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BRIEF REPORT
Complete nucleotide sequence and genome organisation
of grapevine Bulgarian latent virus
Toufic Elbeaino •Michele Digiaro •Frida Fallanaj •
Slobodan Kuzmanovic •Giovanni Paolo Martelli
Received: 1 September 2010 / Accepted: 23 December 2010 / Published online: 18 January 2011
ÓSpringer-Verlag 2011
Abstract The complete genome sequence of grapevine
Bulgarian latent virus (GBLV) has been determined. RNA-1
(7,452 nt in length) contains a single ORF of 6,285 nt,
encoding a polyprotein with conserved motifs characteristic
of the viral protease cofactor (Prot-cofact), the NTP-binding
protein (NTP), the cysteine-like protease (Cyst-Prot) and the
RNA-dependent RNA polymerase (RdRp) of members of
the order Picornavirales and show high aa sequence identity
with blackcurrant reversion virus (BRV, 64%). RNA-2
(5,821 nt) contains a single ORF of 4,500 nt, encoding a
polyprotein in which the conserved motifs of the movement
protein (MP) and coat protein (CP) have been identified. The
GBLV CP aa sequence shows highest homology with that of
blueberry leaf mottle virus (BLMoV, 68%). Both RNAs
have a poly(A) tail and a NCR at the 3’ and 5’ termini,
respectively. The results of this study confirm the classifi-
cation of GBLV as a member of a distinct species in sub-
group C of the genus Nepovirus.
Grapevine Bulgarian latent virus (GBLV) is a species in
the genus Nepovirus (order Picornavirales, family Seco-
viridae)[1] whose members are serologically distantly
related to blueberry leaf mottle virus (BLMoV) [2]. An
isolate belonging to this species has been recovered by sap
inoculation from symptomless Bulgarian grapevines of cv.
Rcatzitelli and characterized physicochemically [3]. This
virus has also been reported to occur in Portugal, the for-
mer Yugoslavia and Hungary [4]. Based on the size and
packaging of RNA-2, nepoviruses have been divided into
three subgroups [5]. The viral genome of GBLV consists of
two functional RNA species (RNA-1 and RNA-2) with a
molecular weight of 2.2 910
6
and 2.1 910
6
Da, respec-
tively [3], and this virus is currently assigned to subgroup
C, together with other grapevine-infecting nepoviruses, i.e.,
blueberry leaf mottle virus (BLMoV), grapevine Tunisian
ringspot virus (GTRSV), cherry leaf roll virus (CLRV),
peach rosette mosaic virus (PRMV) and tomato ringspot
virus (ToRSV) [5].
The amount of available molecular information on
viruses of subgroup C is limited, since it appears that the
complete genome sequences of only two members, black-
currant reversion virus (BRV) and ToRSV, have been
determined, while partial sequences are available for
BLMoV, PRMV and CLRV. We now report the complete
nucleotide sequence of both GBLV RNAs and discuss their
relationship at the molecular level with sequenced mem-
bers of the same subgroup.
GBLV was transmitted by sap inoculation to Chenopo-
dium quinoa plants from young leaves of glasshouse-forced
cuttings of an infected SO4 rootstock (V. berlandieri 9
V. riparia) from Serbia (accession Serb1), purified and
fractionated in 10-40% linear sucrose density gradients as
described [3]. Purified virions were exposed to antisera of
grapevine nepoviruses available at the Department of Plant
Protection and Applied Microbiology, University of Bari,
i.e., BLMoV, CLRV, GBLV, GTRSV, PRMV, ToRSV,
Arabis mosaic virus (ArMV), artichoke Italian latent virus
T. Elbeaino (&)M. Digiaro F. Fallanaj
Istituto Agronomico Mediterraneo di Bari, Via Ceglie 9,
70010 Valenzano, Bari, Italy
e-mail: elbeaino@iamb.it
S. Kuzmanovic
Institute for Plant Protection and Environment, Belgrade,
Serbia
G. P. Martelli
Dipartimento di Protezione delle Piante e Microbiologia
Applicata, Universita
`degli Studi, Via Amendola 165/A,
70126, Bari, Italy
123
Arch Virol (2011) 156:875–879
DOI 10.1007/s00705-010-0908-5
(AILV), grapevine Anatolian ringspot virus (GARSV),
grapevine chrome mosaic virus (GCMV), grapevine defor-
mation virus (GDefV), grapevine fanleaf virus (GFLV),
tomato black ring virus (TBRV), and tobacco ringspot virus
(TRSV), and observed under a Philips Morgagni electron
microscope.
Viral RNAs, extracted from a purified virus preparation
[6], were reverse-transcribed using random hexamers and/
or an oligo(DT) primer according to Gubler and Hoffman
[7]. DOP-PCR was first used to generate internal sequence
fragments with primer DOP4 (5’-CCGACTCGAGNNNN
NNTTTACG-3’) [8] using a ‘‘DOP-PCR Master kit’’
(Roche, Switzerland), following the manufacturer’s
instructions. The 3’ termini of both RNAs were amplified
using GBLV-specific primers designed on DOP-generated
clones in conjunction with an oligo(dT) primer, whereas
the 5’ termini were amplified using the 5’RACE-PCR
System (Roche Diagnostics, USA) with GBLV-specific
antisense primers. Sets of specific GBLV RNA-1 and
RNA-2 sense and antisense primers were then designed for
closing sequence gaps between the DOP clones. All PCR
runs consisted of 35 cycles, with an initial denaturing
temperature of 94°C for 1 min, 58°C for 30 sec and an
elongation time of 1 to 3 min at 72°C, depending on
amplicon size. All amplicons were cloned into the Strata-
Clone PCR cloning vector pSC-A (Stratagene, USA),
introduced into Escherichia coli DH5aor SoloPACK cells
and custom sequenced (Primm Co., Italy). Two specific
primers designed to amplify the CP cistron (ca. 1,566 bp)
of GBLV-Serb1, denoted GBLV-CPs (5’-AGTGCCCTTT
TAGCCGATACCAG-3’) and GBLV-CPa (5’-TCACTT
AAGTGGCGTTACGCT-3’), were used in RT-PCR for
comparative analysis with the corresponding gene of a
GBLV isolate from Bulgaria (GBLV-Blg1).
Nucleotide and protein sequences were analysed with
the assistance of the DNA Strider 1.1 program [9]. Multiple
alignments of nucleotide and amino acid sequences were
obtained using the default options in CLUSTALX 1.8 [10].
A search for homologies with proteins from the Protein
Information Resources database (PIR, release 47.0) was
done with the FASTA [10], BlastX and BlastP programs
[11]. The secondary structure of viral RNAs was analyzed
with the Mfold program [12]. Tentative phylogenetic trees
were constructed using the NJPLOT package [13] with
1000 bootstrap replicates. Protein function analysis was
done using the appropriate software for prediction of gly-
cosylation sites (NetNGlyc 1.0 vers.) [14], transmembrane
helices (TMHMM 2.0 vers.), and cleavage sites (SignalP
3.0, TragetP vers.) [15].
The average yield of purified GBLV-Serb1 from
C. quinoa was ca. 5 mg/kg of tissue. Virions were clearly
decorated by a polyclonal antiserum to the GBLV type
strain [3] and only weakly by the antiserum to BLMoV, but
not by any of the other antisera tested, thus confirming the
identification of the Serb1 as a genuine isolate of GBLV. In
1.2% agarose gel electrophoresis, RNAs migrated as two
distinct bands with an estimated size of 7.5 kbp and
6.0 kbp for RNA-1 and RNA-2, respectively (not shown).
Sequence analysis showed that the GBLV genome is
composed of two single-stranded RNA species, which are
7,452 nt (RNA-1, accession no. FN691934) and 5,821 nt
(RNA-2, accession no. FN691935) long. Both RNAs have
a VPg at their 5’ end and terminate with a poly(A) tail.
RNA-1 has a single ORF containing three in-frame
AUG codons, at positions 88, 103 and 229, none of which
possesses an optimal consensus sequence for initiation of
translation as determined by Kozak [16]orLu
¨tzke et al.
[17]. Translation of RNA-1 ends with an ochre codon
(UAA) at position 6,375. The coding region (CR) consists
of 6,285 nt and expresses a polypeptide that is 2,095 amino
acid (aa) in size, with a predicted M
r
of ca. 234 kDa,
covering 84% of the RNA-1 coding capacity, similarly to
ToRSV RNA-1 (80%) [18].
The 5’ leader sequence of GBLV RNA-1 is 228 nt in
size, i.e., only slightly longer than the corresponding NCR
of RNA-2 (189 nt). Sequence comparison of the RNA-1
and RNA-2 5’ leader sequences revealed very low
sequence homology (35%), contrary to what has been
reported for ToRSV (81%) [19]. By contrast, the 3’ NCR of
RNA-1 (1,077 nt) had a higher (72%) identity with the
comparable RNA-2 region (1,132 nt). In addition, both
sequences showed several nt stretches that were up to 81%
identical to the corresponding regions of BLMoV.
The predicted polyprotein encoded by GBLV RNA-1
showed the typical genome organisation of subgroup C
nepoviruses and the conserved motifs characterizing the
putative viral protease co-factor (Prot-cofact), helicase
(Hel, also called nucleotide triphosphate binding protein,
NTB), genome-linked protein (VPg), cysteine-like protease
(Cyst-Prot) and RNA-dependent RNA-polymerase (RdRp)
core domains of nepoviruses. The initiation of these
domains occurred at nt positions 88, 1813, 3400, 3628,
4303, and their translation yielded polypetides with a
predicted M
r
of 63, 59, 8, 25 and 78 kDa, respectively.
The GBLV Prot-cofact contained three hydrophobic
regions, two of which were clearly predicted to be trans-
membrane helices according to several programs (HMM-
TOP, TMHMM and SOSUI). Based on the ‘‘NetpicoRNA’’
prediction program [20], this domain is putatively cleaved
at aa position 443 (Q/A) into two proteins (X1 and X2),
similarly to ToRSV [21]. X2 protein is highly hydrophobic
and is located upstream of the NTB domain in the
RNA1-encoded polyprotein. Moreover, it shares conserved
sequence motifs with cowpea mosaic virus (CPMV)
32-kDa protein, which is an ER-targeted protein that is
believed to be involved in the assembly of the viral
876 T. Elbeaino et al.
123
replication complex (VRC). Zhang et al.[22] have sug-
gested that ToRSV-X2-NTB is targeted to the endoplasmic
reticulum, possibly acting as a second membrane anchor
for the VRC. Whether this is also the case with the cor-
responding sequence of GBLV remains to be determined.
The entire GBLV RNA-1-encoded amino acid sequence
shared the highest similarity with nepoviruses of subgroup
C, i.e., blackcurrant reversion virus (BRV, 64%), tomato
white ringspot virus (TWRSV, 50%), artichoke yellow
ringspot virus (AYRSV, 46%), ToRSV (44%), PRMV
(40%) and, to a lesser extent, nepoviruses of subgroups A
and B. Nucleotide comparison of the 3’NCR of GBLV and
BLMoV RNA-1 sequences showed 49% identity, which
is higher than that found with other nepoviruses of sub-
group C.
RNA-2 is 5,821 nt in length, excluding the 3’-poly(A) tail.
The sequences of the 5’ and 3’ termini consist of 189 nt and
1,132 nt, respectively. A single ORF of 4,500 nt encoding a
polypeptide of 1,500 aa (167 kDa) constitutes the whole
RNA-2. This ORF begins with a AUG at nt position 190 and
terminates with a UGA at position 4,689, thus including 71%
of the complete RNA-2 sequence. In the 5’-to-3’ direction,
RNA-2 encodes, in order, a product involved in RNA-2
replication referred to in the literature as putative homing
protein (HP), the movement protein (MP) and the capsid
protein (CP). Translation of HP, MP and CP starts at nt
positions 190, 1720 and 3166, yielding polypetides with a
predicted M
r
of 56, 55 and 56 kDa, respectively. Multiple
sequence alignment of GBLV CP with comparable
sequences of subgroup C nepoviruses (BRV, BLMoV)
revealed 68% and 36% identity with CPs of BLMoV and
BRV, respectively. The three conserved motifs and eight
conserved aa stretches described by Le Gall et al.[5] and
Latvala et al.[23] were found in the N-terminus of GBLV
RNA-2 sequence.
Comparative sequence analysis of CPs of the GBLV
isolates from Serbia and Bulgaria showed a divergence of
12% and 4% at the nt and aa level, respectively, which is in
line with what has been reported for other members of the
genus Nepovirus.
Computer-assisted analysis for prediction of cleavage
sites, along with pairwise alignment conducted on the
sequences of subgroup C nepoviruses, identified in GBLV
RNA-1 four dipeptide residues (Q/G, N/A, Q/A and Q/S),
located at aa position 575, 1104, 1180 and 1405, that
putatively cleave the polypeptide at the level of the Prot-
cofact, NTB, VPg, Cyst-prot and RdRp domains, respec-
tively (Fig. 1). In the case of GBLV RNA-2, a dipeptide
(Q/G), which is also reported to be a cleavage site for
ToRSV CP [24], was identified in three locations at aa
position 346, cleaving the HP domain in two proteins
(X3/X4), similar to ToRSV [25], and at aa positions 511
and 993, cleaving the MP and the CP domain, respectively
(Fig. 1).
The structure predicted for the 5’ NCR of GBLV RNA-2
is a potential stem-loop structure capping two 6-bp stems at
the 5’ terminus and two other potential hairpins structures,
with a loop apex located upstream from the initiation codon
Cyst-Pro
VPg POL
NTB Poly(A)-3’
5’-VPg
RNA-1
MP CP
RNA-2
190 4689
88 6375
Poly(A)-3’
N/A Q/A Q/S
Q/G Q/G
7452
5821
5’-VPg
DOP-PCR R1 R4R2 R3
RACE-PCR Oligod (T)
RT-PCR R5 R6 R7
R8 R9
Q/G
63 kDa 59 kDa 8 kDa 25 kDa 78 kDa
DOP-PCR
RACE-PCR
RT-PCR
N1 N2 N3
Oligod (T)
N4 N5
7N6N
56 kDa 55 kDa 56 kDa
1
1
x1 x2
x3 x4
Q/G
Q/A
Cyst-Pro
VPg POL
NTB Poly(A)-3’
5’-VPg
RNA-1
MP CP
RNA-2
190 4689
88 6375
Poly(A)-3’
N/A Q/A Q/S
Q/G Q/G
7452
5821
5’-VPg
DOP-PCR R1 R4R2 R3
RACE-PCR Oligod (T)
RT-PCR R5 R6 R7
R8 R9
Q/G
63 kDa 59 kDa 8 kDa 25 kDa 78 kDa
DOP-PCR
RACE-PCR
RT-PCR
N1 N2 N3
Oligod (T)
N4 N5
7N6N
56 kDa 55 kDa 56 kDa
1
1
x1 x2
x3 x4
Q/G
Q/A
Fig. 1 Schematic representation of cDNA cloning and sequencing
strategy of GBLV RNA-1 and RNA-2. Boxes represent RNA1- and
RNA2-encoded polyproteins with their putative function and their
estimated molecular weights shown above and below the boxes,
respectively. Prot-cofact: protease cofactor (X1 and X2 proteins),
NTB: nucleotide triphosphate binding protein, VPg: viral genome-
linked protein, Cyst-Pro: cysteine-like protease, Pol: polymerase, HP:
homing protein (X3 and X4 proteins), MP: movement protein, CP:
capsid protein. Initiation and termination of protein translation are
represented by dotted lines at the 5’ and 3’ termini of each RNA. Bars
represent the cDNA clones obtained as follows: (1) DOP-PCR clones
represented by R1-4 (RNA1) and N1-3 (RNA2) and generated from
random reverse-transcribed ssRNA of purified virions. DOP-PCR
clones are listed sequentially according to BLAST analysis. (2) RT-
PCR clones represented by R5-7 (RNA1) and N4-5 (RNA2) and
obtained using the sense and antisense virus-specific primers
indicated by arrowheads (nt sequence not shown). (3) Regions
flanking the 3’ termini of both RNA segments were amplified using
an oligo(dT) primer together with virus-specific sense primers
designed on R4 and N3 clones. The 5’RACE-PCR technique was
used to amplify the 5’ caps of both RNAs segments. The RNA-1-
encoded protein is cleaved by the viral protease at 5 sites to release 6
mature proteins and several intermediate precursors. RNA-2-encoded
protein is cleaved at 3 sites to release 4 protein domains and possible
precursors. Putative predicted cleavage sites are indicated above the
boxes
Grapevine Bulgarian latent virus 877
123
(AUG). These structures are reported to be involved in
replication, translation efficiency and the viral infection
cycle, or in host/virus interactions [5,26].
A phylogenetic tree constructed with the amino acid
sequences of CPs of grapevine-infecting nepoviruses con-
firmed the assignment of GBLV to subgroup C, as it clearly
clustered in a clade comprising nepoviruses of this sub-
group (Fig. 2).
In conclusion, to our knowledge, GBLV represents the
third member of nepovirus subgroup C to be completely
sequenced, after ToRSV and BRV. The proposed sero-
logical relationship between GBLV and BLMoV is now
further supported by the high amino acid identity at the CP
level (67%), a value below the threshold set by ICTV for
species demarcation in the genus Nepovirus (75%) but
similar to that reported for other serologically related
nepoviruses, i.e., GFLV and ArMV (68%), GDefV and
ArMV (70%), and GCMV and TBRV (62%). Moreover,
the comparison of GBLV and BLMoV at the molecular
level confirms that enough divergence exists between these
viruses to keep them assigned to separate species rather
than classifying them as strains of the same virus. This
suggests that BLMoV should be moved from EPPO list A2
to EPPO list A1, which includes pests that are never
detected in the Euro-Mediterranean area [27].
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AILV (X87254)
BRSV (NC-003694)
TBRV (NC-004440)
GCMV (NC-003621)
998
1000
BLMoV (U20261)
GBLV (FN691935)
BRV (NC-003502)
1000
ToRSV (NC-003839)
1000
ArMV (AY017339)
GDefV (AAQ56597)
GFLV (NC-003623)
976
1000
1000
0.05
1000
Subgroup A
Subgroup C
Subgroup B
991
CPMV (NC003550)
AILV (X87254)
BRSV (NC-003694)
TBRV (NC-004440)
GCMV (NC-003621)
998
1000
BLMoV (U20261)
GBLV (FN691935)
BRV (NC-003502)
1000
ToRSV (NC-003839)
1000
ArMV (AY017339)
GDefV (AAQ56597)
GFLV (NC-003623)
976
1000
1000
1000
Subgroup A
Subgroup C
Subgroup B
991
CPMV (NC003550)
Fig. 2 Phylogenetic tree generated from the alignment of the overall
coat protein amino acid sequences of members of the genus
Nepovirus belonging to subgroups A, B and C. EMBL or GenBank
accession numbers of the sequences used in phylogenetic analysis are
in parentheses. Cowpea mosaic virus (CPMV) was used as an
outgroup. Bootstrap values are shown at branch points
878 T. Elbeaino et al.
123
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