Stabilization of cauliflower mosaic virus P3 tetramer by covalent linkage

Cauliflower mosaic virus (CaMV) open reading frame (ORF) III encodes a 15 kDa protein (P3) that is indispensable for viral infectivity. Although P3 has been shown to be a prerequisite for CaMV aphid transmission, its role in viral replication remains unknown. We previously showed that P3 forms a tetramer in planta and that P3 tetramer co-sediments with viral coat protein on sucrose gradient centrifugation, suggesting that a tetramer may be the functional form of P3. We presumed that disulfide bonds were involved in tetramer formation because 1) the tetramer was detected by Western blotting after electrophoresis under non-reducing conditions, and 2) the cysteine-X-cysteine motif is well conserved in CaMV P3 and P3 homologues among Caulimoviruses. Therefore we mutated either or both of the cysteine residues of CaMV P3. The mutant viruses were infectious and accumulated to a similar extent as the wild-type. An analysis of mutant proteins confirmed that the wild-type P3 molecules in the tetramer are covalently bound with one another through disulfide bonds. It was also suggested that mutant proteins are less stable than wild-type protein in planta. Furthermore, sedimentation study suggested that the disulfide bonds are involved in stable association of P3 with CaMV virions or virion-like particles, or both. The mutant viruses could be transmitted by aphids. These results suggested that the covalent bonds in P3 tetramer are dispensable for biological activity of P3 under experimental situations and may have some biological significance in natural infection in the field.     

Aphid transmission of cauliflower mosaic virus requires the viral PIII protein

The open reading frame (ORF) III product (PIII) of cauliflower mosaic virus is necessary for the infection cycle but its role is poorly understood. We have used in vitro protein binding ('far Western') assays to demonstrate that PIII interacts with the cauliflower mosaic virus (CaMV) ORF II product (PII), a known aphid transmission factor. Aphid transmission of purified virions of the PII-defective strain CM4-184 was dependent upon added PII, but complementation was efficient only in the presence of PIII, demonstrating the requirement of PIII for transmission. Deletion mutagenesis mapped the interaction domains of PIII and PII to the 30 N-terminal and 61 C-terminal residues of PIII and PII, respectively. A model for interaction between PIII and PII is proposed on the basis of secondary structure predictions. Finally, a direct correlation between the ability of PIII and PII to interact and aphid transmissibility of the virus was demonstrated by using mutagenized PIII proteins. Taken together, these data argue strongly that PIII is a second 'helper' factor required for CaMV transmission by aphids.     

Aphid transmission and a polypeptide are specified by a defined region of the cauliflower mosaic virus genome

Infection of young turnip leaves with an aphid-transmissible isolate, Cabb B-JI, of cauliflower mosaic virus (CaMV) causes synthesis of an Mr 18 000 polypeptide (p18) which co-purifies with virus inclusion bodies. This polypeptide is not detectable in leaves infected with either of two aphid non-transmissible isolates. Campbell and CM4-184. Construction in vitro, of hybrid genomes between Cabb B-JI and Campbell isolates demonstrates that aphid transmissibility and presence of p18 is dependent on the small genome fragment from the BstEII site to the XhoI site. A deletion made in this fragment within open reading frame (ORF) II causes loss of aphid transmissibility and also terminates production of p18. We conclude that aphid transmissibility and the presence of p18 are related to the expression of ORF II of the CaMV genome.     

The avirulence domain of Cauliflower mosaic virus transactivator/viroplasmin is a determinant of viral virulence in susceptible hosts

Cauliflower mosaic virus (CaMV) transactivator/viroplasmin (Tav) is a multifunctional protein essential for basic replication of CaMV. It also plays a role in viral pathogenesis in crucifer and solanaceous host plants. Deletion mutagenesis revealed that N- and C-terminal parts of Tav are not essential for CaMV replication in transfected protoplasts. Two deletion mutants having only minimal defects in basic replication were infectious in turnips but only with highly attenuated virulence. This was shown to be due to delayed virus spread within the inoculated leaves and to the upper leaves. Unlike the wild-type virus, the mutant viruses successfully spread locally without inducing a host defense response in inoculated Datura stramonium leaves, but did not spread systemically. These results provide the first evidence that a Tav domain required for avirulence function in solanaceous plants is not essential for CaMV infectivity but has a role in viral virulence in susceptible hosts.     

The acquisition of a caulimovirus by different aphid species: comparison with a potyvirus

The acquisition and transmission of cauliflower mosaic virus (CaMV) by six aphid species and three clones of aphids was studied and compared with that of turnip mosaic virus (TuMV) with Myzus persicae. Two clones of Aphis fabae were unable to transmit CaMV, but the other species, Acyrthosiphon pisum, Brevicoryne brassicae, Megoura viciae, M. persicae and Rhopalosiphum padi transmitted in a bior multi-phasic manner. There was no statistical evidence of a bimodal transmission pattern. R. padi is recorded as a vector of CaMV for the first time. The transmission efficiency of CaMV varied with time of acquisition and suggested that accumulation of the virus occurred with two peaks of efficiency within the anterior region of the insect gut. The time at which these two peaks occurred varied between the species, but the basic pattern was common to all transmitting aphid species in this study. This pattern contrasted with that of TuMV. The transmission data are discussed in terms of bimodal transmission, the influence of feeding behaviour, the role of a helper protein associated with both TuMV and CaMV and the evidence for site specific attachment of CaMV.     

A second cauliflower mosaic virus gene product influences the structure of the viral inclusion body

We have used electron microscopy of thin sections and experiments on isolated viroplasms to compare the properties of four strains of cauliflower mosaic virus (CaMV), three of which were partially or completely deleted in open reading frame (ORF) II. Our results confirm that this gene is required for aphid transmissibility and show that the product of ORF II influences the firmness with which virions are held within the viroplasm. Analysis of the proteins in the viroplasms showed that a mutant with a partial deletion in ORF II produced a protein smaller than the normal ORF product. This smaller protein was non-functional with respect both to aphid transmissibility and properties of the viroplasms.     

A single amino acid position in the helper component of cauliflower mosaic virus can change the spectrum of transmitting vector species

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.     

Splicing of cauliflower mosaic virus 35S RNA is essential for viral infectivity.

A splicing event essential for the infectivity of a plant pararetrovirus has been characterized. Transient expression experiments using reporter constructs revealed a splice donor site in the leader sequence of the cauliflower mosaic virus (CaMV) 35S RNA and three additional splice donor sites within open reading frame (ORF) I. All four donors use the same splice acceptor within ORF II. Splicing between the leader and ORF II produces an mRNA from which ORF III and, in the presence of the CaMV translational transactivator, ORF IV can be translated efficiently. The other three splicing events produce RNAs encoding ORF I-II in-frame fusions. All four spliced CaMV RNAs were detected in CaMV-infected plants. Virus mutants in which the splice acceptor site in ORF II is inactivated are not infectious, indicating that splicing plays an essential role in the CaMV life cycle. The results presented here suggest a model for viral gene expression in which RNA splicing is required to provide appropriate substrate mRNAs for the specialized translation mechanisms of CaMV.     

Virus Factories of Cauliflower Mosaic Virus Are Virion Reservoirs That Engage Actively in Vector Transmission

Cauliflower mosaic virus (CaMV) forms two types of inclusion bodies within infected plant cells: numerous virus factories, which are the sites for viral replication and virion assembly, and a single transmission body (TB), which is specialized for virus transmission by aphid vectors. The TB reacts within seconds to aphid feeding on the host plant by total disruption and redistribution of its principal component, the viral transmission helper protein P2, onto microtubules throughout the cell. At the same time, virions also associate with microtubules. This redistribution of P2 and virions facilitates transmission and is reversible; the TB reforms within minutes after vector departure. Although some virions are present in the TB before disruption, their subsequent massive accumulation on the microtubule network suggests that they also are released from virus factories. Using drug treatments, mutant viruses, and exogenous supply of viral components to infected protoplasts, we show that virions can rapidly exit virus factories and, once in the cytoplasm, accumulate together with the helper protein P2 on the microtubule network. Moreover, we show that during reversion of this phenomenon, virions from the microtubule network can either be incorporated into the reverted TB or return to the virus factories. Our results suggest that CaMV factories are dynamic structures that participate in vector transmission by controlled release and uptake of virions during TB reaction.     

Interaction between the open reading frame III product and the coat protein is required for transmission of cauliflower mosaic virus by aphids

Transmission of cauliflower mosaic virus (CaMV) by aphids requires two viral nonstructural proteins, the open reading frame (ORF) II and ORF III products (P2 and P3). An interaction between a C-terminal domain of P2 and an N-terminal domain of P3 is essential for transmission. Purified particles of CaMV are efficiently transmitted only if aphids, previously fed a P2-containing solution, are allowed to acquire a preincubated mixture of P3 and virions in a second feed, thus suggesting a direct interaction between P3 and coat protein. Herein we demonstrate that P3 directly interacts with purified viral particles and unassembled coat protein without the need for any other factor and that P3 mediates the association of P2 with purified virus particles. The interaction domain of P3 is located in its C-terminal half, downstream of the P3-P2 interaction domain but overlapping a region which binds nucleic acids. Mutagenesis of P3 which interferes with the interaction between P3 and virions is correlated with the loss of transmission by aphids. Taken together, our results demonstrate that P3 plays a crucial role in the formation of the CaMV transmissible complex by serving as a bridge between P2 and virus particles.     

Cauliflower mosaic virus protein P6-TAV plays a major role in alteration of aphid vector feeding behaviour but not performance on infected Arabidopsis

Emerging evidence suggests that viral infection modifies host plant traits that in turn alter behaviour and performance of vectors colonizing the plants in a way conducive for transmission of both nonpersistent and persistent viruses. Similar evidence for semipersistent viruses like cauliflower mosaic virus (CaMV) is scarce. Here we compared the effects of Arabidopsis infection with mild (CM) and severe (JI) CaMV isolates on the feeding behaviour (recorded by the electrical penetration graph technique) and fecundity of the aphid vector Myzus persicae. Compared to mock-inoculated plants, feeding behaviour was altered similarly on CM- and JI-infected plants, but only aphids on JI-infected plants had reduced fecundity. To evaluate the role of the multifunctional CaMV protein P6-TAV, aphid feeding behaviour and fecundity were tested on transgenic Arabidopsis plants expressing wild-type (wt) and mutant versions of P6-TAV. In contrast to viral infection, aphid fecundity was unchanged on all transgenic lines, suggesting that other viral factors compromise fecundity. Aphid feeding behaviour was modified on wt P6-CM-, but not on wt P6-JI-expressing plants. Analysis of plants expressing P6 mutants identified N-terminal P6 domains contributing to modification of feeding behaviour. Taken together, we show that CaMV infection can modify both aphid fecundity and feeding behaviour and that P6 is only involved in the latter.     

Cauliflower mosaic virus ORF III product forms a tetramer in planta: its implication in viral DNA folding during encapsidation.

Cauliflower mosaic virus (CaMV) open reading frame (ORF) III encodes a 15 kDa protein; the function of which is as yet unknown. This protein has non-sequence-specific DNA binding activity and is associated with viral particles, suggesting that the ORF III product (P3) is involved in the folding of CaMV DNA during encapsidation. In this study, we demonstrated that P3 forms a tetramer in CaMV-infected plants. A P3-related protein with an apparent molecular weight of 60 kDa was detected by Western blotting analysis using anti-P3 antiserum under non-reducing conditions, while only 15 kDa P3 was detected under reducing conditions. Analysis of P3 using viable mutants with a 27-bp insertion in either ORF III or IV revealed that the 60 kDa protein was a tetramer of P3. The P3 tetramer co-sedimented with viral coat protein in multiple fractions on sucrose gradient centrifugation, suggesting that P3 tetramer binds to mature and immature virions. These results strongly suggested that CaMV P3 forms a tetramer in planta and that disulfide bonds are involved in its formation and/or stabilization. The finding of P3 tetramer in planta suggested that viral DNA would be folded compactly by the interaction with multiple P3 molecules, which would form tetramers, while being packaged into the capsid shell.     

The effect of Pymetrozine, a feeding inhibitor of Homoptera, in preventing transmission of cauliflower mosaic caulimovirus by the aphid species Myzus persicae (Sulzer)

Mature turnip plants, mechanically infected as seedlings with the semi-persistent, aphid transmitted caulimovirus, cauliflower mosaic (CaMV), were treated by spraying with either a solution of Pymetrozine plus adjuvant oil, adjuvant oil or water only. At the same time turnip seedlings were sprayed for each of the three treatments. Two h after spraying, Myzus persicae were caged onto an infected turnip plant for each of the three treatments. Twenty four h later, groups of 20 aphids were transferred from the infected plants, to seedlings from each of the three treatments. After 24 h, these were removed and seedlings were later recorded for infection. This acquisition/transmission assay was repeated at 3, 7, 14 and 21 days from treatment. Only aphids exposed to the Pymetrozine treated source plants were shown to move off the plant and failed to transmit CaMV effectively to treated or control seedlings during the 0 and 3 day assays. The majority soon died when transferred to test seedlings. Progressively, more aphids were found to survive and transmit CaMV during the 7 day and 14 day assays. By 21 days no significant effect could be recorded between treatments and controls. Aphids transferred from control treated source plants to Pymetrozine treated seedlings were able to transmit CaMV within all the assays, although higher mortality was recorded in the day 0 assessment when compared to those transferred to control treated seedlings. We conclude from this trial, that a single foliar treatment of 100 mg litre¹ Pymetrozine to CaMV infected turnip plants, effectively reduces the vectoring capability of M. persicae, that feed on these plants, for up to 7 days. However, Pymetrozine failed to stop virus transmission to treated seedlings from the ingress of viruliferous aphids. Pymetrozine was not shown to cause any phytotoxic responses to plants used in this trial.     

VAPA, an innovative "virus-acquisition phenotyping assay" opens new horizons in research into the vector-transmission of plant viruses

Host-to-host transmission--a key step in plant virus infection cycles--is ensured predominantly by vectors, especially aphids and related insects. A deeper understanding of the mechanisms of virus acquisition, which is critical to vector-transmission, might help to design future virus control strategies, because any newly discovered molecular or cellular process is a potential target for hampering viral spread within host populations. With this aim in mind, an aphid membrane-feeding assay was developed where aphids transmitted two non-circulative viruses [cauliflower mosaic virus (CaMV) and turnip mosaic virus] from infected protoplasts. In this assay, virus acquisition occurs exclusively from living cells. Most interestingly, we also show that CaMV is less efficiently transmitted by aphids in the presence of oryzalin--a microtubule-depolymerising drug. The example presented here demonstrates that our technically simple "virus-acquisition phenotyping assay" (VAPA) provides a first opportunity to implement correlative studies relating the physiological state of infected plant cells to vector-transmission efficiency.