise nucleotide identity (%ID)
and comparison with that of other functional ele-
ments of alphabaculovirus genome. An average pair-
wise percent ID (av %ID) of CNE across 1225 al-
phabaculovirus pairs was found to be 73 %. The es-
tablished fact that this value greatly exceeds the
conservation levels of the vast majority of baculovi-
rus genomic functional elements, both coding and
non-coding, strongly suggests that CNE represents
the essential genome element whose overall se-
quence appears to be under strong negative selection
Materials and methods
The nucleotide sequences of CNE, polh and pif2
were extracted from 50 alphabaculovirus genomes
available in GenBank database by using NCBI-
BLAST.
[The] Members of each orthologous group were
aligned by using ClustalW software [11]. [The] %ID
matrix table was built by Alistat software [Eddy S.
SQUID – C function library for sequence analysis
2005. [http://selab.wustl.edu/cgi-bin/selab.pl?mode
=software#squid] 1225-member %ID samples re-
sulted from pairwise comparison of 50 orthologs
were used for the av %ID calculation and subsequent
statistical analysis.
Results
1. CNE has evolved very slowly over the evolu-
tionary time
[The] Phylogenetic studies suggest that the represen-
tatives of genus Alphabaculovirus are pretty diverse
[12]. It is of interest if the level of virus diversifica-
tion is high enough to consider their common ele-
ment CNE as a sequence of extreme conservation.
Earlier for the 27 particular alphabaculovirus
pairs the evolutionary relationships were inferred on
the basis of values of average amino acid identity
(AAI) across all orthologs shared by two genomes
[13–22] (Table S1). CNEs of all members of these
pairs were subjected to [the] pairwise %ID analysis.
It was found that three most unrelated pairs
(LyxyMNPV- AcMNPV, LyxyMNPV- OpMNPV
and LyxyMNPV – MaviMNPV) are characterized
by 33 % AAI and 67–68 % CNE ID. The 33 % AAI
value points that the pairwise amino acid ID of the
majority of orthologous proteins shared by the mem-
133
Extreme evolutionary stability of conserved non-protein coding element of baculovirus genome
bers of aforementioned pairs is close to so called
“twilight zone” (region between 30 % and 20 % ID
where homology may exist but cannot be reliably as-
sumed [23]). This means that the level of divergence
between members of the pair is as high as possible to
provide their relatedness detection on the basis of
protein homology search. Accordingly, the nucleo-
tide similarity search tools are poorly applicable for
the reliable detection of the orthologous genes for
the members of these virus pairs due to the fact that
the sequence similarity degrades more rapidly at the
DNA than at the amino acid level [24]. Herewith, in
spite of so large divergence between the species, the
level of their CNE ID (67–68 %) is still significant
for the nucleotide-based similarity detection. Taking
into account that the baculovirus replication is char-
acterized by a high degree of recombination events,
there is theoretical probability that such high level of
CNE %ID appears to be the result of a horizontal
transfer of CNE between distantly related baculovi-
Table S1. List of 27 virus pairs and their AAI and CNE ID percentage value
N Virus pair Average aminoacid identity for
shared ORFs ( %) CNE identity ( %) Reference
1 HearSNPV-G4-HzSNPV 99 96 [13]
2 HearMNPV- MacoNPV-B 98 98 [14]
3 AcMNPV-BmMNPV 93 90 [15]
4 AcMNPV-RoMNPV 91 94 [16]
5 HearMNPV-MacoNPV-A 90 92 [14]
6 CfDEFMNPV- AgMNPV 88 92 [17]
7 LyxyMNPV- LdMNPV 74 86 [18]
8 CfDEFMNPV- EppoNPV 69 84 [17]
9 CfDEFMNPV- OpMNPV 68 83 [17]
10 CfDEFMNPV- CfMNPV 68 81 [17]
11 HearMNPV- AgseNPV 58 86 [14]
12 AcMNPV- OpMNPV 56 80 [19]
13 AcMNPV - CfDEFMNPV 55 80 [17]
14 OrleNPV - EcobNPV 52 79 [20]
15 OrleNPV - ChchNPV 48 81 [20]
16 HearSNPV-G4- SeMNPV 46 76 [22]
17 AcMNPV- OrleNPV 43 78 [20]
18 AcMNPV - LdMNPV 42 69 [21]
19 HearSNPV-G4- LdMNPV 41 68 [22]
20 HearSNPV-G4- AcMNPV 41 75 [22]
21 HearSNPV-G4- BmNPV 41 78 [22]
22 HearMNPV-HearSNPV-G4 41 76 [14]
23 LyxyMNPV-SeMNPV 37 73 [18]
24 CfDEFMNPV- HearSNPV-G4 35 72 [22]
25 LyxyMNPV- AcMNPV 33 67 [18]
26 LyxyMNPV- OpMNPV 33 67 [18]
27 LyxyMNPv - MaviMNPV 33 68 [18]
Standard abbreviations of full virus names are used in the table
134
V. E. Makarenko, I. M. Kikhno, V. I. Kashuba
%
ID
CNE polhPROM polh pif2 p18PROM
40
20
80
100
60
Fig. 2. Box and whisker plot for distribution of ID values for
CNE sequence compared with those of the most conserved ele-
ments of baculovirus genome. All data were estimated for 1225
alphabaculovirus pairs from 50-member sample. Small black
squares indicate average %IDs, horizontal line within boxes dis-
play median %IDs. Polh promoter region, polh coding region,
pif2 coding region and p18 promoter region are marked as polh-
PROM, polh, pif2 and p18PROM respectively.
ruses. However, it is not the case with the CNE of
viruses above: an analysis of relationship between
pairwise AAIs and CNE %IDs (Fig.1) suggests that
the distribution of variables follows the linear rela-
tionship, and there is no any outlier on the plot in-
dicative of CNE transfer between highly unrelated
alphabaculoviruses.
Thus, based upon the data above, it can be sug-
gested that CNE evolved very slowly from the early
stages of the alphabaculovirus evolution.
2. CNE conservation level is comparable with
those of the most conserved coding regions
In order to quantify the relative CNE conservation
value we compared the degree of CNE conservation,
calculated as an av % ID for the 1225-member sam-
ple resulted from pairwise comparison of 50 CNE ,
with those of the genes coding regions of polh (the
most conserved baculovirus protein) and pif2 (the
most conserved protein among 34 core proteins
shared by all baculoviruses) [25]. In the work pre-
sented here we have not checked for, nor removed
recombinants, assuming that av %ID values cannot
be affected significantly by the presence of some re-
combinants in the 1225-member sample.
CNE, polh and pif2 sequences were extracted
from each of 50 alphabaculovirus genomes available
in the GeneBank database and subsequently aligned.
1225 %ID values displayed in the %ID matrix
table were subjected to statistical analysis. Box and
whisker plots were used to visualize the distribution
ranges of %IDs for each orthologous group (Fig. 2).
The dataset of each group of orthologs was found to
follow a nearly normal distribution (p<0.01). The
pairwise IDs values calculated for 1225 virus pairs
ranged from 62 % to 100 % (av %ID 73 %) for the
CNE, from 79 % to 100 % (av %ID 88 % ) for the
polh, and from 43 % to 100 % (av %ID 65 %) for
the pif2.
The comparison of av %ID of the CNE and re fe-
renced genes showed that CNE tends to be more
conserved than pif2 and less conserved than polh.
Consequently, CNE can be defined as an exceedin-
gly conserved genomic element, the conservation
degree of which is comparable with that of the co-
ding regions of the most conserved baculovirus
protein genes.
60 70 80 90 100
% ID CNE
%
A
A
I
30
40
50
60
70
80
90
100
Fig. 1. Relationship between average amino acid identity
(AAI) and CNE identity (ID) in alphabaculovirus genomes.
The dot plot was created on the basis of 27 virus pairs dataset
listed in TanbleS1.
135
Extreme evolutionary stability of conserved non-protein coding element of baculovirus genome
3. CNE is quite unique among baculovirus non-
protein coding elements in respect of its evolu-
tionary stability
Besides CNE, the baculovirus set of the known non
protein-coding functional elements includes the pro-
moters of different temporal regulation, hrs (homol-
ogous repeat regions, that play roles of enhancers
and origins of replication), so called non-hr origins
of replication (Rewieved in [26]), 5` and 3` UTRs of
mRNAs, ncRNA genes (ncRNA of unknown func-
tion [8] and microRNA precursor genes [27] are
among them). It is well established that the known
alphabaculovirus non-hr origins are not alignable
[28], as well as hrs originating from the genomes of
relatively diverse alphabaculoviruses do not express
extended homology, although they contain short
conserved palindromic segment, a binding site for
ie1 [29]. The nucleotide sequences of baculovirus
genes of miRNA precursors were found not being
conserved even among closely related baculovirus-
es [30].
The question whether CNE is unique among the
alphabaculovirus non protein-coding elements in re-
spect of extended sequence conservation arises from
these data. To clarify this issue we focused on the
search for the functional non protein-coding se-
quences of extended length shared by the alphabacu-
lovirus genomes.
The promoter regions of 44 late and very late
genes shared by all alphabaculoviruses were cho-
sen as a class of elements subjected to the analysis.
The baculovirus late and very late genes are spe-
cific in the conserved motif (A/T/G)TAAG, which
serves as a transcriptional initiator [8]. The 12–18-
bp region encompassing the TAAG has been shown
as a minimal promoter determinant for the basal
transcription from late [31] and very late promoters
[32]. AcMNPV was used as a reference virus to de-
fine the exact extension of the promoter region
(5`UTR in conjunction with overlapping minimal
promoter) of each gene because the genuine tran-
scription start sites of all AcMNPV genes were de-
termined [8]. The regions extracted for the analysis
from the AcMNPV genome were bounded by a
nucleotide upstream from the translation start co-
don and by the 30-bp nucleotide stretch upstream
from the transcription start site TAAG. The pro-
moter regions less than 40 bp in length were ex-
cluded, and finally 37 of 44 regions were chosen
for the analysis, including the regions of polh, lef-
9, ODV-e18 , Ac81, lef-5, vlf-1, 38K, gp41, p33,
alk-exo, p6.9, lef-2, Ac68, vp91, vp59, p143, pp34,
ODV-e25, Ac106, Ac1313, pp31, Ac53, 25K, Ac75,
Ac76, p18, ODV-e28, Ac102, F-protein, ODV-c42,
me53, p78/83, Ac19, calyx, pkip, Ac60. The recent-
ly published phylogenetic tree was used as a guide
to infer the evolutionary distances within alphabac-
uloviruses [33], and 10 viruses belonging to diverse
evolutionary lineages were chosen for the first
round of analysis: AcMNPV, EppoNPV, LeseNPV,
AgMNPV, OrleNPV, LdMNPV, HearSNPV,
TnSNPV, MacoNPV-B, SeMNPV. The promoter
regions of each of 37 genes were extracted from
each of 10 above genomic sequences. The lengths
of extracted sequences were similar to the lengths
of their AcMNPV orthologs if their TAAG was
found at the position close to that of TAAG site in
the AcMNPV promoter region. If the TAAG was
absent in the above position the 5`end of the ex-
tracted sequence was extended up to the first up-
stream TAAG.
The multiple sequence alignment was used to as-
sess the conservation levels of 37 promoter regions.
The consensus sequences derived from the align-
ments served as the main criteria of conservation.
A strong tendency to the extended sequence conser-
vation was clearly displayed by the alignments of
9 promoter regions (polh, ODV-e18, pp34, ODV-e25,
pp31, 25K, p18, Ac102, calyx): long sequence
stretches (from 40 to 140bp) enriched by absolutely
conserved nucleotides were visualized. The most
conserved of them assigned to the polh promoter re-
gion and the longest of them assigned to the p18 pro-
moter region were chosen for the subsequent align-
ing using a sample from 50 viruses. The multiple
alignments demonstrated that the conserved nucleo-
tide stretch of the polh promoter region is represent-
ed by full 5`UTR, whereas the conserved nucleotide
136
V. E. Makarenko, I. M. Kikhno, V. I. Kashuba
stretch of the p18 promoter region includes not only
the full 5`UTR but also a dozen of conserved nucleo-
tides located just upstream the TAAG.
The %ID datasets of the promoter regions of both
genes were found to be distributed nearly normally
(p<0.01). ID values of the polh promoter region var-
ied from 49 % to 100 % (av %ID 74 %) over se-
quence lengths ranging from 39 to 42 bp. ID values
of p18 varied from 35 % to 100 % (av %ID 65 %)
over sequence lengths ranging from 129 to 145 bp
(Fig.2). The comparison of avID values allows the
suggestion that CNE index of this value is nearly the
same as that of the polh promoter region and higher
than that of the p18 promoter region.
Discussion
In this study the relationship between the CNE con-
servation degree and the degree of virus pair diver-
gence has been analyzed. This preliminary analysis
revealed the tendency to strong evolutionary stabil-
ity of CNE. A comparison of the CNE conservation
level with those of the nucleotide sequences of
genes for the structural proteins under strong evolu-
tionary constraint (polh and pif2) and with those of
the most conserved promoter regions of the late
genes (polh and p18) was done to investigate the
observed tendency further. The CNE sequences,
gene coding sequences and gene promoter region
sequences were extracted from 50 alphabaculovirus
genomes available in NCBI database, aligned and
subjected to pairwise comparison. Resulting data al-
lowed us to recognize the alphabaculovirus CNE as
an element of extreme conservation across the 152–
156 bases, being more highly conserved than the
vast majority of both coding and non-coding se-
quences in the alphabaculovirus genomes. CNE is
unique among the previously studied baculovirus
non protein-coding elements (hrs, non-hr oris,
genes of miRNA precursors) because the latter do
not exhibit a tendency of sequence conservation be-
tween distantly related alphabaculoviruses (see ref-
erences in Results, section3).
Of the particular interest are the results of promot-
er region analysis conducted within a framework of
this study: nine baculovirus late genes (polh,
ODV-e18, pp34, ODV-e25, pp31, 25K, p18, Ac102,
calyx) were found to be associated with the promoter
regions of relatively high levels of sequence conser-
vation. ID values of the two most conserved of them
(the polh promoter region including 5’UTR and p18
promoter region including 5’UTR and dozen nucleo-
tides upstream from the transcription start site) were
found to be comparable with the CNE ID value. An
extreme conservation of some baculovirus 5`-UTRs
is quite explicable: 5`UTRs may represent RNA re-
gions that adopt a selectively constrained spatial
structure providing molecule stability and/or inter-
action with proteins.
In contrast, it is difficult to predict whether the re-
vealed phenomenon of extreme conservation of the
alphabaculovirus CNE specifies its similarity with
some eukaryotic CNEs or it indicates the uniqueness
of some viral element providing the alphabaculovi-
rus-specific function(s).
Three alternative (but not mutually exclusive) hy-
potheses may be suggested to explain the above phe-
nomenon.
1. “The overlap hypothesis”. The CNE represents
the region of overlapping several non-coding ele-
ments of extended length. The overlapping elements
may evolve slowly due to the increasing deleterious
effect of mutations affecting more than one element.
However, this assumption is valid for the explana-
tion of overall high conservation degree of the CNE
but the following question remains unanswered:
why is the length of quite variable CNE blocks kept
unaltered (i.e. are any insertions or deletions within
variable regions not allowed)?
2. “The secondary structure hypothesis”. The
CNE nucleotide sequence conservation is dictated
by significance of the secondary structure of this ele-
ment at either DNA or RNA level. This assumption
is supported by the fact of CNE enrichment in the
dyad symmetry elements that specifies CNE as a se-
quence potentially capable of the DNA conforma-
tional changes [34], and by the fact that as shown by
the AcMNPV transcriptome analysis three ncRNA
are likely overlap CNE in the AcMNPV genome [8].
137
Extreme evolutionary stability of conserved non-protein coding element of baculovirus genome
It should be noted, however, that conserved parts of
the overlapping ncRNAs represented by CNE were
shown not to be essential for the AcMNPV life cycle
[7] and this put the necessity of their extreme conser-
vation under the question. In addition, the hypothe-
sis can be considered strictly valid only if the CNE-
overlapping ncRNAs would be identified in all other
alphabaculovirus genomes.
3. “The module hypothesis”. CNE appears to be
the module, consisting of several conserved motifs,
which bind cooperatively acting proteins. These
modules, within which the particular distances be-
tween protein-binding sites provide interaction of
bound proteins, were predicted by bioinformatic ap-
proaches in eukaryotic genomes [35]. Herewith,
such organization has been observed only in very
few cases of transcriptional regulatory elements
[36], among them the most studied is the enhancer
of interferon-β gene that rather represents [a] par-
ticular case as it consists of numerous non spaced
overlapping motifs [37]. While [the] c4-c5 CNE
conserved block was shown to be the only which
bind[s] proteins at 40 hours post infection [9], c2, c3
and c6 blocks remain the candidate binding motifs
for cooperatively acting proteins at some other time
point. It also seems plausible to speculate that two
similar terminal blocks c1 and c7 that bind proteins
in uninfected cell, as well as in early infection [9],
represent such recognition motifs for cooperatively
acting proteins, and the strictly predetermined dis-
tance between them appears to be the prerequisite of
their cooperation and, thus defines the fixed CNE
length.
Further experimental research is necessary to in-
fer the essence of extreme conservation of the alpha-
baculovirus CNE, so the functional and structural
studies of this element are under way.
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Надзвичайна еволюційна стабільність консервативного
некодуючого елементу (CNE) геному бакуловірусів
В. Е. Макаренко, І. В. Кіхно, В. І. Кашуба
Мета. Визначити рівень еволюційної стабільності CNE
(conserved non protein-coding element) – некодуючого функціо-
нального елементу бакуловірусного геному, що грає виключну
роль у патогенезі бакуловірусів. Методи. Для ідентифікації
ортологічних послідовностей в геномах 50 альфабакуловірусів
застосовували NCBI-BLAST. Ортологічні послідовності вирів-
нювали за допомогою алгоритму вирівнювання ClustalW.
Отримували матрицю значень відсотків ідентичності (percent
identity, %ID) для попарних порівняннь за допомогою програ-
ми Alistat. Результати. Середній рівень ID (73 %), розрахова-
ний для 1225-членної вибірки, виявився порівнюваним з таки-
ми показниками кодуючих послідовностей найконсервативні-
ших бакуловірусних генів polh та pif2, а також з показниками
промоторних ділянок polh та p18, що є найконсервативнішими
представниками невеликої групи еволюційно стабільних про-
моторних ділянок пізніх генів. Висновки. CNE є одним з най-
консервативніших елементів геномів альфабакуловірусів.
К л юч ов і с л ов а: бакуловіруси, консервативний некодую-
чий елемент.
Чрезвычайная эволюционная стабильность
консервативного некодирующего элемента (CNE)
геномов бакуловирусов
В. Э. Макаренко, И. В. Кихно, В. И. Кашуба
Цель. Определить уровень эволюционной стабильности CNE
(conserved non protein-coding element) – некодирующего функ-
139
Extreme evolutionary stability of conserved non-protein coding element of baculovirus genome
ционального элемента бакуловирусного генома, который игра-
ет исключительную роль в патогенезе бакуловирусов. Методы.
Для идентификации ортологичных последовательностей в ге-
номах 50 альфабакуловирусов применяли NCBI-BLAST.
Ортологичные последовательности выравнивали при помощи
алгоритма выравнивания ClustalW. Получали матрицу значе-
ний процентов идентичности (percent identity, %ID) для попар-
ных сравнений при помощи программы Alistat. Результаты.
Средний уровень ID (73 %), рассчитанный для 1225-членной
выборки, является сравнимым с таковыми показателями коди-
рующих последовательностей наиболее консервативных баку-
ловирусных генов polh и pif2, а также с показателями промо-
торных участков polh и p18, которые являются самыми консер-
вативними представителями небольшой группы эволюционно
стабильных промоторных участков поздних генов. Выводы.
CNE является одним из наиболее консервативных элементов
геномов альфабакуловирусов.
К л юч е в ы е с л ов а: бакуловирусы, консервативный неко-
дирующий элемент
Received 11.02.2016
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