1. Gene, 161 (1995) 293-294
0 1995 Elsevier Science B.V. All rights reserved. 0378-l 119.19S/$O9.50
GENE 08994
Brief Notes
RNA 4 sequences from cucumber mosaic virus subgroups I and II*
(Recombinant DNA; cDNA; CMV; molecular cloning; nucleotide sequence; coat protein; classification)
Beau J. Anderson, Patricia M. Boyce ** and Christopher L. Blanchard **
School qfScience und Technology. and Centre fbr Conservation Farming. Charles Sturt Uniwrsit!.. Wugga Wugga. NSW3678. iiustrtrlitr
Received by J.L. Slightom: 13 January 1995; Accepted: 1 February 1995: Received at publishers: 13 April 1995
293
SUMMARY
The complete sequences of RNA 4 from cucumber mosaic virus (CMV) strains Ny (subgroup I) and Sn (subgroup
II) have been determined and compared to all other known complete CMV RNA 4 sequences. The identification of a
unique EcoRI site, present only in subgroup-II RNA 4 sequences, provides the basis for a simple method of classifying
CMV isolates.
Cucumber mosaic virus (CMV) is a tripartite single-
stranded positive sense RNA plant virus (Peden and
Symons, 1973). The genome is divided into three species,
RNAs 1,2 and 3. A fourth subgenomic RNA, RNA 4, is
generated from the 3’-half of RNA 3 and encodes the
viral coat protein (CP) (Schwinghamer and Symons,
1975; Davies and Symons, 1988). A very large number of
different strains of CMV have been isolated and divided
into two subgroups (I and II) on the basis of a range of
different criteria including serological typing, symptomo-
logical and host range data, peptide mapping of the CP
and nucleic acid hybridisation (see Palukaitis et al., 1992;
Wahyuni et al., 1992). This division of CMV isolates into
two subgroups is also reflected in the nt sequence varia-
Corrrspondencr to: Dr. B.J. Anderson, School of Science and
Technology, Charles Sturt University, P.O. Box 588, Wagga Wagga,
NSW 2678, Australia. Tel. (61-69) 332-739; Fax (61-69) 332-737:
e-mail: banderso@zac.riv.csu.edu.au
*On request, the authors will supply detailed experimental evidence for
the conclusions reached in this Brief Note.
** Current address: CSIRO Division of Plant Industry, GPO Box 1600.
ACT 2601, Australia. Tel. (61-69) 246-5376.
Abbreviations: aa. amino acid(s); bp. base pair(s); CP, coat protein:
CMV. cucumber mosaic virus; kb, kilobase or 1000 bp; ORF, open
reading frame; nt, nucleotide(s): PCR, polymerase chain reaction; UTR.
untranslated region(s).
SSDI 037% I I 19( 95)00276-6
tion that exists between isolates that have been cloned
and sequenced (see Palukaitis et al., 1992).
The complete sequence of RNA 4 of two CMV strains,
Ny (subgroup I) and Sn (subgroup II) was determined
(Fig. 1) and compared against all other other complete
CMV RNA 4 sequences present in the database (from
strains Kin. Q, Trk7, Sn, WL (subgroup II), Japanese Y,
Y, L, Fny, Ny, C, M, 0, Korean, NT9 and As (subgroup
I); data not shown). The lengths of RNA 4 from Ny-CMV
and Sn-CMV are 1033 and 1041 nt, respectively. The CP
ORF is 657 nt (218 aa) in all CMV strains and encodes
a protein of 24 141 and 24 173 Da in Ny-CMV and
Sn-CMV, respectively.
The length of the 5’ UTR is 69977 nt in subgroup-l
strains and 53363 nt in subgroup-II strains, and is the
most variable region of RNA 4 both between and within
the subgroups. The 3’ UTR is 300&304 nt in subgroup-I
strains and 321-327 nt in subgroup-11 strains, and shows
a considerable degree of variation between the subgroups,
but is reasonably conserved within the subgroups. The
nt sequence of the CP ORF is the most highly conserved
region of the molecule, but again demonstrates subgroup
specific variation. The overall nt sequence identity
between the subgroups is 74.9% (comparing consensus
sequences), and that within the subgroups is 93.6% for

2. 294
Fig. 1. Alignment of RNA 4 sequences from CMV strains Ny (ny) and Sn (sn). Sequences were aligned using the PILEUP and PRETTY programs
of the GCG (Genetics Computer Group, Madison, WI, USA) Sequence Analysis Software package for VAXjVMS computers. Identical nt are shown
in upper case, nt substitutions in lower case and gaps in the sequence are shown as dots. The start and stop codons of the CP ORF are underlined.
The EcoRI site, present in the Sn-CMV sequence, is double underlined. The Ny-CMV and Sn-CMV RNA 4 sequences have GenBank accession Nos.
U22821 and U22822, respectively.
subgro.up II and 69% for subgroup I. This unusually low
degree of homology for the subgroup-I RNA 4 sequences
is due to the divergent nature of the 5’and 3’ UTR of
CMV strains Kor, NT9 and As. However, it is quite clear
that these strains do belong to subgroup I on the basis
of their nt sequence. Our results show that there is a
greater degree of sequence variation in the UTR of the
RNA 4 molecules than has been previously observed
(Palukaitis et al., 1992, and references therein).
All subgroup-II RNA 4 sequences contain a unique
EcoRI site (see Fig. l), which in absent in the subgroup-I
RNA 4 sequences. This could be used as the basis of a
method to classify CMV strains (Wiley et al., 1993).
Previous hybridisation studies (Owens and Palukaitis,
1988; Wahyuni et al., 1992) suggest that RNA 4 sequences
would be a reliable indication of subgroup classification.
REFERENCES
Davies, C. and Symons, R.H.: Further implications for the evolutionary
relationships between tripartite plants viruses based on cucumber
mosaic virus RNA 3. Virology 165 (1988) 216-224.
Owen, .I.and Palukaitis, P.: Characterisation of cucumber mosaic virus,
I. Molecular heterogenicity mapping of RNA 3 in eight CMV
strains. Virology 166 (1988) 495-502.
Palukaitis, P., Roossinck, M.J., Dietzgen, R.G. and Francki, R.I.B.:
Cucumber mosaic virus. Adv. Virus Res. 41 (1992) 281-348.
Peden, K.W.C. and Symons, R.H.: Cucumber mosaic virus contains a
functionally divided genome. Virology 53 (1973) 487-492.
Schwinghamer, M.W. and Symons, R.H.: Fractionation of cucumber
mosaic virus RNA and its translation in a wheat embryo cell-free
system. Virology 63 (1975) 252-262.
Wahyuni, W.S., Dietzgen, R.G., Hanada, K. and Francki, R.I.B.:
Serological and biological variation between and within subgroup
I and II strains of cucumber mosaic virus. Plant Pathol. 41 (1992)
282-297.
Wiley, S., Wilson, C.R., Jones, R.A.C. and Jones, M.G.K.: A polymerase
chain reaction assay for cucumber mosaic virus in lupin seeds. Aust.
J. Agric. Res. 44 (1993) 41-51.
ACKNOWLEDGEMENTS
This work was supported by the Grains Research and
Development Corporation.