Journal o f General Virology (1991), 72, 9-14.
Printed in Great Britain
9
The genome organization of potato virus M RNA
S. K. Zavriev,* K. V. Kanyuka and K. E. Levay
All-Union Research Institute of Agricultural Biotechnology, Moscow 127253, U.S.S.R.
The 8534 nucleotide sequence of the genome of the
carlavirus, potato virus M (PVM), has been determined. The sequence contains six large open reading
frames (ORFs) and non-coding regions consisting of 75
nucleotides at the 5' end, 70 nucleotides followed by a
poly(A) tail at the 3' end and 38 and 21 nucleotides
between three large blocks of coding sequences. The
ORF beginning at the first initiation codon at nucleotide 76 encodes a polypeptide of 223K which, accord-
ing to its primary sequence analysis, seems to be a virus
RNA replicase. The next coding block consists of three
ORFs encoding polypeptides of 25K, 12K and 7K. The
third block consists of two ORFs encoding polypeptides of 34K (PVM coat protein) and llK. The l l K
polypeptide contains a pattern resembling the consensus for a metal-binding nucleic acid-binding 'finger'.
The nucleotide sequence EMBL accession number is
X53062.
Introduction
Ampicillin-resistant transformants were analysed for PVM-specific
inserts by colony hybridization (Maniatis et al., 1982). The insert size of
selected recombinant eDNA clones was determined by restriction
enzyme analysis of plasmid DNA samples. Finally, six clones
containing plasmids with long overlapping eDNA inserts (see Fig. 1)
were isolated and used for sequencing. A nested series of exonuelease
III deletions were generated from the original eDNA clones according
to the Erase-a-base system (Promega Biotec). Competent E. coli XL-1B
cells were used as bacterial hosts. Transformants containing plasmids
with the appropriate size of deletion were grown in 1.5 ml of selective
liquid medium and plasmid DNA was isolated as described by Del Sal
et al. (1988). The extent of the exonuelease III deletions was determined
by restriction analysis. Dideoxynueleotide sequencing of dsDNA
templates was carried out using [~t-32p]dATP or [~-35S]dATP
and T7 phage-derived DNA polymerase (Sequenase; U.S. Biochemical). We have sequenced more than 96% of the eDNA in both
orientations and more than 47% of the sequence was determined by
examining two or three different clones covering each particular
region.
Potato virus M (PVM), a member of the carlavirus
group, has filamentous particles composed of multiple
copies of the coat protein and a monopartite plus-sense
ssRNA of approximately 8 kb in length (Proll et al.,
1981; Wetter & Milne, 1981; Tavantzis, 1984). In our
previous papers we published the nucleotide sequence of
the 3'-proximal 2630 nucleotides of PVM genomic RNA
and demonstrated a similarity between PVM and the
potexviruses in the gene arrangement and encoded
amino acid sequences (Rupasov et al., 1989, 1990). The
nucleotide sequences of the 3' terminal regions of the
genomic RNAs of two other carlaviruses, potato virus S
(PVS) and lily symptomless virus (LSV), were published
by MacKenzie et al. (1989) and Memelink et al. (1990),
respectively. In this article we report the nucleotide
sequence of PVM RNA and outline its genomic
organization.
Methods
R N A sequencingl The 5' terminus of PVM RNA was sequenced using
reverse transcriptase and the 32p-end-labelled synthetic primer 5'
dCTCAAGTATATGCCTGCC 3', complementary to nucleotides 246
to 263 of the final sequence.
Isolation o f virus RNA. PVM (Russian wild strain) was purified from
infected tomato plants as described by ProU et al. (1981). RNA was
isolated from the purified virus preparation using the phenol-SDS
extraction method (Proll et al., 1981) with minor modifications.
cDNA cloning and sequencing. Double-stranded eDNA for the 3'proximal 2630 nueleotides of PVM genomic RNA has been synthesized and sequenced previously (Rupasov et al., 1989). Doublestranded eDNA for the Y-proximal approximately 6000 nucleotides
was synthesized by a similar procedure using a synthetic deoxyoligonucleotide primer complementary to the viral genome as deduced from
the known sequence. The double-stranded cDNA was ligated to a
H/ndlI-cut plasmid pGEM-3Z (Promega) and transformed into
competent Escherichia coli XL-IB cells (Stratagene Gene Cloning
System) as described by Maniatis et al. (1982).
0000-9830 © 1991 SGM
12K
25KI~
223K
_
IlK
_
1000
2000
3000
4000
5000
6000
7000
8000
I
I
I
I
I
|
|
|
"~
pVM 107
3
u
, pVM 9
~pVM 30
pVM~47 L pVM 28
, ,/~/x
[ ~ ] I 9 K poly(A)
t
pVM 33
,
Fig. 1. Schematic representation of six ORFs in PVM genomic RNA,
the ORF in the negative strand, and long overlapping eDNA clones
used for sequencing. All are shown relative to their location in the PVM
genome. The arrow indicates the position of the oligonucleotide primer
used for run-off sequencing of the viral RNA 5' terminus.
o~
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=
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t.Jr3
P V M R N A genome organization
nucleotide sequencing (see Methods). Since the first two
nucleotides could not be unequivocally identified in this
way they are denoted by N in Fig. 2. The sequence of the
PVM RNA Y-terminal 20 nucleotides is very similar to
that of PVS RNA as determined by Monis & DeZoeten
(1990). The overall base composition of the PVM
genomic RNA is 26.5~ A, 24.9~ U, 28.5~ G, 20.1 ~o C.
Analysis of the PVM genome sequence shows the
presence of six potential ORFs (Fig. 1). The first and
longest ORF encodes a polypeptide of 1968 amino acids
of Mr 223316 (PVM 223K protein). Detailed analysis
and the main characteristics of the proteins encoded by
the other five ORFs were given previously (Rupasov et
al., 1989). Analysis of the negative strand sequence of
PVM genomic RNA revealed only one ORF encoding a
putative 179 amino acid long polypeptide of Mr 19019
(19K protein). The ORF of this protein is localized at
positions 923 to 1459 of the negative strand (see Fig. 1).
The most interesting feature of the primary structure of
the 19K protein is that it consists of two domains which
have significant homology to each other (Rupasov et al.,
1990).
P V M R N A nucleotide sequences probably essential for
genome expression
The PVM genome, by analogy to potexviruses, may be
expressed through at least two subgenomic RNAs
(Rupasov et al., 1989). Similarity between nucleotide
sequences preceding the initiation codons (i.e. the
location of the 5' ends of the putative subgenomic RNAs)
is significant for the PVM 25K and 34K protein genes
(Rupasov et al., 1989), but was not observed with the
223K protein gene. It can be supposed that an important
part in PVM genome expression is played by the
sequence
ACAAGNNNN-GAANNGC(Nn)AUG
(consensus) found upstream of the PVM RNA ORFs
(Fig. 3). This sequence is clearly observable upstream of
the 223K protein gene and the genes of the triple block
proteins but is almost or completely absent upstream of
the 34K and I l K protein genes. This sequence is
probably essential for translation of the PVM triple
block proteins, being a signal for either reinitiation of
translation or independent initiation of translation.
ACAA-IInt-GAAAUAC
-
20nt
-
AUG
223K
5961
34
ACAAGuAUIrOGAAGuGC
-
43nt
-
AUG
25K
6651
ACAAGcAuc-GAAc*GC
--
1 8nt
-- A U G
6968
ACAAGcUAG-GAAG--GC
-
I 8nt
-
AUG
12K
7K
Fig. 3. Comparison of sequences upstream of the AUG start codons of
the 223K, 25K, 12K and 7K ORFs in PVM RNA. Gaps are introduced
for best alignment. Conserved nucleotides are indicated in boldface.
Numbers at the left show positions of nucleotides according to the
sequence from Fig. 2.
13
Discussion
The genome organization of carlaviruses, based on the
data presented here and those from the sequencing
studies with PVS and LSV RNAs, shows a strong
similarity in gene arrangement and encoded amino acid
sequences of corresponding polypeptides (Rupasov et al.,
1989; MacKenzie et al., 1989; Memelink et al., 1990).
Analysis of the first ORF at the 5" end, which encodes
a 223K polypeptide, reveals similarities with the large
ORF proteins of potexviruses and tymoviruses (Morozov
et al., 1990). One of the homologous domains of the PVM
223K protein is the GDD domain (Fig. 2, underlined)
which is conserved in the RNA replicase components of
all plant and animal single-stranded plus-sense RNAcontaining viruses (Poch et al., 1989). Recently, a high
level of similarity in the GDD domain region was found
between the replicase proteins of carlaviruses and the
216.5K protein of apple chlorotic leaf spot closterovirus
(German et al., 1990).
The 25K protein of PVM, as well as all analogous
proteins encoded by PVS, LSV and potexviruses, the
58K protein of barley stripe mosaic virus (Gustafson &
Armor, 1986) and the 42K protein of beet necrotic yellow
vein virus (Bouzoubaa et al., 1986), contains the
consensus sequence of NTP-utilizing DNA helicases
(Gorbalenya et al., 1988), including the sequence GKS/T
(Fig. 2, underlined). This characteristic sequence is
duplicated in the PVM proteins, being also present in
one of the domains of the 223K protein (see Morozov et
al., 1990). Thus, the comparison of carlavirus and
potexvirus large replication proteins strengthens the
earlier conclusions regarding the similarity in genome
organization of these virus groups (Rupasov et al., 1989;
Memelink et al., 1990).
The in vitro translation product of the 34K protein gene
is precipitated by antisera against PVM, indicating that
the 34K protein is the virus coat protein (K. V. Kanyuka
& S. K. Zavriev, unpublished observations). The
carlavirus coat protein genes are followed by Y-proximal
ORFs encoding proteins containing a pattern similar to
C X 2 C X 1 2 C X 4 C , which resembles the consensus of a
metal-binding nucleic acid-binding 'finger' (Klug &
Rhodes, 1987; R. Coutts, personal communication).
Analogous 'finger' structures are found in other plant
virus-specific proteins (see Sehnke et al., 1989), particularly in tobraviruses (Hamilton et al., 1987; MacFarlane
et al., 1989). The functional role of such polypeptides is
not yet known. It can be suggested that they take part in
the first steps of virion assembly and/or are involved in
viral RNA replication.
We thank Dr S. Yu. Morozov for his interest and helpful discussions.
This work was partly financed by the Monsanto Company. The
sequence data appear under the EMBL accession no. X53062.
14
S. K. Zavriev, K. V. Kanyuka and K. E. Levay
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