Pelargonium Flower Break Virus Research Articles

Natural occurrence of pelargonium flower break virus infectingPelargonium zonaleplants in South America

AbstractIn 2023, virus‐like symptoms were observed inPelargonium zonaleplants in Brazil. Transmission electron microscope analysis revealed the presence of isometric particles measuring ca. 30 nm in diameter in leaf extracts and ultrathin sections of symptomatic field plants. Molecular assays using PFBV‐specific primers and sequencing of amplicons identified the pelargonium flower break virus (PFBV, genusAlphacarmovirus). Mechanically inoculated plants ofChenopodium quinoaandP. zonaleexhibited symptoms of chlorotic local lesions and systemic chlorotic spots, respectively. This is the first report of PFBV infectingP. zonaleplants in South America (Brazil).

First report of Pelargonium flower break virus (PFBV) in Iran and determining some of its molecular properties

First report of Pelargonium flower break virus (PFBV) in Iran and determining some of its molecular properties

An Update on the Intracellular and Intercellular Trafficking of Carmoviruses.

Despite harboring the smallest genomes among plant RNA viruses, carmoviruses have emerged as an ideal model system for studying essential steps of the viral cycle including intracellular and intercellular trafficking. Two small movement proteins, formerly known as double gene block proteins (DGBp1 and DGBp2), have been involved in the movement throughout the plant of some members of carmovirus genera. DGBp1 RNA-binding capability was indispensable for cell-to-cell movement indicating that viral genomes must interact with DGBp1 to be transported. Further investigation on Melon necrotic spot virus (MNSV) DGBp1 subcellular localization and dynamics also supported this idea as this protein showed an actin-dependent movement along microfilaments and accumulated at the cellular periphery. Regarding DGBp2, subcellular localization studies showed that MNSV and Pelargonium flower break virus DGBp2s were inserted into the endoplasmic reticulum (ER) membrane but only MNSV DGBp2 trafficked to plasmodesmata (PD) via the Golgi apparatus through a COPII-dependent pathway. DGBp2 function is still unknown but its localization at PD was a requisite for an efficient cell-to-cell movement. It is also known that MNSV infection can induce a dramatic reorganization of mitochondria resulting in anomalous organelles containing viral RNAs. These putative viral factories were frequently found associated with the ER near the PD leading to the possibility that MNSV movement and replication could be spatially linked. Here, we update the current knowledge of the plant endomembrane system involvement in carmovirus intra- and intercellular movement and the tentative model proposed for MNSV transport within plant cells.

Pest Identification Guide: Western Flower Thrips Frankliniella occidentalis (Pergande)

Western flower thrips transmit the carmovirus Pelargonium flower break virus (PFBV), the ilarvirus Tobacco streak virus (TSV), the tospoviruses Chrysanthemum stem necrosis virus (CSNV), Groundnut ringspot virus (GRSV), Impatiens necrotic spot virus (INSV), Tomato chlorotic spot virus (TCSV), and Tomato spotted wilt virus (TSWV). This species is primarily a flower feeder, so most damage would be expected on the flower or fruit. Learn to identify the western flower thrips with this two-page illustrated guide written by Jeffrey D. Cluever and Hugh A. Smith, and published by the Department of Entomology and Nematology, May 2016. ENY-2034/IN1127: Pest Identification Guide: Western Flower Thrips Frankliniella occidentalis (Pergande) (ufl.edu)

Analysis of the subcellular targeting of the smaller replicase protein of Pelargonium flower break virus

Replication of all positive RNA viruses occurs in association with intracellular membranes. In many cases, the mechanism of membrane targeting is unknown and there appears to be no correlation between virus phylogeny and the membrane systems recruited for replication. Pelargonium flower break virus (PFBV, genus Carmovirus, family Tombusviridae) encodes two proteins, p27 and its read-through product p86 (the viral RNA dependent-RNA polymerase), that are essential for replication. Recent reports with other members of the family Tombusviridae have shown that the smaller replicase protein is targeted to specific intracellular membranes and it is assumed to determine the subcellular localization of the replication complex. Using in vivo expression of green fluorescent protein (GFP) fusions in plant and yeast cells, we show here that PFBV p27 localizes in mitochondria. The same localization pattern was found for p86 that contains the p27 sequence at its N-terminus. Cellular fractionation of p27GFP-expressing cells confirmed the confocal microscopy observations and biochemical treatments suggested a tight association of the protein to membranes. Analysis of deletion mutants allowed identification of two regions required for targeting of p27 to mitochondria. These regions mapped toward the N- and C-terminus of the protein, respectively, and could function independently though with distinct efficiency. In an attempt to search for putative cellular factors involved in p27 localization, the subcellular distribution of the protein was checked in a selected series of knockout yeast strains and the outcome of this approach is discussed.

ATTEMPTS TO ERADICATE TWO PELARGONIUM VIRUSES (PFBV AND PLPV) BY MERISTEM CULTURE AND SHOOT-TIP CRYOTHERAPY

Attempts to eradicate the Pelargonium flower break virus (PFBV) and Pelargonium line pattern virus (PLPV) by meristem culture and apex droplet-vitrification cryopreservation was carried out using 5 different cultivars. A simple meristem culture did not permit to eliminate PFBV and only 15% of Pelargonium x hortorum 'Stellar Artic' plants regenerated from meristems was PLPV-ELISA-negative. Plants regenerated from cryopreserved apices were tested by DAS-ELISA after a 3-month growing period. Viruses were not detected in 25 and 50% of the tested plants for PFBV and PLPV respectively. Immunolocalisations were carried out for virus localisation in apices from greenhouse plants (control) and vitroplants regenerated after meristem culture or cryopreservation. Immunolocalisations realised on control explants excised from DAS-ELISA positive plants showed that PFBV and PLPV were present in the apices, even in the meristematic dome. However, viral particles were more numerous in the cells of the basal zone than in the more meristematic ones. Immunolocalisations realised on apices from the DAS-ELISA negative cryoregenerated plants showed the viruses were still present. Our results firstly demonstrated that PFBV and PLPV are even present inside meristematic cells and secondly that cryopreservation could decrease their amount in Pelargonium plants but without eliminating them totally. More knowledge on virus behaviour during cryopreservation processes could optimize the management of genetic resources using this conservation method.

An Internal Ribosome Entry Site Directs Translation of the 3′-Gene from Pelargonium Flower Break Virus Genomic RNA: Implications for Infectivity

Pelargonium flower break virus (PFBV, genus Carmovirus) has a single-stranded positive-sense genomic RNA (gRNA) which contains five ORFs. The two 5′-proximal ORFs encode the replicases, two internal ORFs encode movement proteins, and the 3′-proximal ORF encodes a polypeptide (p37) which plays a dual role as capsid protein and as suppressor of RNA silencing. Like other members of family Tombusviridae, carmoviruses express ORFs that are not 5′-proximal from subgenomic RNAs. However, in one case, corresponding to Hisbiscus chlorotic ringspot virus, it has been reported that the 3′-proximal gene can be translated from the gRNA through an internal ribosome entry site (IRES). Here we show that PFBV also holds an IRES that mediates production of p37 from the gRNA, raising the question of whether this translation strategy may be conserved in the genus. The PFBV IRES was functional both in vitro and in vivo and either in the viral context or when inserted into synthetic bicistronic constructs. Through deletion and mutagenesis studies we have found that the IRES is contained within a 80 nt segment and have identified some structural traits that influence IRES function. Interestingly, mutations that diminish IRES activity strongly reduced the infectivity of the virus while the progress of the infection was favoured by mutations potentiating such activity. These results support the biological significance of the IRES-driven p37 translation and suggest that production of the silencing suppressor from the gRNA might allow the virus to early counteract the defence response of the host, thus facilitating pathogen multiplication and spread.

A membrane-associated movement protein of Pelargonium flower break virus shows RNA-binding activity and contains a biologically relevant leucine zipper-like motif

Two small viral proteins (DGBp1 and DGBp2) have been proposed to act in a concerted manner to aid intra- and intercellular trafficking of carmoviruses though the distribution of functions and mode of action of each protein partner are not yet clear. Here we have confirmed the requirement of the DGBps of Pelargonium flower break virus (PFBV), p7 and p12, for pathogen movement. Studies focused on p12 have shown that it associates to cellular membranes, which is in accordance to its hydrophobic profile and to that reported for several homologs. However, peculiarities that distinguish p12 from other DGBps2 have been found. Firstly, it contains a leucine zipper-like motif which is essential for virus infectivity in plants. Secondly, it has an unusually long and basic N-terminal region that confers RNA binding activity. The results suggest that PFBV p12 may differ mechanistically from related proteins and possible roles of PFBV DGBps are discussed.

First Report of Carnation mottle virus in Phalaenopsis Orchids

In November 2003, two Phalaenopsis orchids from two different nurseries with symptoms of chlorotic rings on leaves were observed in Changhua County of central Taiwan. Symptomatic plants were collected and examined for the presence of viruses. Electron microscopic examination of ultrathin sections of leaf tissues from the symptomatic orchids found isometric virions of 32 nm in diameter. Subsequently, an isolate (herein designated as '92-orchid-1') with particles of similar size were isolated from one symptomatic orchid and established in Chenopodium quinoa (3). After indirect ELISA tests using antisera against Carnation mottle virus (CarMV), Cucumber mosaic virus, Cymbidium ringspot virus, Tomato bushy stunt virus, Capsicum chlorosis virus, Impatiens necrotic spot virus, Tomato spotted wilt virus, Tomato ringspot virus, and Lisianthus necrosis virus, this isolate reacted positively with the antiserum produced against CarMV (1). CarMV-TW-infected and healthy C. quinoa were used as positive and negative controls, respectively. To further characterize this virus, the conserved region of the polymerase gene (ORF1RT) of Carmoviruses was amplified with degenerate primer pairs, FJJ2003-17 (5'-TATATCTCGAGCAA(A/C)TAGGGG(G/T)GCCT) and FJJ2003-18 (5'-TATAGGATCCCC(C/T)A(A/T)(A/G)GC(A/T)GTGTTCA), by reverse transcription (RT)-PCR using the total RNA isolated from the leaves of 92-orchid-1-, CarMV-TW-infected, and healthy C. quinoa (3). The 894-nt ORF1RT conserved region of isolate 92-orchid-1 (GenBank Accession No. HQ117873) shared 97.1, 65.6, 61.7, and 63.5% nucleotide identities and 98.3, 70.2, 66.1, and 64.7% amino acid identities with those of CarMV (X02986), Pelargonium flower break virus (NC_005286), Saguaro cactus virus (NC_001780), and Angelonia flower break virus (NC_007733), respectively. The sequence comparison of the ORF1RT conserved region indicated that 92-orchid-1 was a carmovirus related to CarMV. Sequence analyses of the coat protein (CP) gene (GenBank Accession No. HQ117872) amplified with the specific CP primer pairs of CarMV (FJJ2004-53: 5'-ACTGCGCTCGAGCTACTCTGTTGACAGTTCTA, and 2004-54: 5'-ATATATGGATCCCGTCCCGCCGTGTGTGTCTA) showed the isolate shared 95.8 to 98.8% nucleotide identities and 96.8 to 98.9% amino acid identities with those of 40 CarMV isolates. Furthermore, the CP gene shared 96.9, 97.0, and 98.8% nucleotide identities and 98.0, 95.7, and 98.3% amino acid identities with isolates from carnation (GenBank Accession No. AY383566) (1), calla lily (GenBank Accession No. HQ117870) (2), and lisianthus (GenBank Accession No. FJ843021), respectively, in Taiwan. These results suggested that this isolate was CarMV but distinct from the above-mentioned three isolates and designated CarMV-Ph. From 2004 to 2007, a further survey of 280 symptomatic Phalaenopsis plants by ELISA using CarMV polyclonal antibodies (1) found that approximately 4% of those tested were infected. To our knowledge, this is the first report of CarMV in Phalaenopsis orchids and the occurrence has substantial implications for the important nursery and floral industry in Taiwan.

Identification and characterization of RNA-binding activity in the ORF1-encoded replicase protein of Pelargonium flower break virus

Pelargonium flower break virus (PFBV) belongs to the genus Carmovirus (family Tombusviridae) and, as with the remaining members of the group, possesses a monopartite genome of single-stranded, positive-sense RNA that contains five ORFs. The two 5'-proximal ORFs (ORFs 1 and 2) encode two polypeptides of 27 and 86 kDa (p27 and p86), respectively, that show homology with replication proteins. The p27 does not present any motif to explain its presumed involvement in replication, while p86 has the motifs conserved in RNA-dependent RNA polymerases. In this work, we have confirmed the necessity of p27 and p86 for PFBV replication. To gain insights into the function(s) of p27, we have expressed and purified the protein from Escherichia coli and tested its ability to bind RNA in vitro. The results have shown that p27 is able to bind ssRNA with high affinity and in a cooperative fashion and that it is also capable of binding other types of nucleic acids, though to a lesser extent. Additionally, competition experiments suggest that p27 has a preference for PFBV-derived ssRNAs. Using truncated forms of p27, it can be concluded that several regions of the protein contribute to its RNA-binding properties and that this contribution is additive. This study is the first to show nucleic acid-binding ability of the ORF1 product of a carmovirus and the data obtained suggest that this product plays an essential role in selection and recruitment of viral RNA replication templates.

Characterization of the subgenomic RNAs produced by Pelargonium flower break virus: Identification of two novel RNAs species

Pelargonium flower break virus (PFBV), a member of the genus Carmovirus, has a single-stranded positive-sense genomic RNA (gRNA) of 3.9 kb. The 5′ half of the gRNA encodes two proteins involved in replication, the p27 and its readthrough product, p86 (the viral RNA dependent-RNA polymerase, RdRp), and the 3′ half encodes two small movement proteins, p7 and p12, and the coat protein (CP). As other members of the family Tombusviridae, carmoviruses express ORFs that are not 5′-proximal from subgenomic RNAs (sgRNAs). Analysis of double-stranded RNAs extracted from PFBV-infected leaves and Northern blot hybridizations of total RNA from infected plants or protoplasts revealed than PFBV produces four 3′-coterminal sgRNAs of 3.2, 2.9, 1.7 and 1.4 kb, respectively. The 5′ termini of the 1.7 and 1.4 kb sgRNAs mapped 26 and 143 nt upstream of the initiation codons of the p7 and CP genes, respectively, whereas the 5′-ends of the 3.2 and 2.9 kb sgRNAs were located within the readthrough portion of the RdRp gene. The PFBV sgRNAs begin with a motif which is also present at the 5′ terminus of the gRNA and the minus polarity of the regions preceding their corresponding start sites (in the gRNA) may be folded into hairpin structures resembling those found for the sgRNA promoters of other carmoviruses. The results indicate that, besides the sgRNAs involved in the translation of the movement proteins and the CP identified in most carmoviral infections, PFBV produces two additional sgRNAs whose biological significance is currently unknown. The possible participation of the 3.2 and 2.9 kb PFBV sgRNAs in the expression of readthrough portions of the RdRp is discussed.

Inhibition of RNA silencing by the coat protein of Pelargonium flower break virus: distinctions from closely related suppressors

Viral-derived double-stranded RNAs (dsRNAs) activate RNA silencing, generating small interfering RNAs (siRNAs) which are incorporated into an RNA-induced silencing complex (RISC) that promotes homology-dependent degradation of cognate RNAs. To counteract this, plant viruses express RNA silencing suppressors. Here, we show that the coat protein (CP) of Pelargonium flower break virus (PFBV), a member of the genus Carmovirus, is able to efficiently inhibit RNA silencing. Interestingly, PFBV CP blocked both sense RNA- and dsRNA-triggered RNA silencing and did not preclude generation of siRNAs, which is in contrast with the abilities that have been reported for other carmoviral CPs. We have also found that PFBV CP can bind siRNAs and that this ability correlates with silencing suppression activity and enhancement of potato virus X pathogenicity. Collectively, the results indicate that PFBV CP inhibits RNA silencing by sequestering siRNAs and preventing their incorporation into a RISC, thus behaving similarly to unrelated viral suppressors but dissimilarly to orthologous ones.

Complete nucleotide sequence of Nootka lupine vein-clearing virus

The complete genome sequence of Nootka lupine vein-clearing virus (NLVCV) was determined to be 4,172 nucleotides in length containing four open reading frames (ORFs) with a similar genetic organization of virus species in the genus Carmovirus, family Tombusviridae. The order and gene product size, starting from the 5'-proximal ORF consisted of: (1) polymerase/replicase gene, ORF1 (p27) and ORF1RT (readthrough) (p87), (2) movement proteins ORF2 (p7) and ORF3 (p9), and, (3) the 3'-proximal coat protein ORF4, (p37). The genomic 5'- and 3'-proximal termini contained a short (59 nt) and a relatively longer 405 nt untranslated region, respectively. The longer replicase gene product contained the GDD motif common to RNA-dependent RNA polymerases. Phylogenetically, NLVCV formed a subgroup with the following four carmoviruses when separately comparing the amino acids of the coat protein or replicase protein: Angelonia flower break virus (AnFBV), Carnation mottle virus (CarMV), Pelargonium flower break virus (PFBV), and Saguaro cactus virus (SgCV). Whole genome nucleotide analysis (percent identities) among the carmoviruses with NLVCV suggested a similar pattern. The species demarcation criteria in the genus Carmovirus for the amino acid sequence identity of the polymerase (<52%) and coat (<41%) protein genes restricted NLVCV as a distinct species, and instead, placed it as a tentative strain of CarMV, PFBV, or SgCV when both the polymerase and CP were used as the determining factors. In contrast, the species criteria that included different host ranges with no overlap and lack of serology relatedness between NLVCV and the carmoviruses, suggested that NLVCV was a distinct species. The relatively low cutoff percentages allowed for the polymerase and CP genes to dictate the inclusion/exclusion of a distinct carmovirus species should be reevaluated. Therefore, at this time we have concluded that NLVCV should be classified as a tentative new species in the genus Carmovirus, family Tombusviridae.

Angelonia flower mottle, a new disease of Angelonia angustifolia caused by a hitherto unknown carmovirus

), a Central Americanornamental, is receiving increasing attention in the USA and Europe forits attractive flowers. In Germany, a number of accessions from com-mercial companies, subjected to evaluation for varietal release, exhibitedconspicuous flower mottling symptoms, suggestive of virus infection.Carmovirus-like isometric particles (approx. 30 nm in diameter)were detected in plants with symptoms by electron microscopy. Sapinoculation studies revealed that the virus is mechanically transmissibleto Angelonia plants, leading to mottling symptoms on the petals offlowers. When inoculated to Nicotiana hesperis, N. occidentalis, N. gluti-nosa and N. clevelandii , latent infections confined to the site of inoculationsite resulted. An antiserum (DSMZ AS-858) raised against a purifiedvirus preparation, reacted specifically with homologous antigen in westernblot analyses. Virus was detected in flowers and in symptomless leaves.Immunoelectron microscopic decoration tests showed no detectablecross-reaction to several other carmoviruses.Clones of the complete coat protein gene were obtained by RT-PCRfrom viral RNA isolated from purified particles. Sequence analysis of the1053 nucleotide coat protein gene (EMBL Acc. No. AM050058; encodinga predicted 351 amino acid product) confirmed this virus to be a typical,but distinct, carmovirus with 49 and 48% nucleotide sequence identity(37% and 35 % amino acid sequence identity) to Pelargonium flowerbreak virus and Carnation mottle virus respectively.During cultivation of Angelonia, transient mild chlorotic spots appearedon the leaves of infected and non-infected plants, which are suspected tobe stress-induced. Since the flower mottle symptoms consistently correlatewith infection by the carmovirus, it is proposed naming this tentative newcarmovirus species, Angelonia flower mottle virus.*E-mail: s.winter@bba.de Accepted 5 October 2005 at www.bspp.org.uk/ndr where figures relating to this paper can be viewed. © 2006 The Authors Journal compilation © 2006 BSPP

Biological and Molecular Characterization of a Novel Carmovirus Isolated from Angelonia

ABSTRACT A new carmovirus was isolated from Angelonia plants (Angelonia angustifolia), with flower break and mild foliar symptoms, grown in the United States and Israel. The virus, for which the name Angelonia flower break virus (AnFBV) is proposed, has isometric particles, approximately 30 nm in diameter. The experimental host range was limited to Nicotiana species, Schizanthus pinnatus, Myosotis sylvatica, Phlox drummondii, and Digitalis purpurea. Virions were isolated from systemically infected N. benthamiana leaves, and directly from naturally infected Angelonia leaves, using typical carmovirus protocols. Koch's postulates were completed by mechanical inoculation of uninfected Angelonia seedlings with purified virions. Isometric particles were observed in leaf dips and virion preparations from both Angelonia and N. benthamiana, and in thin sections of Angelonia flower tissue by electron microscopy. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis of dissociated purified virus preparations, a major protein component with a molecular mass of 38 kDa was observed. Virion preparations were used to produce virus-specific polyclonal antisera in both Israel and the United States. The antisera did not react with Pelargonium flower break virus (PFBV), Carnation mottle virus (CarMV), or Saguaro cactus virus (SgCV) by either enzyme-linked immunosorbent assay or immunoblotting. In reciprocal tests, antisera against PFBV, CarMV, and SgCV reacted only with the homologous viruses. The complete nucleotide sequence of a Florida isolate of AnFBV and the coat protein (CP) gene sequences of Israeli and Maryland isolates were determined. The genomic RNA is 3,964 nucleotides and contains four open reading frames arranged in a manner typical of carmoviruses. The AnFBV CP is most closely related to PFBV, whereas the AnFBV replicase is most closely related to PFBV, CarMV, and SgCV. Particle morphology, serological properties, genome organization, and phylogenetic analysis are all consistent with assignment of AnFBV to the genus Carmovirus.

INFECTIVITY OF IN VITRO TRANSCRIPTS FROM A FULL-LENGTH cDNA CLONE OF PELARGONIUM FLOWER BREAK VIRUS IN AN EXPERIMENTAL AND A NATURAL HOST

To carry out reverse genetics experiments with Pelargonium flower break virus (PFBV), one of the most important viruses affecting Pelargonium spp., cDNA clones were constructed from which RNA transcripts can be synthesized in vitro. Two populations of overlapping RT-PCR products encompassing the complete PFBV RNA were ligated into pUC18 with the T7 RNA polymerase promoter fused to the 5’ extremity of the viral cDNA. The RNA transcripts derived from one of the resulting clones were infectious when mechanically inoculated to the experimental host Chenopodium quinoa and to the natural host P. zonale inducing local and systemic infections, respectively. The sequence of the infectious cDNA was almost 98% identical to that determined previously for a Spanish isolate of PFBV. This is the first description of a biologically active PFBV cDNA clone, an essential tool for detailed analyses of the viral genome.

High incidence of Pelargonium line pattern virus infecting asymptomatic Pelargonium spp. in Spain

Geraniums (Pelargonium spp.) are traditional ornamental plants largely cultivated in Europe and northern America. Vegetative propagation makes them prone to viral infections, which have detrimental effects on crop production and quality. Asymptomatic samples collected in Spain were tested for a range of viruses using ELISA. The tobamovirus, Tobacco mosaic virus (TMV), the cucumovirus, Cucumber mosaic virus (CMV), and several viruses in the family Tombusviridae, namely, Pelargonium line pattern virus (PLPV), Pelargonium flower break virus (PFBV), and Pelargonium leaf curl virus (PLCV), were detected either singly or in combination in 59.2% of 800 samples. PLPV and PFBV infections were confirmed by dot-blot hybridisation. The most relevant viral infection found on Spanish asymptomatic geraniums was by Pelargonium line pattern virus (PLPV). Symptoms did not develop for 3 years on most of the PLPV infected geranium plants under greenhouse conditions.

Development of a Non-radioactive Dot-blot Hybridisation Assay for the Detection of Pelargonium Flower Break Virus and Pelargonium line Pattern Virus

The ornamental geranium, Pelargonium×hortorum Bailey, is a traditional ornamental plant widely cultivated in Europe and Northern America. Vegetative propagation facilitates rapid spread of viral infections which have detrimental effects on the production and the quality of the crop. A non-radioactive nucleic acid hybridisation method was developed for detection of Pelargonium flower break virus (PFBV) and Pelargonium line pattern virus (PLPV) in infected host plants. This method was significantly more sensitive than the conventional ELISA test when using either purified viral preparations or crude plant extracts. The distribution of the viruses was studied by means of the non-isotopic hybridisation technique. The results indicated that the petioles and the apical blade regions of fully expanded leaves were the best source of test material. The hybridisation procedure enables the detection of PFBV and PLPV in a single assay, and its simplicity allows its application to routine large-scale indexing.

Complete nucleotide sequence and genome organization of Pelargonium flower break virus.

The complete nucleotide sequence of Pelargonium flower break virus (PFBV) has been determined. The genomic RNA is 3923 nucleotides (nt) long and contains five open reading frames (ORFs). The 5'-proximal ORF encodes a 27 kDa protein (p27) and terminates with an amber codon which may be read-through into an in-frame p56 ORF to generate a 86 kDa protein (p86) containing the viral RNA dependent-RNA polymerase motifs. Two small ORFs, located in the central part of the viral genome, encode polypeptides of 7 (p7) and 12 kDa (p12), respectively, which are very likely involved in virus movement. Interestingly, p12 presents a leucine zipper motif that has not been previously reported in related proteins. The 3'-proximal ORF encodes a 37 kDa capsid protein (CP). The p12 ORF is in-frame with the p86 ORF and a double read-through protein of 99 kDa (p99) may be produced. Amino acid sequence comparisons revealed that the proteins encoded by ORFs 2, 3 and 4 are more similar to the corresponding gene products of Carnation mottle virus than to those of other carmoviruses, whereas the p27 and the CP show higher identity with the equivalent proteins of Saguaro cactus virus. Phylogenetic analysis conducted with the different viral products confirmed the assignment of PFBV to the genus Carmovirus.

Characterization and in vitro translation analysis of pelargonium flower break virus.

Pelargonium flower break virus (PFBV) is one of the common viruses in the glasshouses of Western Europe and has been assigned to the genus Carmovirus. A Spanish isolate obtained from nursery-grown Pelargonium zonale plants (PFBV-m) has been characterized. The molecular weight of genomic RNA and coat protein of PFBV-m were determined to be 1.36 x 10(6) (corresponding to approximately 4 kb) and 36,000, respectively. Only genomic-size RNA was encapsidated in PFBV virions; making necessary to purify double-stranded RNA from infected tissue in order to detect putative PFBV subgenomic RNAs. PFBV RNA directed the synthesis of a major polypeptide of 34 kDa and three other relevant polypeptides of estimated sizes 88-90 kDa, 42 kDa and 35-36 kDa. Antisera specific to PFBV immunoprecipitated the in vitro translated 35-36 kDa polypeptide indicating that this polypeptide is the PFBV coat protein. The PFBV in vitro translation pattern was very similar to that of CarMV although the relative levels of translated coat protein differed dramatically between the two viruses, most probably due to the lack of encapsidation of subgenomic PFBV. In vitro translation studies with a different biological clone obtained from the same PFBV-m isolate revealed a prominent additional polypeptide which is postulated to be a truncation of the 5' proximal ORF.