1022-7954/04/4012- © 2004
Russian Journal of Genetics, Vol. 40, No. 12, 2004, pp. 1356–1363. Translated from Genetika, Vol. 40, No. 12, 2004, pp. 1637–1645.
Original Russian Text Copyright © 2004 by Istomina, Snegireva, Shiyan.
The attenuated tomato strain V-69 of tobacco
mosaic virus (TMV) belongs to the tobamovirus group.
As in all other tobamoviruses, the single positive strand
of the viral genomic RNA carries information on repli-
case proteins (126 kDa/183 kDa), transport protein
(TP; 30 kDa), and coat protein (CP; 17.5 kDa). The col-
linear 126- and 183-kDa proteins are translated from a
common 5'-proximal open reading frame in the course
of the cotranslational dissociation of viral particles
immediately after the virus penetration into a plant cell:
the synthesis of the 126-kDa protein is terminated on
the internal termination amber codon (UAG), and the
183-kDa protein is formed if the termination codon is
read through [1, 2]. The TP and CP are translated from
subgenomic mRNAs that are 3'-coterminal and are
transcribed from the negative strand of the TMV RNA
at some distance from the respective subgenomic pro-
moters [3–5]. The main function of the TP is to trans-
port the virus from cell to cell; in addition, it is neces-
sary for the formation of the replicative complex .
The CP is involved in the systemic transport of the virus
in a plant ; it is the only structural protein coating the
genomic RNA and sometimes determining speciﬁc
symptoms . Within the 183-kDa protein gene, an
additional reading frame for the 54-kDa protein trans-
lated from the subgenomic mRNA has been found. This
protein has never been found in plant tissues infected
with the virus .
The TMV V-69 strain is a natural attenuated strain
that was obtained earlier in our laboratory and is used
in practical agronomy for vaccination of tomato .
This strain does not cause noticeable symptoms of the
viral disease in susceptible tomato plants and causes
only slightly manifested symptoms in tobacco plants. It
induces necroses in the leaves of hypersensitive hosts.
The V-69 strain can reproduce in tomato plants carrying
the resistance gene
The TMV V-69 strain repro-
tomato plants retains its attenuated phe-
notype, which extends the ﬁeld of its application; V-69
may be used for protecting susceptible tomato plants
Earlier, we demonstrated [11, 12] that the genome
of the TMV V-69 strain contains 38 nucleotide substi-
tutions compared to the pathogenic tomato strains L .
Six out of 38 substitutions cause changes in the amino
acid sequences of the proteins encoded by the virus,
ﬁve substitutions being located in replicase proteins
(four in the sequence that is common for the 126-kDa
and 183-kDa proteins and one in the 183-kDa one) and
one substitution in the CP .
In this study, we obtained a full-length cDNA copy
of the TMV strain V-69 RNA integrated into the pBS(+)
vector under the control of the promoter of the phage
T7 polymerase. For convenience, all restriction sites
located in the genome itself were eliminated using in
vitro point mutagenesis and one
dIII site in the viral
nucleotide sequence was inactivated. After in vitro tran-
scription, we obtained an RNA that did not cause
noticeable symptoms of the disease in tomato plants
and in tobacco.
The full-length copy may be used for studying the
effects of mutations found in the TMV V-69 strain on
the virus pathogenicity and for constructing a vector for
transit gene expression [14–16].
Construction of a Full-Length cDNA
of Tobacco Mosaic Virus Strain V-69 Genome
E. A. Istomina, P. B. Snegireva, and A. N. Shiyan
Vavilov Institute of General Genetics, Russian Academy of Sciences, 119991 Russia; e-mail: email@example.com
Received June 3, 2004
—The genome of the tobacco mosaic virus (TMV) strain V-69 was sequenced by us earlier. The com-
parison of its nucleotide sequence with those of well-known tobamovirus strains (using published data) dem-
onstrated considerable homology between strain V-69 and tomato strain L of TMV. In this study, full-length
cDNA copy of TMV strain V-69 genomic RNA has been cloned in vector pBS(+) under the control of the T7
promoter and with an
I site at the 3'-terminus. The in vitro transcription of this construction with the use of
phage T7 RNA polymerase in the presence of a cap analog (m
GpppG) has yielded an infectious RNA. This
RNA has induced the same symptoms as those caused by TMV strain V-69 in infected indicator plants. Three
point mutations inactivating one of two
dIII sites have been introduced into the cDNA. The absence of the
dIII site at position 1449 of the viral nucleotide sequence and the corresponding mutations serve as markers
of the construction obtained. The full-length cDNA copy of the TMV strain V-69 RNA can be used both as a
research tool and for constructing a vector for foreign gene expression.