Aphid transmission of cauliflower mosaic virus: The role of the host plant

Transmission of plant viruses is the result of interactions between a given virus, the host plant and the vector. Most research has focused on molecular and cellular virus-vector interactions, and the host has only been regarded as a reservoir from which the virus is acquired by the vector more or less accidentally. However, a growing body of evidence suggests that the host can play a crucial role in transmission. Indeed, at least one virus, Cauliflower mosaic virus, exploits the host''s cellular pathways to form specialized intracellular structures that optimize virus uptake by the vector and hence transmission.Key words: virus, vector, host plant, transmission, interactionsTransmission is a step in a virus''s life cycle that is often neglected. Nonetheless, it is obvious that also this step is obligatory for a virus, as it could not maintain itself without dispersing to other hosts and infecting them. Most plant viruses are transmitted by insects, using two different strategies: “circulant transmission” where the virus, once taken up by the vector during feeding on an infected plant, passes from the intestine via the body lumen to the salivary glands and is finally inoculated with the saliva into a new host plant; the second strategy is “non-circulant transmission” where transmissible virus particles attach only to the exterior mouthpieces of the insect from which they are released into a new host. Whereas the first strategy obviously requires highly specific interactions between the virus and the vector to allow for passage of the virus through the vector, non-circulant transmission was initially thought of as a more or less accidental event, where virus sticks non-specifically to the mouthpieces. However, it becomes more and more evident that also non-circulant transmission is the result of sophisticated interactions between a given virus, a host and a vector. The vectors are most often aphids that, due to their non-destructive feeding behavior, are ideally suited as virus vectors. In fact, once landed on a plant, aphids first probe the prospective food source by short, only seconds lasting intracellular punctures in epidermis and mesophyll cells that do not even kill the punctured cells.¹ After these exploratory punctures and when they judge the plant as suited, the aphids insert their proboscis-like mouthpieces (stylets) into the phloem and feed from its sap for time spans that may exceed several hours. Depending on the tissues they infect, plant viruses can be acquired by aphids during either or only one of the two puncture phases. For example, Luteoviruses are only acquired from the vascular tissues,² whereas Cauliflower mosaic virus is acquired from both tissues.³Cauliflower mosaic virus (CaMV) is one of the best studied viruses on what concerns non-circulant transmission, the most often used transmission mode employed by plant viruses. For its transmission, a transmissible complex must form that attaches to a protein receptor located in the stylets of the aphid.⁴ This complex is not only, as for some viruses, composed of the virus particle, but also, as for many non-circulantly transmitted plant viruses, of a viral helper protein that with one domain interacts with the virus particle and with another with the stylet receptor⁵ (Fig. 1A). The helper protein of CaMV, P2, seems to have no other function but to assist in transmission as CaMV mutants deleted of P2 are perfectly infectious but not transmissible.⁶ A puzzling fact is that P2 may be acquired independently of the virus particle, meaning that it alone can bind to the stylet receptor and that virus particles either attach concomitantly with P2 onto the stylets or later attach to pre-bound P2. This has consequences for the composition of the transmitted viral population as it can be compiled of virus particles originating from the same cell from which P2 was acquired, but also from other cells and even sieve tubes that themselves do not contain P2.³ In fact, this potentially sequential acquisition mode of CaMV by the vector is controlled by the intracellular⁷ and tissue-specific localization of P2 that is only found in epidermis and parenchyma cells.³ In these cells, P2 localizes exclusively in a single viral inclusion, the transmission body, that has been proposed and recently been shown to be specialized for transmission:^8–10 if this structure does not form, CaMV can not be taken up by the aphid, even if functional P2 is present in the infected cell.Open in a separate windowFigure 1(A) The different strategies of non-circulant transmission: Viruses (V) using the capsid strategy (CS) attach directly to a receptor (R) in the tip of the a proboscis forming aphid stylets (blue), whereas in the helper strategy (HS) this interaction is mediated by the viral helper protein (H) that binds the virus particle to the receptor. Note that the helper protein can bind independently of the virus to the stylets. Whether the same receptor is used by different viruses as presented in the schema, is not known. (B) A turnip protoplast transfected with CaMV was double-labelled late in infection for CaMV helper protein P2 (red) and the marker protein for the virus factories P6 (green). It is visible that P2 localizes in a single, large transmission body, whereas the numerous virus factories are devoid of P2 (Colocalization would be revealed by yellowish color (M) in this superposition). (C and D) Turnip protoplasts were cotransfected with CaMV and TBK5-GFP and immunolabelled for P2 (red) and TBK5 was detected by GFP fluorescence. (C) shows a cell early in infection, where P2 and TBK5-GFP colocalize on a network that we identified as the microtubule cytoskeleton (unpublished data). (D) shows a cell later in infection where P2 and TBK5-GFP colocalize, as indicated by the yellowish color, in a transmission body. Note that TBK5-GFP also strongly labels the nucleus (N).This posed the interesting question how the transmission body forms during infection because elucidating this mechanism would show that CaMV hijacks cellular pathways for the sole purpose to ensure its transmission. It was known that besides the single transmission body a second type of viral inclusion bodies is found in infected cells: the numerous “electron-dense inclusions” that are assumed to be the virus factories (Fig. 1B) where all viral synthesis occurs¹¹ and where most virus particles accumulate. However, P2 was never described in the factories, presenting the paradox: if it is translated in the factories why is it not found there? Of different possible scenarios we chose to test the hypothesis that P2 is produced in the factories and then exported. Protoplasts were transfected with CaMV particles and kinetics of P2 accumulation followed by immunofluorescence. The results showed that P2 is indeed translated in the viral factories but then associates temporally with microtubules before finally condensing into a single transmission body. Also the other known components of the transmission body, the viral protein P3 and to a lesser degree, some virus particles, followed the same route from viral factories to the transmission body.Experiments with cytoskeleton drugs confirmed that transient localization of transmission body components with microtubules, but not with actin filaments, is necessary for transmission body formation. The results also indicated that both microtubules and actin filaments are apparently not required for other steps of the intracellular infection cycle because formation of viral factories was only slightly inhibited by the drugs.The results show that CaMV specifically uses the microtubule cytoskeleton to form the transmission body and thus enable vector transmission. Consequently, non-circulant transmission of at least this virus is not a random event where the vector takes up some transmissible complexes by chance. It is rather the result of highly specific interactions, where the virus “intentionally” (ab)uses cellular pathways to optimize acquisition by the vector, and this long before arrival of the latter on an infected plant.A lot of questions remain open, though. Are P2 and the other components of the transmission body actively transported on microtubules, or is their transient colocalization with microtubules part of an alternative transport mode? We started to more closely examine interaction between P2 and microtubules and privileged the hypothesis that the protein might be transported by a motor activity on microtubules. As preliminary data indicated that P2 does not possess an innate translocating activity, we looked for a cellular motor protein and tested as a candidate the kinesin TBK5.¹² This transport protein is, when overexpressed, able to bundle microtubules into a single focus, just as transmission bodies are singular structures in the cell. When healthy protoplasts were cotransfected with TBK5 and CaMV, TBK5 localized transiently with P2 on microtubules and in transmission bodies (Fig. 1C and D). This might be taken as the first evidence that a kinesin might be involved in formation of transmission bodies, but more experimentation is needed to confirm this hypothesis.A by far more important question is: Have also other viruses, whether from the plant or the animal kingdoms, that are noncirculantly (or mechanically, as animal virologists call this mode of transmission) transmitted, developed similar strategies that fine-tune interactions between the host and the virus to prepare and perfect transmission?     

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 cauliflower mosaic virus virion-associated protein is dispensable for viral replication in single cells

Cauliflower mosaic virus (CaMV) open reading frame III (ORF III) codes for a virion-associated protein (Vap), which is one of two viral proteins essential for aphid transmission. However, unlike the aphid transmission factor encoded by CaMV ORF II, Vap is also essential for systemic infection, suggesting that it is a multifunctional protein. To elucidate the additional function or functions of Vap, we tested the replication of noninfectious ORF III-defective mutants in transfected turnip protoplasts. PCR and Western blot analyses revealed that CaMV replication had occurred with an efficiency similar to that of wild-type virus and without leading to reversions. Electron microscopic examination revealed that an ORF III frameshift mutant formed normally structured virions. These results demonstrate that Vap is dispensable for replication in single cells and is not essential for virion morphogenesis. Analysis of inoculated turnip leaves showed that the ORF III frameshift mutant does not cause any detectable local infection. These results are strongly indicative of a role for Vap in virus movement.     

Mutagenesis of cauliflower mosaic virus

A series of insertion mutants of cauliflower mosaic virus (CaMV) DNA has been constructed in vitro. These insertions consist of a short DNA sequence (10 or 22 bp) containing a restriction endonuclease site (SmaI) not represented on the viral DNA. Viral infectivity was analyzed by inoculating plants with the mutated cloned viral DNA and observing symptoms. Insertions within ORFVII, and in one site within the large intergenic region, did not interfere with viral infectivity, whilst insertions within ORFII and at the end of ORFIV retarded the development of viral symptoms. All other insertion mutants analyzed were lethal. CaMV with a deletion of 105 bp within ORFVII was viable. Such viable mutants can be used to construct additional deletions or to insert foreign DNA into the viral genome.     

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.     

Mutation of capsid protein phosphorylation sites abolishes cauliflower mosaic virus infectivity

The cauliflower mosaic virus (CaMV) capsid protein is derived by bidirectional processing of the precapsid protein (CP56). We expressed several derivatives of CP56 in Escherichia coli and used them as substrates for virus-associated kinase and casein kinase II purified from plant cells. Three serine residues located at the N terminus of the mature viral protein CP44 were identified as phosphorylation targets. A mutation of one of them in the viral context had little or no effect on viral infectivity, but a mutation of all three serines abolished infectivity. The mapping of phosphorylation sites in CP44, but not CP39 or CP37, and immunodetection of the Zn finger motif in CP44 and CP39, but not CP37, support the model that CP39 is produced from CP44 by N-terminal processing and CP37 is produced from CP39 by C-terminal processing. We discuss the possible role of phosphorylation in the processing and assembly of CaMV capsid protein.     

Interaction of the cauliflower mosaic virus coat protein with the pregenomic RNA leader

Using the yeast three-hybrid system, the interaction of the Cauliflower mosaic virus (CaMV) pregenomic 35S RNA (pgRNA) leader with the viral coat protein, its precursor, and a series of derivatives was studied. The purine-rich domain in the center of the pgRNA leader was found to specifically interact with the coat protein. The zinc finger motif of the coat protein and the preceding basic domain were essential for this interaction. Removal of the N-terminal portion of the basic domain led to loss of specificity but did not affect the strength of the interaction. Mutations of the zinc finger motif abolished not only the interaction with the RNA but also viral infectivity. In the presence of the very acidic C-terminal domain, which is part of the preprotein but is not present in the mature CP, the interaction with the RNA was undetectable.     

Insertional mutagenesis of the cauliflower mosaic virus genome

A series of small insertions has been introduced into the various translational reading frames of the DNA of a "severe" strain of cauliflower mosaic virus (CaMV). A selectable gene (the kanamycin phosphotransferase gene of Tn903), flanked by a series of symmetrically arranged cloning sites taken from M13mp7, was used to prepare the site-specific mutants. In-phase insertions of 12 or 30 bp, which introduced unique SalI sites into reading regions I, III, IV, V and into the amino-proximal portion of region VI, destroyed infectivity. Insertions in the amino-distal portion of region VI, in the large intergenic region, and in region II retained infectivity. The amino-distal insertions in region VI reduced the severity of symptoms in plants. The insertion in region II destroyed aphid transmissibility. Longer DNA segments when inserted into region II or into the amino-distal portion of region VI destroyed infectivity, but similar insertions in the intergenic region were without effect on virus infection or development.     

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.     

Nucleotide sequence of cauliflower mosaic virus DNA

The complete nucleotide sequence (8024 nucleotides) of the circular double-stranded DNA of cauli-flower mosaic virus has been established. The DNA molecule is known to possess three discrete single-stranded discontinuities, often referred to as “gaps”, two in one strand and one in the other. The sequence data indicate that gap 1, the single discontinuity in the α strand, corresponds to the absence of no more than one or two nucleotides with respect to the complementary β strand. The two discontinuities in the β strand, however, are not authentic gaps since no nucleotides are missing, but are instead regions of sequence overlap: a short sequence (19 residues for gap 2, at least 2 residues for gap 3) at one terminus of each discontinuity, probably the 5′ terminus, is displaced from the double helix by an identical sequence at the other boundary of the discontinuity. Analysis of the distribution of nonsense codons in the DNA sequence is consistent with other evidence that only the α strand is transcribed. The coding region extends around the circular molecule from 4 map units of gap 1, the map origin, to map position 91, and consists of six long open reading frames. Our findings suggest, but do not prove, that the DNA sequence of the open reading frames is colinear with viral protein sequences. The cistron for the viral coat protein, which is probably synthesized in the form of a precursor, has been situated in coding region IV on the basis of its unusual amino acid composition.     

Degradation signals within both terminal domains of the cauliflower mosaic virus capsid protein precursor

Targeted protein degradation plays an important regulatory role in the cell, but only a few protein degradation signals have been characterized in plants. Here we describe three instability determinants in the termini of the cauliflower mosaic virus (CaMV) capsid protein precursor, of which one is still present in the mature capsid protein p44. A modified ubiquitin protein reference technique was used to show that these motifs are still active when fused to a heterologous reporter gene. The N-terminus of p44 contains a degradation motif characterized by proline, glutamate, aspartate, serine and threonine residues (PEST), which can be inactivated by mutation of three glutamic acid residues to alanines. The signals from the precursor do not correspond to known degradation motifs, although they confer high instability on proteins expressed in plant protoplasts. All three instability determinants were also active in mammalian cells. The PEST signal had a significantly higher degradation activity in HeLa cells, whereas the precursor signals were less active. Inhibition studies suggest that only the signal within the N-terminus of the precursor is targeting the proteasome in plants. This implies that the other two signals may target a novel degradation pathway.     

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.     

Expression of the cauliflower mosaic virus capsid gene in vivo

Antisera against the N-terminal and C-terminal parts of the potential ORF IV product were used to analyse extracts from CaMV-infected turnip leaves by immunoblotting. Polypeptides of 87, 83, 82, 60 and 57 kDa were detected. The origin of these proteins is discussed.     

Molecular dissection of the cauliflower mosaic virus translation transactivator.

The cauliflower mosaic virus (CaMV) transactivator (TAV) is a complex protein that appears to be involved in many aspects of the virus life cycle. One of its roles is to control translation from the polycistronic CaMV 35S RNA. Here we report a molecular dissection of TAV in relation to its ability to enhance dicistronic translation in transient expression experiments. We have identified a protein domain that is responsible and sufficient for that activity. This 'MiniTAV domain' consists of only 140 of the 520 amino acids in the full-length sequence. A further domain located outside the MiniTAV, and therefore dispensable for transactivation, is probably involved in interactions with other molecules. This was identified by its ability to compete with wild-type TAV and some of its deletion mutants. We found, furthermore, that the TAV protein binds RNA. Two regions needed for RNA-binding properties were defined outside the MiniTAV domain and RNA binding seems not to be directly involved in the transactivation mechanism.     

Aphid transmission of beet western yellows luteovirus requires the minor capsid read-through protein P74.

Beet western yellows luteovirus is obligately transmitted by the aphid Myzus persicae in a circulative, non-propagative fashion. Virus movement across the epithelial cells of the digestive tube into the hemocoel and from the hemocoel into the accessory salivary glands is believed to occur by receptor-mediated endocytosis and exocytosis. Virions contain two types of protein; the major 22 kDa capsid protein and the minor read-through protein, P74, which is composed of the major capsid protein fused by translational read-through to a long C-terminal extension called the read-through domain. Beet western yellows virus carrying various mutations in the read-through domain was tested for its ability to be transmitted to test plants by aphids fed on agro-infected plants and semi-purified or purified virus preparations. The results establish that the read-through domain carries determinants that are essential for aphid transmission. The findings also reveal that the read-through domain is important for accumulation of the virus in agro-infected plants.