A single amino acid position in the helper component of cauliflower mosaic virus can change the spectrum of transmitting vector species.
ABSTRACT Viruses frequently use insect vectors to effect rapid spread through host populations. In plant viruses, vector transmission is the major mode of transmission, used by nearly 80% of species described to date. Despite the importance of this phenomenon in epidemiology, the specificity of the virus-vector relationship is poorly understood at both the molecular and the evolutionary level, and very limited data are available on the precise viral protein motifs that control specificity. Here, using the aphid-transmitted Cauliflower mosaic virus (CaMV) as a biological model, we confirm that the "noncirculative" mode of transmission dominant in plant viruses (designated "mechanical vector transmission" in animal viruses) involves extremely specific virus-vector recognition, and we identify an amino acid position in the "helper component" (HC) protein of CaMV involved in such recognition. Site-directed mutagenesis revealed that changing the residue at this position can differentially affect transmission rates obtained with various aphid species, thus modifying the spectrum of vector species for CaMV. Most interestingly, in a virus line transmitted by a single vector species, we observed the rapid appearance of a spontaneous mutant specifically losing its transmissibility by another aphid species. Hence, in addition to the first identification of an HC motif directly involved in specific vector recognition, we demonstrate that change of a virus to a different vector species requires only a single mutation and can occur rapidly and spontaneously.
- SourceAvailable from: El-Desouky Ammar[show abstract] [hide abstract]
ABSTRACT: Mutations of K --> E in the highly conserved 'KITC' motif of the potyvirus helper component (HC) protein result in loss of HC function in aphid transmission, presumably because of inability to interact with virions, stylets or both. In this study we show that HC of potato virus C (PVC), a naturally occurring variant of potato virus Y (PVY) that has the K --> E mutation, lacks the ability to be retained in stylets, whereas PVY HC is retained. The K --> E mutation in either PVC or a site-directed mutant of tobacco etch virus (TEV) did not hinder binding to capsid protein, nor did deletion of the N-terminal 107 aa of TEV HC. An additional mutation, F -->, L at aa 10 of TEV HC, which renders HC non-functional but does not affect binding to capsid protein, is reported. Collectively, the results suggest that the N-terminal domain is required for interaction of HC with stylets rather than for binding to virions.Journal of General Virology 12/1998; 79 ( Pt 12):3119-22. · 3.13 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: A new baculovirus expression system was used to express the gene II which encodes the 18-kDa aphid transmission factor (ATF) protein of two isolates of cauliflower mosaic virus (CaMV), the aphid-transmissible (AT+) Cabb B-JI and the aphid-nontransmissible (AT-) CM1841 isolates. ATF activity was shown to be associated with the baculovirus expression product of CaMV gene II from both the AT+ and the AT- isolates. No differences could be detected in the stability of the P18s from AT+ and AT- isolates expressed in the baculovirus system. These results indicate that the AT- phenotype of CM1841 isolate might not be due to a defective P18 but to an insufficient concentration of it in infected plant tissue.Virology 03/1993; 192(2):651-4. · 3.37 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Aphid transmission factor (ATF) activity of cauliflower mosaic virus (CaMV) gene II product was recovered after expression of the gene by a baculovirus recombinant. The expression product, when first acquired by aphids through parafilm membrane, was able to mediate the transmission of two aphid nontransmissible isolates (CM1841, CM4-184) providing the first direct evidence that the product of the gene II is the CaMV ATF. The CaMV ATF in its active conformation has a strong tendency to aggregate and all attempts at solubilizing it resulted in the loss of the ATF activity. The CaMV ATF was also expressed in Escherichia coli, using the pGEX 3X plasmid vector, as a fusion protein to glutathione S-transferase (GST) and was purified. The fusion product (GST-P18), whether purified or not, was not able to complement the transmission of transmission-defective isolates. However, when GST-P18 was added to some extracts from a plant infected with an aphid-transmissible isolate (Cabb B-JI), the transmission was inhibited. This suggests that it could be possible to block the in vitro transmission of CaMV using a molecule analogous to the ATF.Virology 03/1993; 192(2):643-50. · 3.37 Impact Factor
JOURNAL OF VIROLOGY, Nov. 2005, p. 13587–13593
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 21
A Single Amino Acid Position in the Helper Component of
Cauliflower Mosaic Virus Can Change the Spectrum of
Transmitting Vector Species
Aranzazu Moreno,1Euge ´nie He ´brard,2† Marilyne Uzest,2Ste ´phane Blanc,2* and Alberto Fereres1
CSIC-CCMA, C/Serrano 115 dpdo., 28006 Madrid, Spain,1and UMR Biologie et Ge ´ne ´tique des Interactions Plantes-Parasites,
CIRAD-INRA-ENSAM, TA 41/K, Campus International de Baillarguet, 34398 Montpellier cedex 05, France2
Received 29 April 2005/Accepted 10 July 2005
Viruses frequently use insect vectors to effect rapid spread through host populations. In plant viruses, vector
transmission is the major mode of transmission, used by nearly 80% of species described to date. Despite the
importance of this phenomenon in epidemiology, the specificity of the virus-vector relationship is poorly
understood at both the molecular and the evolutionary level, and very limited data are available on the precise
viral protein motifs that control specificity. Here, using the aphid-transmitted Cauliflower mosaic virus (CaMV)
as a biological model, we confirm that the “noncirculative” mode of transmission dominant in plant viruses
(designated “mechanical vector transmission” in animal viruses) involves extremely specific virus-vector
recognition, and we identify an amino acid position in the “helper component” (HC) protein of CaMV involved
in such recognition. Site-directed mutagenesis revealed that changing the residue at this position can differ-
entially affect transmission rates obtained with various aphid species, thus modifying the spectrum of vector
species for CaMV. Most interestingly, in a virus line transmitted by a single vector species, we observed the
rapid appearance of a spontaneous mutant specifically losing its transmissibility by another aphid species.
Hence, in addition to the first identification of an HC motif directly involved in specific vector recognition, we
demonstrate that change of a virus to a different vector species requires only a single mutation and can occur
rapidly and spontaneously.
Most plant viruses require the assistance of another organ-
ism—the vector—to spread from one host to the next (34).
Insects, the main virus vectors, are frequently responsible for
causing severe disease epidemics worldwide. Specialized plant
feeders with piercing-sucking mouthparts from the Homoptera,
particularly aphids, are responsible for transmitting around
half of the ?1,000 plant virus species described so far (18). The
specificity of the relationship between aphids and viruses can
vary widely depending on the type of virus-vector interaction,
but it is generally low for the most frequent noncirculative
interactions. The term noncirculative applies to cases of trans-
mission where the virus is simply retained within (and later
released from) the stylets of the vector while feeding on plants
and never replicates or even circulates within the vector body
(15). In fact, most aphid species tested are capable of trans-
mitting any noncirculative virus with various degrees of effi-
ciency, and there are only very few examples reported in the
literature of a given aphid species being unable to act as the
vector for a specific noncirculative virus [e.g., Lypaphis erysimi
(Kaltenbach) and Tobacco etch virus (39), Nasonovia ribisnigri
Mosley and Lettuce mosaic virus (26), or Brachycaudus heli-
chrysi L. and Cauliflower mosaic virus (19)]. Whether this ap-
parent lack of specificity is a viral adaptation to increase the
chances of transmission by several vector species remains an
The molecular basis of the specificity (or lack of specificity)
between noncirculative viruses and their vectors is poorly doc-
umented. One reported example is that of cucumoviruses,
where minor amino acid changes in the coat protein of Cu-
cumber mosaic virus differentially modify transmission by its
two main vectors, Aphis gossypii (Glover) and Myzus persicae
(Sulzer) (30). In potyviruses, the molecular mechanisms of the
virus-vector interaction have been studied extensively, and
some level of specificity has been reported (36). Although a
highly conserved KITC amino acid motif in the N-terminal
domain of the helper component (HC)-Pro protein is manda-
tory for all potyvirus/aphid interactions, other protein se-
quences that affect the efficiency of transmission in a specific
virus/vector couple remain completely unknown (reviewed in
references 32 and 35).
Similarly, for Cauliflower mosaic virus (CaMV, genus Cauli-
movirus), various aphid species transmit the disease with dif-
ferent efficiencies or even fail to transmit it at all (7, 19, 25).
CaMV is certainly the plant virus for which the molecular
mechanisms of virus/vector interactions have been most thor-
oughly documented. However, although the biological and bio-
chemical properties of the various viral proteins involved in
aphid transmission are well characterized (for a review, see
references 4 and 18), the exact domains or motifs involved,
directly or indirectly, in specific recognition between the virus
and one or more aphid species are still unidentified and thus
remain totally uncharacterized. Among the six viral genes ex-
pressed upon CaMV infection, three (open reading frames
[ORFs] II, III, and IV) are involved in vector transmission.
* Corresponding author. Mailing address: UMR Biologie et Ge ´ne ´-
tique des Interactions Plantes-Parasites, CIRAD-INRA-ENSAM, TA
41/K, Campus International de Baillarguet, 34398 Montpellier cedex
05, France. Phone: 33 (0)4 99 62 48 04. Fax: 33 (0)4 99 62 48 22.
† Present address: IRD, Re ´sistance des plantes aux pathoge `nes,
34394 Montpellier cedex 05, France.
The coat protein (P4), the product of ORF IV, has long been
known to be incapable of direct interaction with the aphid
mouthparts. Instead, nonstructural proteins, such as those en-
coded by ORFs II and III (P2 and P3), create a molecular
“bridge” between virus and vector, thus linking the coat pro-
tein to attachment sites within the aphid mouthparts. P3 has
been demonstrated to form a complex with the virus particle
(20, 33), but it cannot bind putative receptors in aphids (9). P3,
in the form of P3-virion complexes, attaches to P2 (the helper
component of CaMV), which in turn directly recognizes the
putative receptor sites within the aphid stylet (9). Consistently,
P2 is the only viral product that is retained in the stylets when
acquired alone by aphids, and its acquisition prior to that of
P3-virion complexes is mandatory for successful transmission
(9). Biochemical and biological characterization of P2 has re-
vealed a number of remarkable properties, but no information
on motifs or domains that could be directly involved in binding
to the aphid stylet has been reported. While the C-terminal
?-helical domain of P2 (from amino acids [AA] 100 to 159) was
shown to be responsible both for P2-P3 binding (21) and for P2
self-association and polymerization (16), a large N-terminal
region (from AA 1 to 100) remains unexplored regarding
structure, biochemical properties, and biological function. It is
thus tempting to hypothesize that the motif that attaches to the
aphid vector resides on this end of the molecule. However, the
lack of naturally nontransmissible variants of CaMV deficient
in P2-aphid binding (2), together with the intrinsic instability of
the N-terminal domain when isolated from the rest of the
molecule (S. Blanc, unpublished results), has so far precluded
further exploration of this possibility.
In this report, we present a series of converging observations
indicating that a domain of P2 recognizing the attachment sites
within the aphid stylets is located at the N terminus of the
protein. More precisely, we identified a single amino acid po-
sition that can either abolish transmission or differentially af-
fect transmission efficiency by various aphid species and
thereby change the spectrum of vector species for CaMV.
Interestingly, we also obtained evidence that changes at this
amino acid position can occur spontaneously when a particular
aphid species is used as a vector after a series of successive
passages in host plants, indicating potential for very rapid ad-
aptation to new vector species.
MATERIALS AND METHODS
Virus, host plants, and vectors. The aphid-transmissible isolate Cabb-S of
CaMV (12) was used as the standard isolate and is hereafter referred to as the
wild type. CaMV Cabb-S and the mutant derivatives described below were
propagated by serial mechanical inoculation in turnip plants (Brassica rapa L. cv.
The aphid species used were previously characterized as being good vectors
(Brevicoryne brassicae L. and Myzus persicae Sulzer), poor vectors (Macrosiphum
euphorbiae Thomas and Nasonovia ribisnigri Mosley), or nonvectors (Brachycau-
dus helichrysi L.) with respect to transmission of CaMV (7, 19, 25). Laboratory
colonies of all aphid species were initiated from a single viviparous aptera
collected on cauliflower (B. brassicae), pepper (M. persicae), lettuce (N. ribisnigri
and M. euphorbiae), or Senecio vulgaris L. (B. helichrysi) plants in central Spain.
All aphid colonies, except N. ribisnigri, were reared in environmental growth
chambers at temperatures of 23°C (day) and 18°C (night) and a photoperiod of
14 h of light and 10 h of dark. The clone of N. ribisnigri was reared at a constant
temperature of 12°C and a photoperiod of 14 h of light and 10 h of dark. B.
brassicae and M. persicae colonies were reared on Brassica rapa cv. Just Right,
while M. euphorbiae and N. ribisnigri were cultured on lettuce (cv. Cazorla) and
B. helichrysi was cultured on chrysanthemum (Chrysanthemum coronarium L.).
Plasmid construction and mutagenesis. Clone pCa37 is the reference clone for
the CaMV isolate Cabb-S (12); clone ?II-S, where the entire coding sequence of
gene II is replaced by the unique restriction site SpeI, was described elsewhere
CaMV mutants with a substitution at amino acid position 6 of P2 were created
by PCR-directed mutagenesis. Gene II was PCR amplified on the template
pCa37 with reverse and forward primers containing an SpeI restriction site at
their 5? extremities. The PCR products were later digested by SpeI and directly
cloned at the corresponding site in plasmid ?II-S. Eight different forward prim-
ers were used, each containing a mutation inducing an amino acid change at
position 6 of the P2 protein sequence. The primers were designed to substitute
either glycine (G; codon, GGA), lysine (K; codon, AAA), glutamic acid (E;
codon, GAA), asparagine (N; codon, AAT), methionine (M; codon, ATG),
threonine (T; codon, ACA), tyrosine (Y; codon, TAT), or histidine (H; codon,
CAT) for the original glutamine (Q; wild-type codon, CAA) and, after cloning,
yielded mutant CaMV clones designated Q6G, Q6K, Q6E, Q6N, Q6M, Q6T,
Q6Y, and Q6H, respectively.
Plasmid Top-S, containing the full genome-length CaMV Cabb-S sequence
with an engineered early stop codon at amino acid position 6 of P2, was described
previously (13). This plasmid is not infectious when inoculated into turnip plants
unless the stop codon reverts to a coding nucleotide triplet; that infectious
revertants appear spontaneously upon Top-S inoculation has been reported
previously (13). Several infectious revertants were characterized and are de-
scribed in Results. To ensure that these revertants were not a mixed population,
viral genomes were extracted from an infected plant as described previously (13)
and cloned in pUC19 using the unique SalI restriction site in the CaMV se-
quence. The nature of the reversion of the stop codon at amino acid position 6
of P2 was determined by sequencing the various clones produced. The clones
were then inoculated back into plants, and the properties determined for each
revertant were verified on individual clones.
To express P2Rev5 using a baculovirus/insect cell expression system, a DNA
fragment of 621 bp released upon digestion of pTop-S-Rev5 (see Results) with
BamHI and BglII was cloned between the BglII sites in the transfer plasmid p119
(17) to produce p119-P2Rev5. To create a fusion between P2Rev5 and green
fluorescent protein (GFP), the GFP gene was extracted from plasmid pEGFP-C1
(8) by Eco47III-PstI double digestion and inserted into pUC19 using XbaI and
PstI sites to yield pUC-GFP. The P2Rev5 coding sequence was PCR amplified
from the plasmid pTop-S-Rev5 with forward and reverse primers containing
BamHI and BglII sites at their 5? extremities, respectively, with the reverse
primer omitting the stop codon of P2Rev5, and the PCR product was inserted
into the BamHI site of the plasmid pUC-GFP, yielding pUC-P2Rev5-GFP. The
sequence encoding a P2Rev5-GFP fusion was then extracted from pUC-P2Rev5-
GFP by a double BamHI-BglII digestion and inserted into p119 at the BglII
cloning site to generate p119-P2Rev5-GFP.
Insect cell culture maintenance and infection with p119-P2Rev5 and p119-
P2Rev5-GFP baculovirus recombinants, as well as production and purification of
recombinant proteins, were as previously described (16). Plasmids containing the
full-length CaMV genome or mutant derivatives were mechanically inoculated
onto host plants as described previously (13).
Protein analysis and microscopy. Accumulation of P2 in turnip plants infected
with Top-S-Rev5 or the CaMV Q6x mutant series was verified by total protein
extraction and 13.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis,
followed by P2- or P4-specific immunodetection as previously described (13).
Far-Western experiments to detect interaction between P2, P3, and P3-virion
complexes were done precisely as previously described (21). Briefly, membranes
from Western blots of P2 and mutant derivative proteins were incubated either
with P3 alone or with a mixture of P3 and virions. The interaction of P3 and
P3-virion complexes with various forms of P2 on the membrane is revealed using
antibodies against P3 and antibodies against virions, respectively. Electron-lu-
cent inclusion bodies in Top-S-Rev5-infected plants were observed using elec-
tron microscopy, as described previously (9). The paracrystals of P2Rev5 pro-
duced in the baculovirus/insect cell expression system were observed by negative
staining electron microscopy as described previously (5). The association be-
tween P2Rev5-GFP and the microtubular network in insect cells was observed
using an epifluorescence microscope. In this case, insect cells were infected with
the corresponding baculovirus recombinant for 48 h and observed live, directly in
the culture medium, without further processing.
Transmission tests. Infected turnip plants, used as virus source plants, were
selected for consistency between batches and uniformity of symptom appear-
ance. Transmission tests were performed essentially as described previously (11).
Groups of 25 to 30 young-adult aphid apterae were placed inside plastic cages for
1 h for preacquisition starving. Aphids were then released on the upper side of
an infected leaf for virus acquisition. In all cases, the aphids were placed on the
13588 MORENO ET AL.J. VIROL.
last expanded leaf showing vein-clearing symptoms. After a 5-min acquisition
access period, groups of five aphids were transferred onto 15-day-old seedlings of
Brassica rapa cv. Just Right, used as test plants, for a 3-hour inoculation period.
Turnip test plants were finally sprayed with imidacloprid (Confidor, Bayer) and
transferred to an aphid-free growth chamber at 26°C (day) and 20°C (night), with
a photoperiod of 16 h of light and 8 h of dark, where they were checked regularly
for symptom appearance for 3 to 5 weeks.
Transmission tests were conducted using all possible combinations of the
selected five aphid species and the eight CaMV variants described above, as well
as wild-type Cabb-S. Eleven replicates of six plants each were used for each virus
variant-aphid species combination.
Statistical analysis. The ratio corresponding to the number of infected plants
divided by the total number of test plants for each of the treatments used in the
study (all combinations of virus variant-vector species) was subjected to a pair-
wise comparison using a chi-square test. Furthermore, the transmission rate
obtained in each of the individual tests was transformed by arc sine ?x (where
x is the observed transmission rate) to reduce heterocedasticity. The transformed
data were subjected to an analysis of variance as a factorial design, where the two
factors under study were the type of virus variant and the species of aphid.
Multiple mean comparisons were made between treatments using the Tamhane
T2 test, which allows reliable pairwise comparisons based on a t test, in cases
where the variance between treatments was not the same. All statistical analyses
were made using the statistical package SPSS (version 12.0) for personal com-
Identification of a residue in P2 involved in interaction with
the aphid vector. We recently described the CaMV clone
Top-S, which has an engineered stop codon at amino acid
position 6 of P2 and which is not infectious unless this codon
reverts to an amino acid coding triplet (13). Because the amino
acid at position 6 lies in the uncharacterized N-terminal region
of P2, which is possibly involved in mediating the P2-aphid
interaction (see the Introduction), we decided to inoculate
host plants with clone Top-S and systematically test spontane-
ous revertants for P2 accumulation and aphid transmissibility.
The fifth revertant analyzed, designated Top-S-Rev5, failed to
be transmitted by aphids despite significant P2 accumulation
(data not shown, but see also CaMV mutant Q6Y in Fig. 2 and
3), and cloning and sequencing of the viral DNA revealed a
single nucleotide change, resulting in introduction of Y at
amino acid position 6 of P2 in place of the stop codon.
To understand the reason for the lack of aphid transmissi-
bility of Top-S-Rev5, and hence likely explain why P2Rev5 is
biologically inactive, we examined all of the well-characterized
biological or biochemical features of wild-type P2 for this par-
ticular mutant. P2Rev5 was expressed in the baculovirus/insect
cell expression system, and its lack of biological activity was
confirmed by aphid transmission testing (not shown) as previ-
ously described (3). Large amounts of P2Rev5 accumulated as
paracrystals (Fig. 1a), similar to those obtained previously with
wild-type P2 (5), thus indicating that the overall structure and
the polymerization of the molecule is not affected. In live Sf9
insect cells, paracrystals of P2Rev5 were clearly associated with
microtubules (not shown), as described earlier for wild-type P2
(6), and a P2Rev5-GFP fusion expressed in the same system
confirmed that the affinity of wild-type P2 for the microtubular
network of the host cell was not abolished in P2Rev5 (Fig. 1b).
Figure 1c shows that, in infected plant cells, P2Rev5 forms
electron-lucent inclusion bodies similar to those formed by
wild-type P2 (9, 10). Finally, we demonstrated that the capacity
of P2 to bind P3 and P3-virion complexes (21) is also unaltered
in P2Rev5 (Fig. 1d). Overall, the characterization of mutant
Top-S-Rev5 presented in Fig. 1 indicates that none of the
previously described properties of P2 are significantly affected
by the mutation “Rev5,” and thus the loss of aphid transmis-
sion is most likely due to the loss of an uncharacterized func-
tion of P2, such as its capacity to recognize and bind aphid
An independent observation later supported this hypothesis.
Indeed, by maintaining the wild-type Cabb-S isolate through 10
serial aphid transmissions using B. brassicae as a vector, we
obtained a variant that was very poorly transmitted by M.
persicae, a species known to transmit wild-type CaMV Cabb-S
with high efficiency (see Fig. 3). The transmission efficiencies
were tested as described in Materials and Methods, using 28
test plants for each vector species, and were 31.7% and 5.5%
for B. brassicae and M. persicae, respectively. We purified the
viral DNA from the final series of infected plants, and DNA
sequencing revealed a single mutation leading to a change
from Q to H at amino acid position 6 of P2. This CaMV variant
confirmed that the amino acid residue at position 6 of P2 is
indeed involved in aphid recognition and, together with the
results obtained with Top-S-Rev5, supports the view that its
substitution by other residues could either totally abolish trans-
mission or cause some alteration in vector specificity.
The amino acid residue at position 6 of P2 determines the
spectrum of vector species of CaMV. To further characterize
this phenomenon and test the above hypothesis, we created a
series of eight mutant clones of CaMV, all with an amino acid
change at position 6 of P2. Plants inoculated with these eight
different CaMV variants showed symptoms of infection 2 to 3
weeks after inoculation, and all accumulated virions and P2
(Fig. 2). Variations in the detected amounts of P2 and virions
in Fig. 2 are similar to variations we routinely observed with a
single CaMV clone. The fact that they do not correlate with
transmission efficiencies reported in Table 1 and Fig. 3 confirm
that they are not significant.
Aphid transmission tests were then performed with all pos-
sible combinations between wild-type or mutant CaMV vari-
ants and the various aphid species used in this study, and the
results are summarized in Table 1 and Fig. 3. The first striking
observations were that none of the mutations had a positive
effect on transmission efficiency and that different amino acids
at this position had various impacts on the spectrum of aphid
species that could successfully transmit the virus. Variant Q6Y
was never transmitted, whatever the aphid species, confirming
the results obtained with Top-S-Rev5. All of the other variants
were transmitted by at least one aphid species and fell into
three distinct categories: (i) variants that had no effect or only
a minor effect on transmission and behaved as wild-type CaMV
(Q6M, Q6N, and Q6T); (ii) variants for which the transmission
rate by all vector species was dramatically reduced (Q6G, Q6K,
and Q6E); and (iii) variants with transmission rates that were
affected differentially depending on the vector species (best
exemplified by Q6H). Most strikingly for the variant Q6H, the
transmission rate was dramatically and specifically reduced
with M. persicae, and to a lesser extent M. euphorbiae, whereas
it was barely modified, if at all, for other species. For all CaMV
mutants, virus DNA was PCR amplified from aphid-inoculated
plants, and ORF II was sequenced, confirming the absence of
both back mutation to wild type or other sequences at amino
acid position 6 of P2 and second-site reversions.
VOL. 79, 2005VECTOR SPECIFICITY IN NONCIRCULATIVE TRANSMISSION13589
On examination of the data obtained with different vector
species in Fig. 3 and Table 1, it appears that the transmission
rate observed with a poor vector species is not greatly sensitive
to changes at amino acid position 6 of P2. For example, N.
ribisnigri (dotted line in Fig. 3) transmitted all mutants except
Q6Y and Q6K at a rather constant rate, as indicated by the
lack of statistically significant differences between mutants in
Table 1. In contrast, transmission by very efficient vectors, such
as B. brassicae and, particularly, M. persicae (plain line in Fig.
3), is greatly affected by some of the mutations (statistical
significances in Table 1).
The amino acid at position 6 of P2 could be directly involved
in aphid binding. Numerous studies on the noncirculative
transmission of plant viruses have repeatedly shown that HC
(for instance, HC-Pro in potyvirus or P2 in caulimovirus) can
be acquired alone, and prior to virus particles, by the vector (3,
9, 14, 24, 38). This simple fact definitively demonstrates that
HCs are viral proteins directly recognizing attachment sites in
the vector mouthparts. In potyviruses, a conserved KITC motif
located in the N-terminal domain of HC-Pro has been shown
FIG. 1. Characterization of biochemical and biological properties of mutant P2Rev5. (a) Crude extracts of Sf9 insect cells infected with a
baculovirus recombinant expressing P2Rev5, observed by negative staining and electron microscopy. White arrows indicate P2Rev5 paracrystal
bundles. (b) Live Sf9 insect cell infected with a baculovirus recombinant expressing a P2Rev5-GFP fusion observed by epifluorescence microscopy.
(c) Plant cell infected with CaMV Top-S-Rev5. The cell contains both electron-dense (ed) and electron-lucent (el) inclusion bodies; virions are
indicated by black arrows. (d) Far-Western experiments revealing P2-P3 interaction. Ten micrograms of wild-type P2, P2157m(a negative control
that can no longer bind P3 [21, 37]), and P2Rev5 were loaded in lanes 1, 2, and 3, respectively. The proteins are specifically revealed with an anti-P2
serum (3) in the left panel and tested for P3 and P3-virion binding capacity in the middle and right panels, respectively (see Materials and
Methods). Molecular mass marker positions 6.5, 16.5, and 25 kDa are shown on the right. Bars represent 100, 1,000, and 500 ?m in panels a, b,
and c, respectively.
FIG. 2. Detection of the coat protein (P4) and P2 in plants infected
with various mutant derivatives of CaMV. The identity of the amino
acid substituted for Q at position 6 of P2 is indicated at the top. The
upper panel shows immunostaining of the coat protein (P4), whereas
the lower panel is P2 specific. The molecular weight scale (in thou-
sands) is indicated on the right.
13590MORENO ET AL.J. VIROL.
to be involved in aphid binding (1). However, whether this
domain directly recognizes the putative receptor of the vector
or indirectly affects the binding capacity of HC-Pro remains
unclear. Here, in the caulimovirus CaMV, we provide the first
identification of a key amino acid of P2 (Q at amino acid
position 6) that is specifically involved in aphid recognition. A
number of arguments support the hypothesis that we have
characterized an element directly involved in the recognition
of, and binding to, the putative receptor(s) in the insect. (i)
The CaMV variant Top-S-Rev5 produces a mutation (equiva-
lent to P2Q6Y) that is not active in transmission when ex-
pressed either in infected plants or in the baculovirus/insect
cell system. Interestingly, P2Rev5 (Q6Y) has biochemical
properties otherwise similar to wild-type P2. If the effect of the
Q6Y mutation on the biological activity of P2 were a structural
and/or indirect effect, one or more of the other features tested
in Fig. 1 would likely also be affected. (ii) Figure 3 and Table
1 show that mutant Q6H is poorly transmitted by Myzus persi-
cae, whereas the effect of this mutation on transmission by
other vector species is less, with some species not being af-
fected at all (i.e., B. brassicae). If the Q6H mutation induced a
change in a distinct domain of P2 governing the interaction
with the aphid, this change should nonspecifically and similarly
affect the interaction with all aphid species. (iii) Based on the
properties of amino acids (22, 23), we could not identify any
structural or biochemical trends predicting the impact of the
various residues tested on the biological activity of P2 (trans-
mission rate in Fig. 3), either in size (cf. Q, N, and T versus E,
which is isometric of Q), polarity (cf. Q/N/T and M versus H),
charge (cf. K and E), or the presence of an aromatic ring (cf.
Y and H). Again, if AA 6 of P2 were acting indirectly, residues
with related biochemical properties would be expected to have
comparable effects. Although direct evidence is still lacking, we
believe that the involvement of AA 6 of P2 in recognizing
FIG. 3. Transmission rates of different CaMV mutants by five aphid species. Means are presented together with standard errors. Bb, Brevicoryne
brassicae; Mp, Myzus persicae; Me, Macrosiphum euphorbiae; Bh, Brachycaudus helichrysi; and Nr, Nasonovia ribisnigri. Note that N. ribisnigri
(dotted line) transmitted all variants at similar rates, while transmission by M. persicae (plain line) varied drastically depending on the type of virus
variant tested. For statistical significances of the observed differences in transmission rates, see Table 1.
TABLE 1. Pairwise comparisons of the transmission rates between the different variants of CaMV by each of the
aphid species used in the studya
No. of plants infected/total no. of plants tested with:
M. persicaeB. brassicae M. euphorbiaeN. ribisnigri B. helychrisi
0/62 a (a)
8/66 bd (ac)
0/65 a (a)
4/62 ad (a)
30/66 c (bc)
36/66 c (bc)
28/66 c (cd)
38/64 c (bd)
2/62 a (a)
3/64 a (a)
3/64 a (a)
23/62 c (b)
31/64 bc (b)
36/66 b (b)
38/65 b (b)
37/64 b (b)
0/62 a (a)
0/66 a (a)
0/66 a (a)
2/62 a (a)
6/66 b (ab)
11/64 b (ab)
9/66 b (ab)
17/63 b (b)
0/62 a (a)
1/66 a (a)
1/66 a (a)
2/62 a (a)
2/66 a (a)
3/66 a (a)
1/65 a (a)
7/64 a (a)
0/62 a (a)
2/66 a (ab)
3/66 a (ab)
10/62 b (b)
6/65 ab (ab)
4/66 ab (ab)
5/66 ab (ab)
11/64 b (ab)
aProportions (number of plants infected/total number of plants tested) followed by the same letter within each column indicate no significant differences (P ? 0.05)
according to pairwise comparisons using a chi-square test or a Fisher’s exact test when expected values were lower than 5. The same letters in parentheses within each
column indicate no significant differences (P ? 0.05) on the basis of a multiple-pairwise-comparison Tamhane T2 test. WT, wild type.
VOL. 79, 2005 VECTOR SPECIFICITY IN NONCIRCULATIVE TRANSMISSION13591
receptor sites within the aphid stylets is more specific and is
most likely direct. In this case, direct perturbation of the in-
terface would likely be due to charge repulsion (as exemplified
with E and K) or steric clashes (Y versus H), with only G
potentially interfering through a distinct mechanism, such as a
local destabilization of the protein structure.
The only other available example of a change of a single or
a few amino acids apparently impacting the specificity of a
noncirculative virus-aphid relationship comes from Cucumber
mosaic virus (CMV) (30). However, it was later demonstrated
that this effect is due to a change in the stability of CMV
virions rather than to a differential interaction with putative
receptors in the two aphid species tested (27, 28). It is impor-
tant to note that CMV is transmitted according to the “capsid
strategy” (for a review, see reference 31), where the coat pro-
tein is able to interact directly with putative receptors in the
stylets, whereas CaMV has adopted the more frequently used
“helper strategy,” in which an additional viral product, the HC,
links the virus particles to these receptors. The mutations we
have engineered in P2 are thus independent of the virions and
cannot alter their stability.
Another important outcome of our results is that they pro-
vide invaluable tools for future attempts to isolate the putative
receptor(s) of noncirculative plant viruses in insect vectors. We
again stress the fact that a large number of plant virus genera
are transmitted in a noncirculative manner, most often accord-
ing to the helper strategy (18), and that it is very well possible
that many virus species use the same or similar receptors.
Unfortunately, even the chemical nature of these putative re-
ceptors remains a mystery. So far, CaMV P2 is the only viral
molecule that can recognize them to be efficiently overpro-
duced, easily purified, and biologically active in a heterologous
expression system (16). The mutants described in the present
study will be very useful as specific affinity targets in the search
for receptors of noncirculative viruses.
Specificity of CaMV aphid transmission. The aphid species
selected for our study covered the main vectors of CaMV that
occur in the field (M. persicae and B. brassicae) plus two species
(M. euphorbiae and N. ribisnigri) that are commonly found
landing on Brassica fields in several growing regions of Spain
(26) and the United Kingdom (7). The latter two species have
a high potential for spreading the virus, even if they are unable
to reproduce and colonize the crop, because CaMV can be
transmitted after brief superficial probes (29). For the pur-
poses of comparison, we also included a species that has been
reported to be a nonvector of CaMV: B. helichrysi (19). Sur-
prisingly, the results of our study showed that B. helichrysi was
able to transmit CaMV, although with low efficiency (Table 1).
These apparently divergent results could be due to the low
number of replicates used by previous authors or to differences
in the transmission abilities of the different aphid clones and
virus isolates used in the two studies.
The fact that the transmission rates obtained with poor vec-
tor species are not very sensitive to changes at amino acid
position 6 of P2, while those obtained with good vector species
are markedly affected, is difficult to explain. One possibility
could be that the interaction between P2 and the putative
receptors is complex and consists of a nonspecific weak bind-
ing, strengthened by more specific and precisely tuned adap-
tation to particular vector species. In this hypothesis, the poor
vectors would transmit with low efficiency due solely to the
nonspecific interaction with P2, whereas the good vectors
would engage additional specific residues of P2 (possibly in-
cluding that at position 6), leading to more stable binding.
One striking observation was that, when compared to wild-
type CaMV Cabb-S, none of the mutations tested had a pos-
itive effect on transmission efficiency, whatever the aphid spe-
cies. Although the panel of amino acids tested covers a wide
range of biochemical properties, the transmission rates of all
variants were either unchanged or reduced. This might indicate
that the Q at amino acid position 6 of P2 is optimal for the
interaction of CaMV with its vectors and that the virus has
evolved to maximize its transmission by a wide range of vector
species. Consistently, all of the CaMV isolates that have been
collected from the field and sequenced to date (available in the
GenBank database) have a glutamine at position 6 of P2. This
speculation infers frequent contact in nature between CaMV
populations and several alternating vector species. Indeed, un-
der our experimental conditions, when only one vector species
(B. brassicae) was involved in transmission through several
serial passages, we rapidly produced a spontaneous variant,
Q6H, that was no longer “optimized” and had almost lost its
transmissibility by one of its best vectors (M. persicae).
Whether the above interpretation is correct or not, both the
fact that we induced important changes in the transmission
performance of a spectrum of vector species by mutating a
single amino acid of the CaMV HC and the spontaneous
appearance of a CaMV mutant at the very same position when
using a single vector species certainly demonstrate that adap-
tation of a plant virus to fluctuations in vector populations is
most likely rapid and likely to occur under field conditions.
We thank D. Gargani and Marc Ravallec for technical assistance in
electron microcopy, P. Travo for fluorescence microscopy, and M.
Duque for assistance in aphid rearing and transmission testing. We are
very grateful to Takii Ltd. for generously providing seeds of Brassica
rapa cv. Just Right.
This work was supported by the Plan Nacional de I?D?I from the
Ministerio de Educacio ´n y Ciencia (AGL-2000-2006) and by the bilat-
eral INRA-CSIC grant HF2003-0318.
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