Virology 394 (2009) 1–7

Contents lists available at ScienceDirect

Virology
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y v i r o

Rapid Communication

Permutation of the active site of putative RNA-dependent RNA polymerase in a newly

identified species of plant alpha-like virus☆
Sead Sabanadzovic a,⁎, Nina Abou Ghanem-Sabanadzovic a, Alexander E. Gorbalenya b
a
Department of Entomology and Plant Pathology, Mississippi State University, Mississippi State, MS 39762, USA
b
Molecular Virology Laboratory, Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands

a r t i c l e i n f o a b s t r a c t

Article history: To direct the genome synthesis, RNA viruses without a DNA stage in the replication cycle use RNA-dependent
Received 4 May 2009 RNA polymerase (RdRp). All RdRps have conserved right hand-like shape that includes characteristic
Returned to author for revision 5 June 2009 A → B → C sequence motifs forming the active site. Recently, the structural permutation of the RdRp active
Accepted 4 August 2009
site (C → A → B) has been described in few double-stranded RNA birnaviruses and a subset of positive-
Available online 29 September 2009
stranded RNA tetraviruses distantly related to Picorna-like viruses. Here we describe a permuted RdRp in the
Keywords:
newly identified plant alpha-like virus with 6.5 kb-long polyadenylated genome, dubbed Grapevine virus Q
Virus (GVQ). The multi-domain layout and sequence similarities place GVQ into the genus Marafivirus of the
Evolution family Tymoviridae. In contrast to other tymovirids, GVQ has 21 amino acid residues corresponding to the
RdRp motif C relocated upstream of the motif A in the putative RdRp. This unique sequence characteristic was
Palm sub-domain extensively verified and identified in several GVQ isolates infecting wild and cultivated Vitis and Rubus spp.
Internal permutation © 2009 Elsevier Inc. All rights reserved.
Marafivirus
Tymoviridae
Grapevine
Blackberry

Introduction underlies remarkable structural conservation. Indeed, all viral RdRps

Family Tymoviridae is currently composed of three genera
(Tymovirus, Marafivirus and Maculavirus), which accommodate
viruses reported from cultivated and wild monocotyledonous and
dicotyledonous plants as well as one entomovirus (Martelli et al.,
2002; Dreher et al., 2005; Katsuma et al., 2005). Genomes of viruses in
the family Tymoviridae slightly differ in number and organization of
cistrons, but all encode a large polyprotein essential for viral
replication (Dreher et al., 2005). This polyprotein contains signature
amino acid motifs of viral methyltransferase (MTR), endopeptydase/
protease (PRO), helicase (HEL) and RNA-dependent RNA-poly-
merases (RdRp) as in other “alpha-like” phytoviruses (Goldbach et
al., 1991).
Diversed RdRps are the most conserved among virus-encoded
proteins and share several conserved signature motifs in a particular
order (Poch et al., 1989; Koonin, 1991; Koonin and Dolja, 1993) that
☆ Sequences reported in this paper have been deposited in the NCBI GenBank
database and have been assigned the accession numbers FJ977041-FJ977044.
⁎ Corresponding author. Department of Entomology and Plant Pathology, Mississippi
State University, 100 Twelve Lane, Mississippi State, MS 39762, USA. Fax: +1 662 325
8837.
E-mail address: ss501@msstate.edu (S. Sabanadzovic).
studied to date have conserved “right hand-like” shape with three
conserved sub-domains referred to as finger, palm and thumb
(Hansen et al., 1997; van Dijk et al., 2004). The palm sub-domain is
the most conserved part of viral RdRps and comprises four out of eight
conserved motifs described by Koonin (1991) that correspond to
motifs A → B → C → D reported by Poch et al. (1989). Motifs A and C
contain spatially juxtaposed and conserved Aspartic (Asp) residues
involved in a two-metal (Mg2+ and/or Mn2+) mechanism of catalysis
(Arnold et al., 1999; Crotty et al., 2003; Ng et al., 2008). Motif B
determines whether RNA or DNA will be synthesized by selecting
NTPs and dNTPs (Hansen et al., 1997).
Due to their functional importance, it would be expected that the
canonical order of motifs is universally conserved across the “RdRp
universe.” Surprisingly, Gorbalenya et al. (2002) reported the non-
canonical organization of RdRp motifs in viral replicases of two
entomoviruses with +ssRNA genomes, Thosea assigna virus (TaV)
and Euprosterna elaeasa virus (EeV), and in two dsRNA viruses
(Infectious pancreatic necrosis virus—IPNV and Infectious bursal disease
virus—IBDV) belonging to the family Birnaviridae (Table 1s, Supple-
mentary material). In these RdRps, the motif C is located upstream of
the motif A to form a non-canonical C → A → B arrangement
associated with a unique connectivity of major structural elements
underneath of the active site (Gorbalenya et al., 2002). Subsequently
the permutation was verified in two studies of the IBDV RdRp

0042-6822/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.virol.2009.08.006
2 Rapid Communication
structure (Pan et al., 2007, Garriga et al., 2007). Phylogenetic analysis
of RdRps of TaV/EeV/birnaviruses and representative ssRNA viruses
(nidoviruses and picorna-like viruses) with canonical polymerases
showed that internally permuted RdRps form a monophyletic deeply
rooted clade. This clustering is due to extensive similarities outside
the permuted motifs rather than the permutation per se (Gorbalenya
et al., 2002). The RdRps with the C → A → B order were referred to as
“internally permuted” or “non-canonical” and that terminology will
be also used in this text.
During a screening for viruses infecting native Vitis and Rubus spp.,
which was carried out in 2007/2008, we have identified a novel virus,
named Grapevine virus Q (GVQ), most resembling members of the
genus Marafivirus (family Tymoviridae) (Sabanadzovic and Abou
Ghanem-Sabanadzovic, 2009). Surprisingly, bioinformatics analysis
of the newly determined GVQ genome sequence revealed the
C → A → B order of RdRp motifs that is likely to have evolved
independently from the previously reported permuted RdRp of TaV/
EeV/birnaviruses. These data are detailed below.
Results and discussion
Identification of GVQ
Using the “universal” tymovirid primer set RD that was designed
on viral RdRp (Sabanadzovic et al., 2000) a single amplicon was
generated in two tested muscadine grapes. The amplicon size was
considerably larger than that of the PCR product amplified from the
positive controls used in tests—Grapevine fleck virus (GFkV) (Saba-
nadzovic et al., 2001) and Poinsettia mosaic virus (Bradel et al., 2000),
which prompted further studies. The same specimens did not generate
amplicons with other primer sets used for the purposes of the survey.
Sequence analyses of multiple clones showed that the amplicons
from both muscadines were of the uniform size of 450 bp and 97%
identical, indicating that they were generated from very closely
related templates present in both samples. These amplicons were 63
nucleotides larger than the sequences of known tymovirids used for
primers design (387 bp). Consistently with the primer design, Blast-
mediated comparison of the amplicon sequences (that belong to GVQ)
with the GenBank/EMBL databases showed most significant similar-
ities with marafiviruses, tymoviruses and maculaviruses of the family
Tymoviridae (E values ranging from 2e−24 to 2e−41). Accordingly,
multiple sequence alignment of the analyzed region involving typical
tymovirids identified the 21 amino acid (aa) insertion upstream of the
RdRp motif A in the newly determined sequences. We noted that the
characteristic 21 aa insertion in the GVQ RdRp had a high similarity
with the RdRp motif C universally located downstream in all
tymovirus sequences. Combined with the lack of a counterpart to
motif C in the expected position of the GVQ genome (see below), this
observation indicated that GVQ has the permuted C → A → B order of
RdRp sequence motifs (Fig. 1).
Given that the permutation affects the RdRp active site and has not
been described for either plant or alpha-like viruses, we extensively
verified the GVQ RdRp sequence. We confirm that the permutation
was observed independent of the type of template used (dsRNA or
total RNA), denaturation conditions (heat, DMSO, methyl mercuric
hydroxide), reverse transcriptases or cloning strategies (RT-PCR
based or classic dscDNA synthesis employing primer walking
technique). One Vitis and one blackberry source of GVQ (see below),
co-infected with typical marafiviruses and/or maculaviruses encod-
ing a canonical RdRp, were used to analyze sequences of the two PCR
products. Comparison of sequences co-amplified from the same
specimen showed they were clearly different, eliminating the
possibility that the longer PCR product is an artifact produced during
manipulation/processing (RNA extraction, reverse transcription, PCR)
of the nucleic acids of the virus with “canonical” RdRps (Fig 1s;
Supplementary material).
Genome sequences
To characterize GVQ, its complete genome sequence was deter-
mined using isolate MG-02 (muscadine grape) as a template.
Excluding the poly(A) tail, it is composed of 6481 nt and includes
two ORFs, a long ORF1 accounting for 96% of the whole genome and a
relatively short overlapping putative ORF2 (Fig. 2A). The ORF1
encodes for a 2081-amino acid-long polyprotein of an estimated
molecular mass of ca 229.5 kDa (p230). The N-terminal three fourths
portion of this polyprotein includes signature motifs of viral MTR
(Rozanov et al., 1992), PRO (Gorbalenya et al., 1991), HEL (Gorbalenya
et al., 1988) and RdRps (Koonin, 1991; Poch et al., 1989; Koonin and
Dolja, 1993) implicated in virus replication. It is predicted to be
autoproteolytically processed between the Hel and RdRp domains by
the Pro-mediated activity to generate two proteins, with the N-
terminal one including MTR-Pro-Hel domains (Bransom and Dreher,
1994; Rozanov et al., 1995). The genome region downstream of the
RdRp is likely involved in formation of two carboxy co-terminal coat
proteins of the estimated Mr of 23000 and 21000. The genome
contains the “tymo/marafibox” signature sequences reported to be a
transcription promoter for subgenomic RNA synthesis followed by a
CAA “consensus box” (Ding et al., 1990). Amino acid identities of GVQ
with the closest known tymovirids approximate 65% in the conserved
genome products used in the comparison (MTR, HEL, CP). Computer-
assisted sequence analysis revealed the presence of an additional
putative ORF, potentially encoding a proline/serine rich protein (total
43%) of unknown function with the calculated mass of 27 kDa (p27).
We performed phylogenetic analysis using neighbor-joining,
maximum likelihood and Bayesian algorithms. In trees involving the
MTR-Pro-HEL and CP domains, GVQ consistently appeared as a sister
lineage to GRVFV, forming a major sub-clade within marafiviruses
(Figs. 2B, E). The clustering of GVQ with marafiviruses is also evident
in the RdRp tree (Fig. 2C), although the relationships of GVQ as well as
GRVFV and MRFV are not fully resolved in this tree. This uncertainty is
not due to the presence of the permuted motif C in the GVQ RdRp,
which along with other poorly conserved regions was removed from
the alignment submitted to the analysis. Importantly, the trees of
marafiviruses, including GVQ, produced for the MTR-Pro-HEL protein
and for an unprocessed replicase polyprotein (including MTR-Pro-
HEL and RdRp domains) were congruent (Figs. 2B, D). This result
indicates that the phylogenetic signal generated by the RdRp domain
of marafiviruses is relatively weak. In contrast, the RdRp dominates
over the MTR-Pro-HEL protein in the signal produced for some
lineages of tymoviruses (Figs. 2B–D). Taken together, these observa-
tions imply that the observed difference between the MTR-Pro-HEL
and RdRp trees in relation to marafiviruses is relatively minor,
indicating that the RdRp motif C permutation is not likely to be
associated with another gross evolutionary event in the GVQ lineage.
Virus occurrence and variability
We further proceeded to study the occurrence of GVQ in other
grapevine sources. For this analysis we used new degenerate primers
that were designed for broad tymovirid detection. Specifically, a pair
of primers designed for the RdRp region (TymZ-F and TymZ-R) clearly
distinguished between tymovirids with the “canonical” and permuted
RdRps (Fig. 3A) by generating amplicons of 344 bp and 407 bp,
respectively. Mixed infections were characterized by the presence of
both bands (Fig. 3B).
Amplicons of 407 bp were found in two samples of Vitis
rotundifolia, single specimens of Vitis aestivalis and in an unknown
cultivar of Vitis vinifera. The same primer set was also applied for
screening native Rubus germplasm present in the Great Smoky
Mountain National Park. Besides the detection of a new “canonical”
marafivirus in multiple blackberry accessions (Blackberry virus S,
BlVS; Sabanadzovic and Abou Ghanem-Sabanadzovic, in press), two
 Rapid Communication 3

Fig. 1. Alignment of the deduced amino acid sequences of RNA-dependent RNA polymerases of Grapevine virus Q and representatives of the three genera in the family Tymoviridae:
Tymovirus (Turnip yellow mosaic virus; TYMV), Maculavirus (Grapevine fleck virus; GFkV) and Marafivirus (Maize rayado fino virus—MRFV; Oat blue dwarf virus—OBDV; Citrus sudden
death-associated virus—CSDaV; Grapevine asteroid mosaic associated virus—GAMaV; Grapevine rupestris vein feathering virus—GRVFV). Conserved motifs A, B and C are boxed. A
21-aa-long insertion located upstream of motif A containing GDD tripeptide, uniquely present in GVQ genome is reported in red. Positions of the primer sets RD and TymZ are
indicated by solid- and dashed-line arrows, respectively. Direct comparison of C motifs of these viruses is presented separately.

distinct bands of 344 bp and 407 bp were evident in the specimen
GSM-9. Sequence analyses showed that the lower band indeed
originated from BlVS genome (Fig. 3B and Fig. 2s; Supplementary
material). Analyses of 407 bp long PCR products from all plant
specimens showed that they are (nearly) identical to the respective
region of the GVQ genome (muscadine isolate).
Another GVQ-specific primer set, designed to the CP region, was
used to determine the full sequence of viral coat proteins of several
isolates identified in the prior analysis. In these isolates the amino acid
conservation varied from 93% to 99% depending on the compared pair
(Table 3s and Fig. 3s; Supplementary material).
Biochemical and biological implications of internal permutation in the
GVQ RdRp
Alignment of permuted RdRp sequences encoded by GVQ and
TaV/EeV shows the colinearity of characteristic motifs C (GDD), A
(DX4-5D) and B (GX2-3TX3N) (Fig. 3C) implying that the GVQ RdRp is
likely to have a fold conserved in the permuted RdRps of other
viruses (Gorbalenya et al., 2002, Pan et al., 2007, Garriga et al., 2007).
In this fold, major structural elements represented by motifs C, A and
B form the active site at one side and are unconventionally,
compared to canonical RdRps, interconnected at the opposite side.
In the IBDV RdRp, this structural organization enables regulation of
the active site access (Garriga et al., 2007). A multiple alignment of
RdRps of marafiviruses includes only two regions containing gaps,
both of which are associated with the GVQ motif C (Fig. 1).
Compared to other marafiviruses, the motif C in the GVQ RdRp is
flanked by extra short sequences of two/three and five residues,
respectively, which may be insertions accompanying the motif C
permutation (reported in blue in Fig. 1). Interestingly, in silico
conversion of the canonical RdRp of poliovirus into the permuted
form required insertion of additional residues in the proximity of
motif C (Gorbalenya et al., 2002). Thus, it is tempting to speculate
4 Rapid Communication

Fig. 2. (A) Schematic representation of GVQ genome with nucleotide coordinates. Boxes depict ORFs and lines represent untranslated genomic regions. (B–E) Phylograms depicting
the relationships of Grapevine virus Q with the members of the family Tymoviridae for the MTR-Pro-Hel protein (B), RdRp (C), replicase polyprotein, (D) and coat protein (E). The
trees were generated using ML algorithm; similar trees were produced using neighbor-joining method or Bayesian inference. Bootstrap values larger than 50 obtained in 100 replics
are indicated at internal branch points. Internal branches with less than 50% support were collapsed. Congruent portions of replicase vs. MTR-Pro-Hel trees and replicase vs. RdRp
trees are highlighted. Viruses used to construct trees, acronyms and RefSeq/GenBank accession numbers are: Anagyris vein yellowing virus (AVYV; NC_011559), Bombyx mori
macula-like latent virus (BmMLV, AB186123), Citrus sudden death associated virus (CSDaV, NC_006950), Eggplant mosaic virus (EMV, J04374), Grapevine rupestris vein feathering
virus (GRVFV, AY128949), Grapevine fleck virus (GFkV, NC_003347), Kennedya yellow mosaic virus (KYMV, NC_001746), Maize rayado fino virus (MRFV, AF265566), Nemesia ring
necrosis virus (NeRNV, NC_011538), Oat blue dwarf virus (OBDV, U87832), Ononis yellow mosaic virus (OYMV, J04375), Physalis mottle virus (PhyMV, Y16104), Poinsettia mosaic virus
(PnMV, NC_002164) and Turnip yellow mosaic virus (TYMV, NC_004063). The definitive/tentative members of the genera Tymovirus, Marafivirus and Maculavirus reported in black,
red and blue, respectively. Poinsettia mosaic virus, an unassigned species in the family, is reported in green.

that the extra residues flanking motif C in GVQ have been accepted
to accommodate the new loop connectivity underneath the permut-
ed motif C β-hairpin.
Apart of the common fold, the permuted RdRps of GVQ and other
viruses can be contrasted. First, GVQ is a plant virus while all other
viruses with permuted RdRps are of the animal origin. All other
unique aspects of the GVQ RdRp are related to the phylogenetic
position of GVQ among tymoviruses of the alpha-like supergroup (see
above) and outside of the lineage formed by TaV/EeV/birnaviruses
that has affinity to Picorna-like supergroup. This deep phylogenetic
separation of the permuted RdRps, identified previously and reported
in this study, is most compatible with parallel origin of the RdRp
permutation in two lineages of RNA viruses. Although available data
are limited, RdRps in these two lineages seem to initiate the RNA
synthesis differently, either relying or not on (protein) priming (Ball,
2007). Thus, the RdRp permutation of the RdRp active site is
compatible with functioning of different types of RdRps. An
intermediate position of the GVQ lineage in the tymovirus tree
 Rapid Communication
Fig. 3. (A) Diagrammatic representation of canonical and internally permuted active
sites of the palm sub-domains of viral RdRps and differences in sizes of PCR
products generated with primers set TymZ. IP= internally permuted, Can = canonical. (B)
Agarose gel electrophoresis of PCR products generated by TymZ primers. Canonical
marafiviruses gave uniform product of 344 bp, while GVQ infected samples generated 407-
bp-long amplicons. Viruses used in this gel are: Poinsettia mosaic virus (PnMV, lane 1),
Blackberry virus S (BlVS, lane 2), Grapevine fleck virus (GFkV, lane 3). Grapevine virus
Q-infected samples are in lanes 5 and 6. Blackberry samples GSM-9 with mixed
infection (BlVS + GVQ) is in lane 4. Negative control (tymovirids-free muscadine) is in
lane 7. (C) Comparison of internally permuted regions in +ssRNA viruses: EeV and TaV
and GVQ. All three motifs appear conserved among phylogenetically distant viruses.
Inter-motif distances are indicated in brackets.
strongly indicates that the most recent ancestor of GVQ with the
permuted RdRp is likely to have emerged from a tymovirus with a
canonical RdRp which predated the origin of the GVQ ancestor. This
reconstruction contrasts with the uncertainty concerning the origin of
the permuted RdRps of the TaV/EeV/birnaviruses due to deep rooting
of this lineage in a tree with many families of RNA viruses (Gorbalenya
et al., 2002).
Mechanistically, generation of an internal permutation in a protein
is a complex three-point event that may partially be responsible for
the scarcity of permuted RdRps in viruses (Gorbalenya et al., 2002). It
could be generated through tandem duplication of motifs A, B and C in
an ancestral canonical RdRp to form the A → B → C → A' → B' → C'
intermediate that is subsequently truncated to form the permuted
C→A'→B' constellation (Gorbalenya et al., 2002). The postulated
intermediate must be short-lived to explain why it has not been
identified so far. Alternatively, it could be envisioned that multiple
template switching during replication of the canonical parent might
lead to the generation of permuted progeny.
Regardless of the mechanism, permuted progeny is a mutant that
must be competitive to survive. Recent study on the functional
implications of the permuted C motif, using IBDV RdRp as a model,
showed unique and strong dependency and stimulation of this
enzyme by Co 2+ ions (Letzel et al., 2007). These properties
discriminate the IBDV RdRp from canonical enzymes including
those encoded by Poliovirus (Arnold et al., 1999), Hepatitis virus C-
(Ranjith-Kumar et al., 2002) or sapovirus- (Fullerton et al., 2007) all of
which are stimulated by Mn2+ and/or Mg2+ ions. In IBDV, permuted
motif C includes an “ADN” aa tripeptide that replaces “GDD”
commonly found in canonical RdRps and internally permuted
polymerases of TeV, EeV and GVQ (von Einem et al., 2004; Xu et al.,
2004; Shwed et al., 2002; Letzel et al., 2007; Gorbalenya et al., 2002,
present work). The precise genetic determinant, GDD-to-ADN
mutation or motif permutation or both, that controls the Co2+ ion
affinity is yet to be mapped in the IBDV RdRp. In this respect, GVQ may
5
provide an attractive alternative model for further studies of
biological properties associated with permuted RdRps, when reverse
genetics for this virus is established.
The presence of two bands among PCR products in our analyses
was reminiscent of the data published by Shi et al. (2003) who
reported two PCR bands of 353 bp and 416 bp in selected grapevine
samples using a single primer pair originally designed for the specific
detection of GFkV. The authors tentatively assigned bands as variants
GFkV353 and GFkV416 of Grapevine fleck virus and did not further
investigate the genetic basis of the variability. Comparison of limited
amino acid sequences reported in the paper (see Shi et al., 2003; Fig.
2B) with our data showed that GFkV416 is indeed an isolate of GVQ
(94% aa identity).
Finally, while reviewing this manuscript before final submission to
the publisher, we noticed newly deposited sequences of a marafivirus
denominated Grapevine Syrah virus 1 (GSyV-1; Al Rwahnih et al.,
2009). Comparison of GVQ and GSyrV-1 revealed over 98% sequence
similarity including conservation of the permuted RdRp, which was
not observed/reported in the original publication describing GSyV-1
(Al Rwahnih et al., 2009). Thus, we propose that GVQ, GSyrV-1 and,
likely, GFkV416 are different isolates of a single marafi virus species
distinguished by the signature organization of RdRp motifs.
Materials and methods
Screening for viruses
Plant materials initially collected for the purpose of screening for
possible viruses in native Vitis spp present in the Southeastern United
States were tested in RT-PCR with degenerate primers for members of
the family Closteroviridae (Tian et al., 1996), Flexiviridae (Dovas and
Katis, 2003), Tymoviridae (Sabanadzovic et al., 2000) and members of
the genus Nepovirus (Digiaro et al., 2007) which covered the most
common taxa of viruses reported in grapevines.
Virus sources
The primary plant material for this investigation was collected in
2007/2008 from an apparently healthy muscadine (Vitis rotundifolia
Michx.) accession MG-02, which tested positive for tymovirids during
initial assessment of the viruses infecting Vitis spp, and was used for
the complete molecular characterization of the virus. Mature vines of
field-grown muscadine were collected and used for double stranded
RNA analyses and/or leaf petioles early in the spring for total RNA
extractions.
Additionally, a total of 47 samples belonging to native and
cultivated grapevines and blackberries were analyzed in this study.
Samples positive for GVQ were used to partially sequence viral RdRp
and coat protein (CP).
Cloning, sequencing and data analyses
Double-stranded RNAs were extracted from phloem scrapings of
the accession MG-02 by selective chromatography through CF-11
columns in the presence of an appropriate buffer containing 16%
ethyl alcohol as described (Valverde et al., 1990). In addition,
extracted nucleic acids were treated with RQ DNase and RNase A
(in high salt conditions) in order to eliminate possible traces of DNA
and ssRNA molecules prior to use as a template for further molecular
work.
Complementary DNA (cDNA) was synthesized applying a slightly
modified protocol of Froussard (1992) involving cDNA synthesis and
its enrichment via PCR, size selection and directional cloning into an
EcoRI-cut pUC119/EcoRI plasmid (TaKaRa, South Korea). Ligation mix
was transferred into Escherichia coli DH5α cells and selected plasmids
were custom sequenced at MWG Biotech facility (Huntsville, AL,
6 Rapid Communication
USA). Initial sequence data were mapped with the help of Lasergene
software (DNAStar, Madison, WI) and specific primers were designed
in order to generate the rest of genomic data via PCR. For
determination of the viral 3′ end, an oligo dT-generated cDNA was
amplified using oligo dT and RD1 primer as described (Abou Ghanem-
Sabanadzovic et al., 2003). The 5′ end was determined according to
the protocol enclosed in the 5′/3′ RACE kit (Roche Diagnostics, USA)
using GVQ-specific primers (Table 2s, Supplementary material).
Sequences were analyzed and compared with GenBank using on-
line resources (Altschul et al., 1997; Marchler-Bauer et al., 2007).
Alignments of polyprotein sequences containing MTR, Pro, Helicase,
and RdRp domains and CP sequences of a selected viruses of the Ty-
moviridae were produced using MUSCLE (Edgar, 2004) with subse-
quent removal of columns containing gaps using GBlocks (Castresana,
2000). These alignments were submitted to phylogenetic analyses
using different methods/software: Neighbour Joining (Phylip; Felsen-
stein, 1989), Maximum-Likehood (PhyML; Guindon and Gascuel,
2003) and Bayesian inference (MrBayes; Huelsenbeck and Ronquist,
2001) applying a 50% majority consensus rule.
RT-PCR
The primer set TymZ (Table 2s; Supplementary material) was
applied in a study on GVQ incidence in Vitis and Rubus germplasm. It
was designed to amplify 344 bp-long amplicon in canonical
marafiviruses and 407 bp in GVQ. Total RNAs were extracted
combining the protocols of Foissac et al. (2005) and commercial
RNeasy Plant Minikit (Qiagen) (Sabanadzovic and Abou Ghanem-
Sabanadzovic, in press), reverse transcribed with and submitted to
PCR with primers TymZ under annealing temperature of 52 °C and an
extension time of 45 s. Nucleic acids extracted from Poinsettia mosaic
virus-infected poinsettias (PnMV; Bradel et al., 2000) and Grapevine
fleck virus-infected grapevines (GFkV; Sabanadzovic et al., 2001) were
used as positive controls.
In total, 35 samples belonging to different species/cultivars of
native and cultivated Vitis spp. were tested with the degenerate
primer set TymZ. Furthermore, the same primers were used to test 12
samples of native Rubus germplasm from the Great Smoky Mountains
National Park for tymovirus/marafivirus/maculavirus infections as
part of the general virus survey.
Another primer set, designed to amplify a 721-nt-long genomic
segment encompassing the viral coat protein gene (GVQ-CP set, Table
2s; Supplementary material), was used to investigate the variability of
GVQ genome among different isolates. PCR conditions were similar to
those described for TymZ set.
Further analyses
In order to further check the sequence data in the RdRp region,
several additional cloning experiments were performed. In these
experiments we adopted several different strategies/conditions: (i)
random primer cloning and PCR using different templates (total
RNA or dsRNA), reverse transcriptases (Thermoscript, M-MLV or
AMV), primer sets; (ii) primer walking technique using virus
specific primers (see Fig 1s; Supplementary material). All generated
products were cloned in a proper plasmid vector and sequenced as
described. In all tests at least one sample containing canonical
marafi/maculavirus was always processed in the same way as GVQ-
infected ones.
Acknowledgments
This work was supported partially by Special Research Initiative
funds from the Mississippi Agricultural and Forestry Experiment
Station and Discover Life in America Inc granted to S.S. Approved for
publication as a Journal Article No J-11620 of the Mississippi
Agricultural and Forestry Experiment Station, Mississippi State
University.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.virol.2009.08.006.
References
Abou Ghanem-Sabanadzovic, N., Sabanadzovic, S., Martelli, G.P., 2003. Sequence
analysis of the 3′end of three Grapevine fleck virus-like viruses from grapevine.
Virus Genes 27, 11–16.
Al Rwahnih, M., Daubert, S., Golino, D., Rowhani, A., 2009. Deep sequencing analysis of
RNAs from a grapevine showing Syrah decline symptoms reveals a multiple virus
infection that includes a novel virus. Virology, doi:10.1016/j.virol.2009.02.028.
Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J.,
1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search
programs. Nucleic Acids Res. 25, 3389–3402.
Arnold, J.J., Ghosh, S.K., Cameron, C.E., 1999. Poliovirus RNA dependent RNA polymerase
(3Dpol): Divalent cation modulation of primer, template, and nucleotide selection.
J. Biol. Chem. 274, 37060–37069.
Ball, L.A., 2007. Virus replication strategies, In: Fields, B.N. Knipe D.M., Howley, P.M.
(Eds.), Field Virology, 5th Ed. Wolters Kluwer Health/Lippincott Williams &
Wilkins, Philadelphia, pp. 119–141.
Bradel, B.G., Preil, W., Jeske, H., 2000. Sequence analysis and genome organisation of
poinsettia mosaic virus (PnMV) reveal closer relationship to marafiviruses than to
tymoviruses. Virology 271, 289–297.
Bransom, K.L., Dreher, T.W., 1994. Identification of the essential cysteine and histidine
residues of the turnip yellow mosaic virus protease. Virology 198, 148–154.
Castresana, J., 2000. Selection of conserved blocks from multiple alignments for their
use in phylogenetic analysis. Mol. Biol. Evol. 17, 540–552.
Crotty, S., Gohara, D., Gilligan, D.K., Karelsky, S., Cameron, C.E., Andino, R., 2003.
Manganese-dependent polioviruses caused by mutations within the viral poly-
merase. J. Virol. 77, 5378–5388.
Digiaro, M., Elbeaino, T., Martelli, G.P., 2007. Development of degenerate and species-
specific primers for the differential and simultaneous RT-PCR detection of grapevine-
infecting nepoviruses of subgroups A, B and C. J. Virol. Methods 141, 34–40.
Ding, S.W., Howe, J., Keese, P., Mackenzie, A., Meek, D., Osorio-Keese, M., Skotnicki, M.,
Srifah, P., Torronen, M., Gibbs, AJ., 1990. The tymobox, a sequence shared by most
tymoviruses: its use in molecular studies of tymoviruses. Nucleic Acids Res. 18,
1181–1187.
Dovas, C.I., Katis, N.I., 2003. A spot nested RT-PCR method for the simultaneous
detection of members of the Vitivirus and Foveavirus genera in grapevine. J. Virol.
Methods 107, 99–106.
Dreher, T.W., Edwards, M.C., Gibbs, A.J., Haenni, A-L., Hammond, R.W., Jupin, I., Koenig,
R., Sabanadzovic, S., Abou Ghanem-Sabanadzovic, N., Martelli, G.P., 2005. Family
Tymoviridae. In: Fauquet, C.M., Mayo, M., Maniloff, J., Desselberger, U., Ball, L.A.
(Eds.), Virus Taxonomy (Eight Report of the ICTV). Elsevier/Academic Press,
London, pp. 1061–1074.
Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high
throughput. Nucleic Acids Res. 32, 1792–1797.
Felsenstein, J., 1989. PHYLIP—phylogeny inference package. Cladistics 5, 164–166.
Foissac, X., Svanella-Dumas, L., Gentit, P., Dulucq, M.-J., Marais, A., Candresse, T., 2005.
Polyvalent degenerate oligonucleotides reverse transcription-polymerase chain
reaction: a polyvalent detection and characterization tool for trichoviruses,
capilloviruses, and foveaviruses. Phytopathology 95, 617–625.
Froussard, P., 1992. A random-PCR method (rPCR) to construct whole cDNA library
from low amounts of RNA. Nucleic Acids Res. 20, 2900.
Fullerton, S.W., Blaschke, M., Coutard, B., Gebhardt, J., Gorbalenya, A., Canard, B., Tucker,
P.A., Rohayem, J., 2007. Structural and functional characterization of sapovirus
RNA-dependent RNA polymerase. J. Virol. 81, 1858–1871.
Garriga, D., Navarro, A., Querol-Audi, J., Abaitua, F., Rodríguez, J.F., Verdaguer, N.,
2007. Activation mechanism of noncanonical RNA-dependent RNA polymerase.
Proc. Natl. Acad. Sci. U.S.A. 104, 20540–20545.
Goldbach, R., Le Gall, O., Wellink, J., 1991. Alpha-like viruses in plants. Semin. Virol. 2,
19–25.
Gorbalenya, A.E., Koonin, E.V., Donchenko, A.P., Blinov, V.M., 1988. A novel superfamily
of nucleoside-triphosphate-binding motif containing proteins which are probably
involved in duplex unwinding in DNA and RNA replication and recombination.
FEBS Lett. 235, 16–24.
Gorbalenya, A.E., Koonin, E.V., Lai, M.M.C., 1991. Putative papain-related thiol proteases
of positive-strand RNA viruses. FEBS Lett. 288, 201–205.
Gorbalenya, A.E., Pringle, F.M., Zeddam, J.L., Luke, B.T., Cameron, C.E., Kalmakoff, J.,
Hanzlik, T.N., Gordon, K.H., Ward, V.K., 2002. The palm subdomain-based active site
is internally permuted in viral RNA-dependent RNA polymerases of an ancient
lineage. J. Mol. Biol. 324, 47–62.
Guindon, S., Gascuel, O., 2003. A simple, fast, and accurate algorithm to estimate large
phylogenies by maximum likelihood. Syst. Biol. 52, 696–704.
Hansen, J.L., Long, A.M., Schultz, S.C., 1997. Structure of the RNA dependent RNA
polymerase of poliovirus. Structure 5, 1109–1122.
Huelsenbeck, J.P., Ronquist, F., 2001. MRBAYES: Bayesian inference of phylogenetic
trees. Bioinformatics 17, 754–755.
 Rapid Communication
Katsuma, S., Tanaka, S., Omuro, N., Takabuchi, L., Daimon, T., Imanishi, S., Yamashita, S.,
Iwanaga, M., Mita, K., Maeda, S., Kobatashi, M., Shimada, T., 2005. Novel macula-like
virus identified in Bombyx mori cultured cells. J. Virol. 79, 5577–5584.
Koonin, E.V., 1991. The phylogeny of RNA-dependent RNA polymerases of positive-
strand RNA viruses. J. Gen. Virol. 72, 2197–2206.
Koonin, E.V., Dolja, V.V., 1993. Evolution and taxonomy of positive strand RNA viruses:
implications of comparative analysis of amino acid sequences. Crit. Rev. Biochem.
Mol. Biol. 28, 375–430.
Letzel, T., Mundt, E., Gorbalenya, A.E., 2007. Evidence for functional significance of the
permuted C motif in Co2+-stimulated RNA dependent RNA polymerase of
infectious bursal disease virus. J. Gen. Virol. 88, 2824–2833.
Marchler-Bauer, A., Anderson, J.B., Derbyshire, M.K., DeWeese-Scott, C., Gonzales, N.R.,
Gwadz, M., Hao, L., He, S., Hurwitz, D.I., Jackson, J.D., Ke, Z., Krylov, D., Lanczycki, C.J.,
Liebert, C.A., Liu, C., Lu, F., Lu, S., Marchler, G.H., Mullokandov, M., Song, J.S., Thanki,
N., Yamashita, R.A., Yin, J.J., Zhang, D., Bryant, S.H., 2007. CDD: a conserved domain
database for interactive domain family analysis. Nucleic Acids Res. 35, 237–240.
Martelli, G.P., Sabanadzovic, S., Abou-Ghanem Sabanadzovic, N., Edwards, M.C., Dreher,
T., 2002. The family Tymoviridae. Arch. Virol. 147, 1837–1846.
Ng, K.K.-S., Arnold, J.J., Cameron, C.E., 2008. Structure-function relationships among
RNA-dependent RNA polymerases. Curr. Top. Microbiol. Immunol. 320, 137–156.
Pan, J., Vakharia, V.N., Tao, Y.J., 2007. The structure of a birnavirus polymerase reveals a
distinct active site topology. Proc. Natl. Acad. Sci. U.S.A. 104, 7385–7390.
Poch, O., Sauvaget, I., Delarue, M., Tordo, N., 1989. Identification of four conserved motifs
among the RNA-dependent polymerase encoding elements. EMBO J. 8, 3867–3874.
Ranjith-Kumar, C.T., Kim, Y.C., Gutshall, L., Silverman, C., Khandekar, S., Sarisky, R.T.,
Kao, C.C., 2002. Mechanism of de novo initiation by the hepatitis C virus RNA-
dependent RNA polymerase: role of divalent metals. J. Virol. 76, 12513–12525.
Rozanov, M.N., Koonin, E.V., Gorbalenya, A.E., 1992. Conservation of the putative
methyltransferase domain: a hallmark of the ‘sindbis-like’ supergroup of positive-
strand RNA viruses. J. Gen. Virol. 73, 2129–2134.
Rozanov, M.N., Drugeon, G., Haenni, A.-L., 1995. Papain-like proteinase of turnip yellow
mosaic virus : a prototype of a new viral proteinase group. Arch. Virol. 140, 273–288.
7
Sabanadzovic, S., Abou Ghanem-Sabanadzovic, N., 2009. Grapevine virus Q: the
first phytovirus with inverted RdRp motifs. Phytopathology 99(Supplement),
S112.
Sabanadzovic, S., Abou Ghanem-Sabanadzovic, N., in press. Identification and molecular
characterization of a marafivirus in Rubus spp. Arch. Virol. doi:10.1007/s00705-
009-0510-x.
Sabanadzovic, S., Abou-Ghanem, N., Castellano, M.A., Digiaro, M., Martelli, G.P., 2000.
Grapevine fleck virus-like viruses in Vitis. Arch. Virol. 145, 553–565.
Sabanadzovic, S., Abou Ghanem-Sabanadzovic, N., Saldarelli, P., Martelli, G.P., 2001.
Complete nucleotide sequence and genomic organization of Grapevine fleck virus.
J. Gen. Virol. 82, 2009–2015.
Shi, B.J., Habili, N., Symons, R.H., 2003. Nucleotide sequence variation in a small region
of the Grapevine fleck virus provides evidence for two sequence variants of the
virus. Ann. Appl. Biol. 142, 349–355.
Shwed, P.S., Dobos, P., Cameron, L.A., Vakharia, V.N., Duncan, R., 2002. Birnavirus VP1
proteins form a distinct subgroup of RNA dependent RNA polymerases lacking a
GDD motif. Virology 296, 241–250.
Tian, T., Klaassen, V.A., Soong, J., Wisler, G., Duffus, J.E., Falk, B.W., 1996. Generation of
cDNAs specific to lettuce infectious yellows closterovirus and other whitefly-
transmitted viruses by RT-PCR and degenerated oligonucleotide primers
corresponding to the closterovirus gene encoding the heat shock protein 70
homolog. Phytopathology 86, 1167–1172.
Valverde, R.A., Nameth, S.T., Jordan, R.L., 1990. Analysis of double-stranded RNA for
plant virus diagnosis. Plant Dis. 74, 255–258.
van Dijk, A.A., Makeyev, E.V., Bamford, D.H., 2004. Initiation of viral RNA-dependent
RNA polymerization. J. Gen. Virol. 85, 1077–1093.
von Einem, U.I., Gorbalenya, A.E., Schirrmeier, H., Behrens, S.E., Letzel, T., Mundt, E.,
2004. VP1 of infectious bursal disease virus is a RNA-dependent RNA polymerase. J.
Gen. Virol. 85, 2221–2229.
Xu, H.T., Si, W.D., Dobos, P., 2004. Mapping the site of guanylylation on VP1, the protein
primer for infectious pancreatic necrosis virus RNA synthesis. Virology 322,
199–210.