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MOLECULAR PLANT PATHOLOGY (2002) 3(6), 419–429
Pathogen profile
Blackwell Science, Ltd
Cauliflower mosaic virus: still in the news
M U R I E L H A A S , M A R I N A B U R E A U , A N G È L E G E L D R E I C H , P I E R R E YO T A N D M A R I O KE L L E R *
Institut de Biologie Moléculaire des Plantes CNRS, Université Louis Pasteur, 12 rue du Général Zimmer, 67084 Strasbourg Cedex, France
SUMMARY
Taxonomic relationship: Cauliflower mosaic virus (CaMV) is
the type member of the Caulimovirus genus in the Caulimoviridae
family, which comprises five other genera. CaMV replicates its
DNA genome by reverse transcription of a pregenomic RNA and
thus belongs to the pararetrovirus supergroup, which includes
the Hepadnaviridae family infecting vertebrates.
Physical properties: Virions are non-enveloped isometric
particles, 53 nm in diameter (Fig. 1). They are constituted by 420
capsid protein subunits organized following T = 7 icosahedral
symmetry (Cheng, R.H., Olson, N.H. and Baker, T.S. (1992)
Cauliflower mosaic virus: a 420 subunit (T = 7), multilayer structure. Virology, 16, 655– 668). The genome consists of a doublestranded circular DNA of approximately 8000 bp that is embedded
in the inner surface of the capsid.
Viral proteins: The CaMV genome encodes six proteins, a
cell-to-cell movement protein (P1), two aphid transmission
factors (P2 and P3), the precursor of the capsid proteins (P4), a
polyprotein precursor of proteinase, reverse transcriptase and
ribonuclease H (P5) and an inclusion body protein/translation
transactivator (P6).
Hosts: The host range of CaMV is limited to plants of the
Cruciferae family, i.e. Brassicae species and Arabidopsis thaliana,
but some viral strains can also infect solanaceous plants. In nature,
CaMV is transmitted by aphids in a non-circulative manner.
I N T RO D U C T I O N
Cauliflower mosaic virus (CaMV) was the first plant virus to be
discovered to contain DNA instead of RNA as genetic material. Its
DNA was the first plant viral genome to be completely sequenced
(Franck et al., 1980). Furthermore, in the 1980s CaMV DNA was
cloned into plasmids in an infectious form and was thought to
hold great promise as a virus–based vector for expressing foreign
Correspondence: E-mail: mario.keller@ibmp-ulp.u-strasbg.fr
© 2002 BLACKWELL SCIENCE LTD
Fig. 1 Electron micrograph of CaMV virions. Courtesy of J. Menissier de
Murcia, Ecole Supérieure de Biotechnologie de Strasbourg.
genes in host plants. However, after some successes, i.e. expression of dihydrofolate reductase (Brisson et al., 1984), metallothionein (Lefebvre et al., 1987) and interferon (De Zoeten et al.,
1989), this strategy was abandoned because the CaMV genome
could tolerate only small insertions. Nevertheless, CaMV DNA
is used worldwide in plant biotechnology, as its 35S promoter
mediates the expression of associated genes at a high level in
most types of plant tissues and is therefore a very useful tool both
for fundamental research and commercial applications (for review,
Scholthof et al., 1996).
CaMV was also in the limelight because its DNA genome is
replicated by the reverse transcription of an RNA intermediate. It
appears that all the plant viruses containing a double-stranded
DNA use a reverse transcriptase for replication and are consequently classified in the Caulimoviridae family (Hull et al., 2000a).
This property is shared by the Hepadnaviruses which infect vertebrates (for review, Rothnie et al., 1994). Together, these viruses
form the so-called pararetrovirus supergroup, to distinguish them
from the true retroviruses. The two major differences are that:
(i) retroviruses contain RNA instead of DNA like pararetroviruses,
and (ii) the proviral DNA of retroviruses resulting from reverse
transcription of the RNA genome is integrated into the host
DNA, whereas the DNA of pararetroviruses behaves as a free
chromosome in the nucleus of the host cell. However, a few cases
of integration of Caulimoviridae DNA, probably by illegitimate
recombination, have recently been reported (for review, Hull
et al., 2000b). In spite of these differences, pararetroviruses and
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M. HAAS et al.
19S
35S
retroviruses share some structural and functional features
indicating that they are phylogenetically related.
This article intends to highlight some virus, vector and host
aspects of recent studies on CaMV. The evolution of CaMV and its
standing relative to other Caulimoviridae are not discussed.
∆1
VII
D N A S T R U C T U RE
The CaMV genome consists of a double-stranded circular DNA
molecule of approximately 8000 bp length. It exists inside the
virus particle in an open circular form, due to the presence of
single-stranded interruptions at specific sites on both (+) and (–)
DNA strands; their number and position vary depending on the
CaMV strains. These sequence discontinuities (called ∆) which
are remnants of the reverse transcription process, have a triplestranded structure whose overlapping strand may have a ribonucleotide sequence at its 5 ′ end. They are repaired by host
enzymes in the nucleus of the host cell to yield a supercoiled DNA
molecule. The latter becomes associated with histones to form a
minichromosome harbouring 42 ± 1 nucleosomes. Methylation
at specific restriction sites of the viral genome appears to occur
in an all-or-none manner 1 week after the infection of host plants
(Tang and Leisner, 1998). CaMV DNA can be subject to recombination events that seem to arise preferentially during reverse
transcription of the pregenomic RNA rather than at the DNA level
(Vaden and Melcher, 1990).
The CaMV genome has seven major open reading frames (ORF
I to VII) which are all located on the (–) DNA strand, and two
intergenic regions of about 700 bp and 150 bp, respectively,
containing regulatory sequences. The ORFs are separated or
overlap by a few nucleotides, except for ORF VI, which lies
between the two intergenic regions (Fig. 2).
TRA N S C R I P T I O N A N D S P L I C I N G
The CaMV minichromosome is transcribed unidirectionally by
the cellular RNA polymerase II into two major capped and polyadenylated transcripts, the 35S and 19S RNAs. These RNAs are
transcribed from their own promoters which are localized in the
large and small intergenic regions, respectively.
The 35S promoter is very strong and constitutive; if it is associated with genes, it mediates their expression in all types of cells
and at all developmental stages of the plant. It contains the
typical promoter motifs recognized by RNA polymerase II and
enhancers which confer on it a high transcriptional activity.
Dissection of the promoter/enhancer region revealed several
domains and subdomains whose synergistic interactions play an
important role in defining tissue-specific expression (Benfey
et al., 1990a,b). Several plant nuclear proteins binding to specific
domains of the 35S promoter are involved in transcriptional
regulation, i.e. transcription factors ASF-2 and TGA1a (Jupin and
VI
I
II
∆3
III
IV
V
∆2
Fig. 2 Schematic diagram of the CaMV genome. Thin lines represent the
double-stranded circular DNA (8 kbp) with sequence discontinuities (∆1–3).
Major ORFs shown by coloured arrows code for the cell-to-cell movement
protein (I), aphid transmission factors (II and III), the precursor of the capsid
proteins (IV), the precursor of aspartic proteinase, reverse transcriptase and
RNase H (V), and an inclusion body protein/translational transactivator (VI). The
solid black lines of the inner circle are the long and small intergenic regions
which contain the 35S and 19S promoters, respectively. The two external
arrowed lines correspond to the 35S and 19S RNAs.
Chua, 1996; Lam and Chua, 1989; Ruth et al., 1994) but none of
the viral proteins have been implicated.
The 35S RNA covers the total genome plus about 180 nt, so it
is terminally redundant. The redundancy is due to the fact that
RNA polymerase II ignores at its first passage the polyadenylation signal located approximately 180 nt downstream from the
transcription start site (Sanfaçon and Hohn, 1990). The polyadenylation signal consists of an AAUAAA sequence which determines the cleavage of the CaMV transcripts 13 nt downstream
and cis-acting upstream elements that increase the efficiency of
the 3′ processing (Sanfaçon et al., 1991). The 35S RNA serves
both as a polycistronic messenger RNA for the synthesis of
proteins P1 to P5 and as template for reverse transcription. Several
spliced versions of the 35S RNA, representing up to 70% of
the total viral RNA could be detected in CaMV-infected plants.
Analysis of these processed RNAs revealed a splice donor site in
the leader region of 35S RNA and three additional sites within the
3′ terminal part of ORF I (Kiss-Laszlo et al., 1995). All four donors
use a single acceptor site which is located inside ORF II. The splicing events generate mRNAs where ORF III is the first major
coding sequence or where ORF I and II are fused in-frame. Whether
the ratio of spliced to unspliced RNA is controlled as it is in
retroviruses is not known. The splicing of 35S RNA is essential for
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et al., 2000). Recently, it has been shown that the function of P6
depends on its association with polysomes and the eukaryotic
initiation factor eIF3 (Park et al., 2001). P6 physically interacts
with the g subunit of eIF3 and three proteins of the 60S ribosomal
subunit, namely L18 (Leh et al., 2000), L24 (Park et al., 2001) and
L13 (M. Bureau, unpublished data). Both L18 and L13 interact
with the P6 miniTAV domain (recently renamed MAV) which
corresponds to the minimal sequence required for translational
transactivation (De Tapia et al., 1993), whereas L24 and the eIF3
subunit g interact with a region located immediately downstream
from the miniTAV: the two latter cellular proteins compete with
each other for interaction with P6. The interactions between L24/
eIF3 and P6 are crucial for the translational transactivation mechanism, since CaMV is no longer infectious when point mutations
in P6 impair these interactions. Park et al. (2001) have demonstrated by pull-down assays that P6 interacts with eIF3 on both
40S and 60S ribosomal subunits and hence proposed that P6
mediates the efficient recruitment of eIF3 to polysomes, thus
allowing translation of polycistronic mRNA by a reinitiation
process. In a model reconciling all their data, Park et al. (2001)
assumed that P6 interacts with the 40S ribosomal subunit-bound
eIF3 which was not removed during the translation of a small
ORF. After the termination step, the ribosomal complex recruits a
ternary initiation complex, resumes scanning and finally reinitiates translation at the first long ORF (I) of the 35S RNA. During
the elongation phase, the P6–eIF3 complex is translocated to the
60S subunit via L18, and at the termination step, it shuttles again
to the 40S subunit to prevent release of this ribosomal subunit so
that it can then reinitiate translation of the next ORF. Concerning
the interaction between P6 and ribosomal protein L24, which
probably involves other P6 molecules than those interacting with
eIF3, the authors proposed that it might enhance the recycling of
the 60S subunit during translation of the 35S RNA. A recent study
performed in a mammalian system demonstrated that the ribosomal protein L18 binds to the double-stranded RNA-activated
protein kinase (PKR) and negatively regulates its activity (Kumar
et al., 1999). PKR belongs to the eIF-2α kinase family which is
involved in several metabolic pathways, among which the translation of mRNAs bearing small ORFs in their leader sequence (for
review see Dever, 2002) Therefore, the P6–L18 interaction might
be also involved in the regulation of a plant PKR-like activity. The
exploration of this possibility will first require investigation of the
role of PKR in plants.
Kinetic studies performed in planta (Maule et al., 1989) and
in turnip protoplasts (Kobayashi et al., 1998) showed that the
expression of CaMV proteins is differentially regulated during the
viral cycle: P1, P5 and P6 are synthesized earlier than P3, whereas
P2 and P4 are late-accumulating proteins. This expression pattern
might be related to the position of the ORFs on these RNAs
and/or to the appearance kinetic of the different viral mRNAs
throughout the infectious cycle.
Fig. 3 The multiplication cycle of CaMV. The mains steps of the viral cycle are:
(i) aphid-mediated entry of the virus into the host cell, (ii) NLS mediated
transport of CaMV particles to the nuclear pore, (iii) import of the viral DNA into
the nucleus, (iv) reparation of DNA sequence discontinuities and association
with histones to form a minichromosome, (v) transcription of the viral DNA
by cellular RNA polymerase II, (vi) translation of the 19S RNA and 35S
RNA and spliced versions, (vii) replication of the genome and morphogenesis
of viral particles in the electron-dense viroplasms, and (viii) cell-to-cell
movement of virus particles through tubules, targeting to the nucleus
and aphid uptake.
F U N C T I O N S O F Ca M V P R OT E I N S
One or more functions have been associated with all the ORF
products (P1 to P6) except for ORF VII, whose corresponding
protein has never been detected in infected plants (Fig. 3). P1
(40 kDa) is a cell-to-cell movement protein which forms tubules
through the plasmodesmata, allowing CaMV particles to move
from one cell to another (Perbal et al., 1993). A central domain of
P1 is needed for targeting the protein to the cell periphery (Huang
et al., 2001a), whereas most of the protein, except for the Cterminal region is required for tubule formation (Thomas and
Maule, 1999). The N- and C-termini, which are the most variable
MOLECULAR PLANT PATHOLOGY (2002) 3(6), 419–429 © 2002 BLACKWELL SCIENCE LTD