Occurrence of a DNA sequence of a non-retro RNA virus in a
host plant genome and its expression: evidence for recombination
between viral and host RNAs
Edna Tanne1, Ilan Sela*
Virus Laboratory, The Hebrew University of Jerusalem, Faculty of Agricultural, Food and Environmental Quality Sciences,
Robert H. Smith Institute for Plant Sciences and Genetics, Rehovot 76100, Israel
Received 8 September 2004; returned to author for revision 26 October 2004; accepted 9 November 2004
Available online 8 January 2005
This study demonstrates that sequences homologous to those of the non-retro RNA virus (Potato virus Y; PVY) are integrated into the
genome of several grapevine varieties. The integrated PVY-coat-protein-like cistron is expressed in the grapevine as indicated by Southern
and Western blot analyses as well as by RNase protection assay. In addition, genome-walking studies showed that one PVY-like sequence is
flanked by 41-bp direct repeats and is embedded in authentic grapevine sequences, flanked by inverted repeats. Rearranged PVY-like
sequences were also found in tobacco. It is suggested that nonhomologous recombination of a potyviral RNA with RNA of a
retrotransposable element took place at some point in evolution. The initial integration locus was probably within a grapevine gene
homologous to a pentatricopeptide repeat-carrying protein, and was later transposed to other locations. The current location is reminiscent of
a MITE-type retroelement, indicating transposition history. Because grapevine cultivars are propagated asexually, without going through a
meiotic phase, the chance for DNA recombination is minimal and the foreign integrated sequence may be better conserved, enabling it to be
expressed correctly in the recipient genome.
D 2004 Elsevier Inc. All rights reserved.
Keywords: PVY; Integration; Grapevine; Transposition; RNA recombination
Viral DNA sequences can become incorporated in host
genomes, and partial sequences of viral DNAs have been
have been found in the host nucleus, either integrated into the
host DNA, or in the form of episomes (Jakowitsch et al.,
1999; Lockhart et al., 2000; Mette et al., 2002; Ndowora et
al., 1999; Richert-Poggeler et al., 2003). Sequences of the
single-stranded DNA geminiviruses have also been found
integrated in their host plant’s genome (Ashby et al., 1997;
Bejarano et al., 1996; Harper et al., 1999). However, the only
viruses with RNA genomes known to integrate a DNA
version of their genome into host chromosomal DNA are the
retroviruses. In this case, the RNA genomes are reverse-
transcribed and the resultant DNA is inserted into the host
DNA by a virus-encoded integrase; these reactions are
required for normal replication (for example, Flint et al.,
2004; Goff, 1992; Hu and Temin, 1990).
Potyviruses are a large, polyphagous group of plant
viruses that carry an RNA genome of sense orientation.
Potyviral RNA is replicated via an RNA replicase, and does
not undergo reverse transcription or genome integration
(Vitis vinifera) varieties have been reported to react with
antiserum against the potyvirus potato virus Y (PVY). Dot
0042-6822/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
* Corresponding author. Fax: +972 8 9473402.
E-mail address: firstname.lastname@example.org (I. Sela).
1Permanent address: Department of Virology, Agricultural Research
Organization, Volcani Center, Bet Dagan 50250, Israel.
Virology 332 (2005) 614–622
blot analysis of nucleic acids extracts also reacted positively
with a potyviral probe (Naveh, 1987; Tanne et al., 1989).
However, potyvirus particles or potyviral-associated inclu-
sion bodies (such as pinwheels) have never been found in the
grapevines. Here we demonstrate that grapevine genomic
DNA carries the entire gene of a potyviral coat protein (CP)
and the potyviral 3VUTR. Potyviral-homologous sequences
were also found in tobacco DNA, albeit in a rearranged form.
Transposable elements (TE) are divided into two general
classes. Class I elements are propagated by a mechanism
that involves RNA intermediates. Some active Class I
elements (retroviruses and retrotransposons) carry long
terminal repeats (LTRs) at both ends. Such elements
typically encode their own reverse transcriptase and
integrase, allowing the production of a DNA copy from
an RNA transcript and its insertion at another site in the
genome. Other types of class I elements lack LTRs and are
either transposed by another mechanism (LINES) or do not
encode transposition proteins (SINES) but depend on those
of other elements, such as LINES. Class I elements are
flanked on either side by direct repeats of host sequences of
various length, formed at the time of their integration into
the host DNA.
Class II TEs are mobilized by a bcut and pasteQ
mechanism. Active class II transposons carry terminal
inverted repeats (TIRs), and are also flanked bydirect repeats
two open reading frames (ORFs), one of which specifies a
transposase, responsible for mobilization. Many types of
inactive (nonautonomous) class II elements have evolved,
comprising an array of repetitive sequences dispersed
throughout the host genome. One such group, miniature
inverted-repeat transposable elements (MITEs), has been
a very high copy number (for example: Fechotte and
MounchOes, 2000; Le et al., 2000; Santiago et al., 2002;
Zhang et al., 2001). MITEs are TIR-carrying short dispersed
sequences (b500 bp). These elements do not carry any ORF;
however, they are located next to transcribed sequences
(Wessler et al., 1995; Yang et al., 2001). Sequence analysis
has indicated that MITEs have probably evolved from active
class II transposons, as some degree of homology was found
with transposase-carrying elements (Fechotte and MounchOes,
2000; Le et al., 2000). In maize, a class II group of size range
several nonautonomous members and one active element
(PIFa; Zhang et al., 2001). MITEs are structurally similar to
thedefective PIF elements.However,theirhighcopynumber
suggests a replicative mechanism of propagation and trans-
position (Fechotte and MounchOes, 2000).
The data presented in this paper suggest that recombi-
nation between viral RNA and the RNA of a host cell
retrotransposable element may have taken place. RNA
recombination between various segments of viral RNAs is a
well-recorded phenomenon, and various mechanisms have
been proposed to account for these recombination events
(Nagy and Simon, 1997, and references therein). However,
all known recombination events occurred between segments
of RNAs of the same virus, or related viruses (some recent
examples: Adams et al., 2003; Oberste et al., 2004; Wu et
al., 2003). A major mechanism leading to viral RNA
recombination appears to be template switching by the viral
replicase (Nagy and Simon, 1997). Indeed, mutation in the
viral replicase is reported to reduce RNA recombination
(Cheng and Nagy, 2003). This may explain the restriction of
RNA recombination to segments of RNAs of the same virus
or between bstrainsQ of the same virus, as these RNAs are
all recognized by the same replicase. A similar mechanism
is also suggested for the appearance of defective interfering
viral RNAs (e.g., Bar-Joseph et al., 1997; Shapka and Nagy,
2004). AU rich regions, and RNA promoters for sub-
genomic RNAs seem to have a role in RNA recombination
(Shapka and Nagy, 2004), and host factors also influence
recombination (Dzianott and Bujarski, 2004). Splicing and
processing of RNAs (such as of ribosomal RNAs) are also
forms of RNA recombination, in which case parts of the
very same transcripts are ligated together. Indications of
non-replicative viral RNA recombination (Gallei et al.,
2004) and virus-host RNA recombination (Charini et al.,
1994; Meyers et al., 1989; Monroe and Schlesinger, 1983)
have been reported. To the best of our knowledge,
recombination between host derived retrotransposable
elements and (non-retro) viral RNAs has not yet been
The data presented in Table 1 support previous findings
(Naveh, 1987; Tanne et al., 1989) by showing that several
grapevine varieties reacted in an ELISA test with an
antiserum against PVY. Previously, we demonstrated that
nucleic acid extracts from several grapevine varieties react
with a PVY probe upon dot-blot hybridization assays
(Tanne et al., 1989). The absence of any potyvirus particles
in the plants analyzed suggested the possible occurrence of
PVY-homologous sequences in the host genome. We
ELISA results following reaction of saps from several grapevines with
antiserum to PVY
Grapevine variety or cloneELISA reading A405
Rouge de Loire
E. Tanne, I. Sela / Virology 332 (2005) 614–622
isolated defective interfering particles of Sindbis virus contain a
cellular tRNA sequence at their 5V end. Proc. Natl. Acad. Sci. U.S.A.
Nagy, P.D., Simon, A.E., 1997. New insights into the mechanisms of RNA
recombination. Virology 235, 1–9.
Naveh, L., 1987. Detection of viruses associated with leafroll disease in
grapevine by ELISA and molecular hybridization techniques. M.Sc.
Thesis. The Hebrew University of Jerusalem. Faculty of Agriculture.
Ndowora, T., Dahlal, G., LaFleur, D., Harper, G., Hull, R., Olszewski,
N.E., Lockhart, B., 1999. Evidence that badnavirus infection in Musa
can originate from integrated pararetroviral sequences. Virology 255,
Neuve ´glise, C., Feldmann, H., Bon, E., Gaillardin, C., Casaregola, S., 2002.
Genomic evolution of the long terminal repeat retrotransposons in
Hemiascomycetous yeasts. Genome Res. 12, 930–943.
Oberste, M.S., Penaranda, S., Pallansch, M.A., 2004. RNA recombination
plays a major role in genome change during circulation of coxsacki B
viruses. J. Virol. 78, 2948–2955.
Richert-Poggeler, K.R., Noreen, F., Schwartzacher, T., Harper, G., Hohn,
T., 2003. Induction of infectious petunia vein clearing (pararetro) virus
from endogenous provirus in petunia. EMBO J. 22, 4836–4845.
Sambrook, J., Russel, D.W., 2001. Molecular Cloning: A Laboratory
Manual. 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring
Santiago, N., Herra ´iz, C., Gon ˇi, R.J., Messeguer, X., Casacuberta, J.M.,
2002. Mol. Biol. Evol. 19, 2285–2293.
Shapka, N., Nagy, P.D., 2004. The AU-rich RNA recombination hot spot
sequence of brome mosaic virus is functional in tombusviruses:
implications for the mechanism of RNA recombination. J. Virol. 78,
Tanne, E., Naveh, L., Sela, I., 1989. Serological and molecular evidence for
the complexity of the leafroll disease of grapevine. Plant Pathol. 38,
Towbin, H., Staehlen, T., Gordon, J., 1979. Electrophoretic transfer of
proteins from polyacrylamide gels to nitrocellulose sheets: procedure
and some applications. Proc. Natl. Acad. Sci. U.S.A. 76, 4350–4354.
Wessler, S.R., Bureau, T.E., White, S.E., 1995. LTR-retrotransposones and
MITEs: important players in evolution of plant genomes. Curr. Opin.
Genet. Dev. 5, 814–821.
Wu, H.Y., Guy, J.S., Yoo, D., Vlasak, R., Urbach, E., Brian, D.A., 2003.
Common RNA replication signals exist among group 2 coronaviruses:
evidence for in vivo recombination between animal and human
coronavirus molecules. Virology 315, 174–183.
Yang, G., Dong, J., Chandrasekharan, M.B., Hall, T.C., 2001. Kiddo, anew
transposable element family closely associated with rice genes. Mol.
Genet. Genomics 266, 417–422.
Zeitoune, S., Livneh, O., Kuznetzova, L., Stram, Y., Sela, I., 1999. T7 RNA
polymerase drives transcription of a reporter gene from T7 promoter,
but engenders post-transcriptional silencing of expression. Plant Sci.
Zhang, X., Feschotte, C., Zhang, Q., Jiang, N., Eggleston, W.B., Wessler, S.,
2001. P instability factor: an active maize transposon system associated
with the amplification of Tourist-like MITES and a new superfamily of
transposases. Proc. Natl. Acad. Sci. U.S.A. 98, 12572–12577.
Zickler, D., Klencker, N., 1999. Meiotic chromosomes: integrating,
structure and function. Annu. Rev. Genet. 33, 603–745.
E. Tanne, I. Sela / Virology 332 (2005) 614–622