Phosphorylation of the termini of Cauliflower mosaic virus precapsid protein is important for productive infection

Cauliflower mosaic virus (CaMV) coat protein precursor (pre-CP) has 489 amino acids (p57) and is processed by the viral proteinase into three major forms: p44, p39, and p37. The N- and C-terminal extensions of pre-CP are released during maturation by the virus-encoded proteinase. We showed that these extensions are phosphorylated at several sites by host casein kinase II (CKII). We have identified the phosphorylated amino acids using an in vitro phosphorylation assay and tested the effect of mutation of these sites on viral infectivity. Mutation of serines S66, S68, and S72 to alanine in the N-terminal extension abolished phosphorylation of the protein in vitro. Also, mutation of all S and T residues in the C-terminus (450 to 489) made this region insensitive to CKII. Amino acid substitutions also were introduced into a full-length infectious clone of CaMV. Mutated forms of the virus with S66, S68, and S72 substituted with A or D showed a delay in symptom development and affected the infectivity of the virus. However, a mutant with an A substitution of all the S and T residues of the C-terminal extension of CP was not infectious. These results suggest that phosphorylation of the N- and C-termini of CaMV pre-CP plays an important role in the initiation of viral infection.     

Biochemical characterization of the helper component of Cauliflower mosaic virus

The helper component of Cauliflower mosaic virus is encoded by viral gene II. This protein (P2) is dispensable for virus replication but required for aphid transmission. The purification of P2 has never been reported, and hence its biochemical properties are largely unknown. We produced the P2 protein via a recombinant baculovirus with a His tag fused at the N terminus. The fusion protein was purified by affinity chromatography in a soluble and biologically active form. Matrix-assisted laser desorption time-of-flight mass spectrometry demonstrated that P2 is not posttranslationally modified. UV circular dichroism revealed the secondary structure of P2 to be 23% alpha-helical. Most alpha-helices are suggested to be located in the C-terminal domain. Using size exclusion chromatography and aphid transmission testing, we established that the active form of P2 assembles as a huge soluble oligomer containing 200 to 300 subunits. We further showed that P2 can also polymerize as long paracrystalline filaments. We mapped P2 domains involved in P2 self-interaction, presumably through coiled-coil structures, one of which is proposed to form a parallel trimer. These regions have previously been reported to also interact with viral P3, another protein involved in aphid transmission. Possible interference between the two types of interaction is discussed with regard to the biological activity of P2.     

Cauliflower mosaic virus as a gene expression vector for plants

It is possible to replace the CaMV (cauliflower mosaic virus) ORF (open reading frame) II with foreign sequences without interfering with virus viability. Such recom-binants can induce the synthesis of substantial amounts of a foreign protein in infected plants and confer new properties to these plants. However, so far only three genes have been successfully cloned and expressed in this way. The expression mechanism of CaMV demands precise replacement of ORF II and probably certain structural features of the viral 35S RNA, which should not be disturbed by inserted sequences. Since these features are largely unknown, it cannot at present be pre-dicted whether an insert will be tolerated. It is more likely that larger inserts will disturb the viral gene expression mechanism than smaller ones.     

The plant gene CCD1 selectively blocks cell death during the hypersensitive response to Cauliflower mosaic virus infection

The P6 protein of Cauliflower mosaic virus (CaMV) W260 elicits a hypersensitive response (HR) on inoculated leaves of Nicotiana edwardsonii. This defense response, common to many plant pathogens, has two key characteristics, cell death within the initially infected tissues and restriction of the pathogen to this area. We present evidence that a plant gene designated CCD1, originally identified in N. bigelovii, can selectively block the cell death pathway during HR, whereas the resistance pathway against W260 remains intact. Suppression of cell death was evident not only macroscopically but also microscopically. The suppression of HR-mediated cell death was specific to CaMV, as Tobacco mosaic virus was able to elicit HR in the plants that contained CCD1. CCD1 also blocks the development of a systemic cell death symptom induced specifically by the P6 protein of W260 in N. clevelandii. Introgression of CCD1 from N. bigelovii into N. clevelandii blocked the development of systemic cell death in response to W260 infection but could not prevent systemic cell death induced by Tomato bushy stunt virus. Thus, CCD1 blocks both local and systemic cell death induced by P6 of W260 but does not act as a general suppressor of cell death induced by other plant viruses. Furthermore, experiments with CCD1 provide further evidence that cell death could be uncoupled from resistance in the HR of Nicotiana edwardsonii to CaMV W260.     

A Distinct Subpopulation of Cauliflower mosaic virus (CaMV) in South Iran

Cauliflower mosaic virus (CaMV) with a high incidence and widespread distribution on Brassica crops in Iran reduces the yield and quality of these crops. The complete sequences of three open reading frames (ORFs) 2, 4 and 6 coding for aphid transmission (AT), coat protein (CP) and inclusion body protein/translation transactivator (TAV) genes, respectively, were determined for two Iranian CaMV isolates from Kerman (south Iran). They induced latent or mild mottle (L/MMo) infection in Brassica oleracea var. capitata so are considered as the (L/MMo) biotype. Clear recombination breakpoints were detected between ORF2 and ORF6 in two Kerman isolates using concatenate fragments. Phylogenetic analysis revealed three Iranian CaMV subpopulations in which the two Kerman isolates in the new subgroup C were added to the two previously reported Iranian subpopulations A (central and west Iran) and B (north‐east Iran). Also three regions of pairwise identity were detected which representing: 97.1–100, 93.8–97.1 and 90.6–93.8% for subgroups A, C and B, respectively. Our analysis showed the high variability of Iranian CaMV population and provided valuable new information for understanding the diversity and evolution of caulimoviruses. Furthermore, star phylogeny was found in the subgroup C with overall lack of nt diversity and high haplotype diversity as evidence of a recent population expansion after a genetic bottleneck although this may have been modified subsequently by clinal genetic drift. The appearance of new genetic types demonstrates a high potential of risks and should be considered in the planning of efficient control programmes.     

Agroinfiltration of Cauliflower mosaic virus gene VI elicits hypersensitive response in Nicotiana species

Cauliflower mosaic virus strain W260 induces hypersensitive response (HR) in Nicotiana edwardsonii and systemic cell death in N. clevelandii. In contrast, the D4 strain of Cauliflower mosaic virus evades the host defenses in Nicotiana species; it induces chlorotic primary lesions and a systemic mosaic in both hosts. Previous studies with chimeric viruses had indicated that gene VI of W260 was responsible for elicitation of HR or cell death. To prove conclusively that W260 gene VI is responsible, we inserted gene VI of W260 and D4 into the Agrobacterium tumefaciens binary vector pKYLX7. Agroinfiltration of these constructs into the leaves of N. edwardsonii and N. clevelandii revealed that gene VI of W260 elicited HR in N. edwardsonii 4 to 5 days after infiltration and cell death in N. clevelandii approximately 9 to 12 days after infiltration. In contrast, gene VI of D4 did not elicit HR or cell death in either Nicotiana species. A frameshift mutation introduced into gene VI of W260 abolished its ability to elicit HR or cell death in both Nicotiana species, demonstrating that the elicitor is the gene VI protein.     

Differential induction of symptoms in Arabidopsis by P6 of Cauliflower mosaic virus

The gene VI protein (P6) of Cauliflower mosaic virus (CaMV) functions as a virulence factor in crucifers by eliciting chlorotic symptoms in infected plants. The ability to induce chlorosis has been associated previously with P6 through gene-swapping experiments between strains and through the development of transgenic plants that express P6. The primary role that has been identified for P6 in the CaMV infection cycle is to modify the host translation machinery to facilitate the translation of the polycistronic CaMV 35S RNA. This function for P6 has been designated as the translational transactivator (TAV) function. In the present study, we have characterized an unusual variant of P6, derived from CaMV strain D4, that does not induce chlorosis upon transformation into Arabidopsis thaliana. The level of D4 P6 produced in transgenic Arabidopsis line D4-2 was comparable to the amount found in transgenic plants homozygous for W260 and CM1841 P6, two versions of P6 that induce strong chlorotic symptoms and stunting in Arabidopsis. A complementation assay proved that P6 expressed in the D4-2 line was functional, as it could support the systemic infection of a CM1841 mutant that contained a lethal frame-shift mutation within gene VI. This complementation assay allowed us to separately assess the contribution of CM1841 gene VI to symptom development versus the contribution of other CM1841 genes. Furthermore, a previous study had shown that the TAV activity of D4 P6 was comparable to that of W260 P6. That comparative analysis of TAV function, coupled with the characterization of the D4-2 transgenic line in the present paper, indicates that the TAV function of P6 may play only a minor role in the development of chlorotic symptoms.     

The Temporal Evolution and Global Spread of Cauliflower mosaic virus,a Plant Pararetrovirus

Cauliflower mosaic virus (CaMV) is a plant pararetrovirus with a double-stranded DNA genome. It is the type member of the genus Caulimovirus in the family Caulimoviridae. CaMV is transmitted by sap inoculation and in nature by aphids in a semi-persistent manner. To investigate the patterns and timescale of CaMV migration and evolution, we sequenced and analyzed the genomes of 67 isolates of CaMV collected mostly in Greece, Iran, Turkey, and Japan together with nine published sequences. We identified the open-reading frames (ORFs) in the genomes and inferred their phylogeny. After removing recombinant sequences, we estimated the substitution rates, divergence times, and phylogeographic patterns of the virus populations. We found that recombination has been a common feature of CaMV evolution, and that ORFs I–V have a different evolutionary history from ORF VI. The ORFs have evolved at rates between 1.71 and 5.81×10⁻⁴ substitutions/site/year, similar to those of viruses with RNA or ssDNA genomes. We found four geographically confined lineages. CaMV probably spread from a single population to other parts of the world around 400–500 years ago, and is now widely distributed among Eurasian countries. Our results revealed evidence of frequent gene flow between populations in Turkey and those of its neighboring countries, with similar patterns observed for Japan and the USA. Our study represents the first report on the spatial and temporal spread of a plant pararetrovirus.     

Association of the P6 Protein of Cauliflower mosaic virus with Plasmodesmata and Plasmodesmal Proteins

The P6 protein of Cauliflower mosaic virus (CaMV) is responsible for the formation of inclusion bodies (IBs), which are the sites for viral gene expression, replication, and virion assembly. Moreover, recent evidence indicates that ectopically expressed P6 inclusion-like bodies (I-LBs) move in association with actin microfilaments. Because CaMV virions accumulate preferentially in P6 IBs, we hypothesized that P6 IBs have a role in delivering CaMV virions to the plasmodesmata. We have determined that the P6 protein interacts with a C2 calcium-dependent membrane-targeting protein (designated Arabidopsis [Arabidopsis thaliana] Soybean Response to Cold [AtSRC2.2]) in a yeast (Saccharomyces cerevisiae) two-hybrid screen and have confirmed this interaction through coimmunoprecipitation and colocalization assays in the CaMV host Nicotiana benthamiana. An AtSRC2.2 protein fused to red fluorescent protein (RFP) was localized to the plasma membrane and specifically associated with plasmodesmata. The AtSRC2.2-RFP fusion also colocalized with two proteins previously shown to associate with plasmodesmata: the host protein Plasmodesmata-Localized Protein1 (PDLP1) and the CaMV movement protein (MP). Because P6 I-LBs colocalized with AtSRC2.2 and the P6 protein had previously been shown to interact with CaMV MP, we investigated whether P6 I-LBs might also be associated with plasmodesmata. We examined the colocalization of P6-RFP I-LBs with PDLP1-green fluorescent protein (GFP) and aniline blue (a stain for callose normally observed at plasmodesmata) and found that P6-RFP I-LBs were associated with each of these markers. Furthermore, P6-RFP coimmunoprecipitated with PDLP1-GFP. Our evidence that a portion of P6-GFP I-LBs associate with AtSRC2.2 and PDLP1 at plasmodesmata supports a model in which P6 IBs function to transfer CaMV virions directly to MP at the plasmodesmata.Through the years, numerous studies have focused on the characterization of viral replication sites within the cell, as well as how plant virus movement proteins (MPs) modify the plasmodesmata to facilitate cell-to-cell movement (for review, see Benitez-Alfonso et al., 2010; Laliberté and Sanfaçon, 2010; Niehl and Heinlein, 2011; Ueki and Citovsky, 2011; Verchot, 2012). It is accepted that plant virus replication is associated with host membranes, and at some point, the viral genomic nucleic acid must be transferred from the site of replication in the cell to the plasmodesmata. This step could involve transport from a distant site within the cell, or alternatively, it may be that replication is coupled with transport at the entrance of the plasmodesmata (Tilsner et al., 2013). However, even with the latter model, there is ample evidence that the viral proteins necessary for replication or cell-to-cell movement utilize intracellular trafficking pathways within the cell to become positioned at the plasmodesma. These pathways may involve microfilaments, microtubules, or specific endomembranes that participate in macromolecular transport pathways, or combinations of these elements (Harries et al., 2010; Schoelz et al., 2011; Patarroyo et al., 2012; Peña and Heinlein, 2012; Tilsner and Oparka 2012; Liu and Nelson, 2013).The P6 protein of Cauliflower mosaic virus (CaMV) is one viral protein that had not been considered to play a role in viral movement until recently. P6 is the most abundant protein component of the amorphous, electron-dense inclusion bodies (IBs) present during virus infection (Odell and Howell, 1980; Shockey et al., 1980). Ectopic expression of P6 in Nicotiana benthamiana leaves resulted in the formation of inclusion-like bodies (I-LBs) that were capable of intracellular movement along actin microfilaments. Furthermore, treatment of Nicotiana edwardsonii leaves with latrunculin B abolished the formation of CaMV local lesions, suggesting that intact microfilaments are required for CaMV infection (Harries et al., 2009a). A subsequent paper showed that P6 physically interacts with Chloroplast Unusual Positioning1 (CHUP1), a plant protein localized to the chloroplast outer membrane that contributes to movement of chloroplasts on microfilaments in response to changes in light intensity (Oikawa et al., 2003, 2008; Angel et al., 2013). The implication was that P6 might hijack CHUP1 to facilitate movement of the P6 IBs on microfilaments. Silencing of CHUP1 in N. edwardsonii, a host for CaMV, slowed the rate of local lesion formation, suggesting that CHUP1 contributes to intracellular movement of CaMV (Angel et al., 2013).In addition to its role in intracellular trafficking, the P6 protein has been shown to have at least four other distinct functions in the viral infection cycle. P6-containing IBs induced during virus infection are likely virion factories, as they are the primary site for CaMV protein synthesis, genome replication, and assembly of virions (Hohn and Fütterer, 1997). Second, P6 interacts with host ribosomes to facilitate reinitiation of translation of genes on the polycistronic 35S viral RNA, a process called translational transactivation (Bonneville et al., 1989; Park et al., 2001; Ryabova et al., 2002). The translational transactivator region of P6 (Fig. 1) defines the essential sequences required for translational transactivation (DeTapia et al., 1993). Third, P6 is an important pathogenicity determinant. P6 functions as an avirulence determinant in some solanaceous and cruciferous species (Daubert et al., 1984; Schoelz et al., 1986; Hapiak et al., 2008) and is a chlorosis symptom determinant in susceptible hosts (Daubert et al., 1984; Baughman et al., 1988; Goldberg et al., 1991; Cecchini et al., 1997). Finally, P6 has the capacity to compromise host defenses, as it is a suppressor of RNA silencing and cell death (Love et al., 2007; Haas et al., 2008), and it modulates signaling by salicylic acid, jasmonic acid, ethylene, and auxin (Geri et al., 2004; Love et al., 2012; Laird et al., 2013). Domain D1 of P6 has been shown to be necessary but not sufficient for suppression of silencing and salicylic acid-mediated defenses (Laird et al., 2013).Open in a separate windowFigure 1.CaMV and host constructs used for confocal microscopy or coimmunoprecipitation (co-IP). A, Structure of CaMV P6 and Arabidopsis (Arabidopsis thaliana) Soybean Response to Cold (AtSRC2.2) proteins. The functions of P6 domains D1 to D4 tested for interaction with AtSRC2.2 are indicated by the shaded boxes. The Mini TAV is the minimal region for the translational transactivation function. The NLSa sequence corresponds to the nuclear localization signal of influenza virus. The NLS sequence corresponds to the nuclear localization signal of human ribosomal protein L22. B, Structure of P6 (Angel et al., 2013), AtSRC2.2, PDLP (Thomas et al., 2008), and CaMV MP fusions developed for confocal microscopy and/or co-IP. aa, Amino acid.Because P6-containing IBs are the site for virion accumulation and they are capable of movement, they may be responsible for delivering virions to the CaMV MP located at the plasmodesmata (for review, see Schoelz et al., 2011). The vast majority of CaMV virions accumulate in association with P6-containing IBs. Furthermore, P6 physically interacts with the CaMV capsid and MP, as well as the two proteins necessary for aphid transmission, P2 and P3 (Himmelbach et al., 1996; Ryabova et al., 2002; Hapiak et al., 2008; Lutz et al., 2012). Recent studies have indicated that P6 IBs serve as a reservoir for virions, in which the virions may be rapidly transferred to P2 electron-lucent IBs for acquisition by aphids (Bak et al., 2013). It stands to reason that P6 IBs may also serve as a reservoir for CaMV virions to be transferred to the CaMV MP in the plasmodesmata.CaMV virions move from cell to cell through plasmodesmata modified into tubules through the function of its MP (Perbal et al., 1993; Kasteel et al., 1996). However, studies have suggested that CaMV virions do not appear to directly interact with the MP. Instead, the MP interacts with the CaMV P3 protein (also known as the virion-associated protein [VAP]), which forms a trimeric structure that is anchored into the virions (Leclerc et al., 1998; Leclerc et al., 2001). Electron microscopy studies have indicated that MP and VAP colocalize with virions only at the entrance to or within the plasmodesmata, and it has been suggested that the VAP/virion complex travels to the plasmodesmata independently from the MP (Stavolone et al., 2005). Consequently, there is a need for a second CaMV protein such as P6 to fulfill the role of delivery of virions to the plasmodesmata (Schoelz et al., 2011).Additional studies have shown that the CaMV MP is incorporated into vesicles and is trafficked on the endomembrane system to reach the plasmodesma (Carluccio et al., 2014). These authors suggest that the CaMV MP is recycled in a vesicular transport pathway between plasmodesmata and early endosome compartments. The CaMV MP interacts with µA-Adaptin (Carluccio et al., 2014) and Movement Protein-Interacting7 (Huang et al., 2001), two proteins shown to have a role in vesicular trafficking. Once the MP arrives at plasmodesmata, it interacts with the Plasmodesmata-Localized Protein (PDLP) proteins, which comprise a family of eight proteins associated with plasmodesmata (Amari et al., 2010). In addition to its interaction with CaMV MP, PDLP1 interacts with the 2B protein of Grapevine fan leaf virus (GFLV) at the base of tubules formed by the 2B protein. Furthermore, an Arabidopsis transfer DNA (T-DNA) mutant line in which three PDLP genes had been knocked out (pdlp1-pdlp2-pdlp3) responded to GFLV and CaMV inoculation with a delayed infection (Amari et al., 2010). This has led to the suggestion that the PDLPs might act as receptors for the MPs of the tubule-forming viruses such as GFLV and CaMV (Amari et al., 2010, 2011).To better understand the function of the P6 protein during CaMV intracellular movement, we have utilized a yeast (Saccharomyces cerevisiae) two-hybrid assay to identify host proteins that interact with CaMV P6. We show that P6 physically interacts with a C2-calcium-dependent protein (designated AtSRC2.2). AtSRC2.2 is a membrane-bound protein that is capable of forming punctate spots associated with plasmodesmata. The localization of AtSRC2.2 with plasmodesmata led to an analysis of interactions between P6 I-LBs, AtSRC2.2, PDLP1, and the CaMV MP and also revealed that a portion of P6 I-LBs are found adjacent to plasmodesmata. These results provide further evidence for a model in which P6 IBs are capable of delivery of virions to plasmodesmata for their transit to other host cells.