Autophagy Inhibits Intercellular Transport of Citrus Leaf Blotch Virus by Targeting Viral Movement Protein.

Autophagy is an evolutionarily conserved cellular-degradation mechanism implicated in antiviral defense in plants. Studies have shown that autophagy suppresses virus accumulation in cells; however, it has not been reported to specifically inhibit viral spread in plants. This study demonstrated that infection with citrus leaf blotch virus (CLBV; genus Citrivirus, family Betaflexiviridae) activated autophagy in Nicotiana benthamiana plants as indicated by the increase of autophagosome formation. Impairment of autophagy through silencing of N. benthamiana autophagy-related gene 5 (NbATG5) and NbATG7 enhanced cell-to-cell and systemic movement of CLBV; however, it did not affect CLBV accumulation when the systemic infection had been fully established. Treatment using an autophagy inhibitor or silencing of NbATG5 and NbATG7 revealed that transiently expressed movement protein (MP), but not coat protein, of CLBV was targeted by selective autophagy for degradation. Moreover, we identified that CLBV MP directly interacted with NbATG8C1 and NbATG8i, the isoforms of autophagy-related protein 8 (ATG8), which are key factors that usually bind cargo receptors for selective autophagy. Our results present a novel example in which autophagy specifically targets a viral MP to limit the intercellular spread of the virus in plants.

Partial biological and molecular characterization of a novel citrivirus from Nandina domestica.

We report the complete genome sequence of a novel virus isolated from Nandina domestica 'Firepower' in Auckland, New Zealand. It was mechanically transmitted to Nicotiana species, although all of these infections were symptomless. The complete genome of the new virus is 8892 nucleotides (nt) long, excluding the 3' poly(A) tail, contains three open reading frames (ORF), and is most closely related to citrus leaf blotch virus (CLBV) Actinidia isolate (CLBV-Act; 72% nt sequence identity), a member of the genus Citrivirus. Replicase and coat proteins, encoded by genome ORFs 1 and 3 respectively, shared 81-83% and 76-79% amino acid (aa) sequence identity, respectively, with CLBV-Act. Computer-based analysis suggests that this novel virus is the result of recombination between CLBV-Act and an unknown virus, highlighting the importance of this phenomenon for betaflexivirus evolution.

ICTV Virus Taxonomy Profile: Alphaflexiviridae.

The family Alphaflexiviridae includes viruses with flexuous filamentous virions that are 470-800 nm in length and 12-13 nm in diameter. Alphaflexiviruses have a single-stranded, positive-sense RNA genome of 5.5-9 kb. They infect plants and plant-infecting fungi. They share a distinct lineage of alphavirus-like replication proteins that is unusual in lacking any recognized protease domain. With a single exception, cell-to-cell and long-distance movement is facilitated by triple gene block proteins in plant-infecting genera. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Alphaflexiviridae, which is available at www.ictv.global/report/alphaflexiviridae.

Novel Divavirus (the family Betaflexiviridae) and Mitovirus (the family Narnaviridae) species identified in basil (Ocimum basilicum).

Transcriptome data obtained from a plant sample often contain a large number of reads that are derived from associated RNA virus genomes that were co-isolated during RNA preparation. These virus-derived reads can be assembled into a novel plant RNA genome sequence. Here, a basil (Ocimum basilicum) transcriptome dataset was analyzed to identify two new RNA viruses, which were named Ocimum basilicum RNA virus 1 (ObRV1) and Ocimum basilicum RNA virus 2 (ObRV2). A phylogenetic analysis of the ObRV1 RNA-dependent RNA polymerase (RdRp) motif indicated that ObRV1 is a novel species of the genus Divavirus of the family Betaflexiviridae. ObRV1 is the fourth divavirus species to be identified. The ObRV2 RdRp motif showed sequence similarity to viruses of the genus Mitovirus of the family Narnaviridae, which infect fungal mitochondria. Although most of the known mitoviruses do not produce a functional RdRp using the plant mitochondrial genetic code, the ObRV2 encodes a full-length RdRp using both the fungal and plant mitochondrial genetic codes.

Dissecting interplays between Vitis vinifera L. and grapevine virus B (GVB) under field conditions.

Plant virus infections are often difficult to characterize as they result from a complex molecular and physiological interplay between a pathogen and its host. In this study, the impact of the phloem-limited grapevine virus B (GVB) on the Vitis vinifera L. wine-red cultivar Albarossa was analysed under field conditions. Trials were carried out over two growing seasons by combining agronomic, molecular, biochemical and ecophysiological approaches. The data showed that GVB did not induce macroscopic symptoms on 'Albarossa', but affected the ecophysiological performances of vines in terms of assimilation rates, particularly at the end of the season, without compromising yield and vigour. In GVB-infected plants, the accumulation of soluble carbohydrates in the leaves and transcriptional changes in sugar- and photosynthetic-related genes seemed to trigger defence responses similar to those observed in plants infected by phytoplasmas, although to a lesser extent. In addition, GVB activated berry secondary metabolism. In particular, total anthocyanins and their acetylated forms accumulated at higher levels in GVB-infected than in GVB-free berries, consistent with the expression profiles of the related biosynthetic genes. These results contribute to improve our understanding of the multifaceted grapevine-virus interaction.

A complex virome unveiled by deep sequencing analysis of RNAs from a French Pinot Noir grapevine exhibiting strong leafroll symptoms.

We have characterized the virome of a grapevine Pinot Noir accession (P70) that displayed, over the year, very stable and strong leafroll symptoms. For this, we have used two extraction methods (dsRNA and total RNA) coupled with the high throughput sequencing (HTS) Illumina technique. While a great disparity in viral sequences were observed, both approaches gave similar results, revealing a very complex infection status. Five virus and viroid isolates [Grapevine leafroll-associated viruse-1 (GLRaV-1), Grapevine virus A (GVA), Grapevine rupestris stem pitting-associated virus (GRSPaV), Hop stunt viroid (HSVd) and Grapevine yellow speckle viroid 1 (GYSVd1)] were detected in P70 with a grand total of eleven variants being identified and de novo assembled. A comparison between both extraction methods regarding their power to detect viruses and the ease of genome assembly is also provided.

Localization and subcellular association of Grapevine Pinot Gris Virus in grapevine leaf tissues.

Despite the increasing impact of Grapevine Pinot gris disease (GPG-disease) worldwide, etiology about this disorder is still uncertain. The presence of the putative causal agent, the Grapevine Pinot Gris Virus (GPGV), has been reported in symptomatic grapevines (presenting stunting, chlorotic mottling, and leaf deformation) as well as in symptom-free plants. Moreover, information on virus localization in grapevine tissues and virus-plant interactions at the cytological level is missing at all. Ultrastructural and cytochemical investigations were undertaken to detect virus particles and the associated cytopathic effects in field-grown grapevine showing different symptom severity. Asymptomatic greenhouse-grown grapevines, which tested negative for GPGV by real time RT-PCR, were sampled as controls. Multiplex real-time RT-PCR and ELISA tests excluded the presence of viruses included in the Italian certification program both in field-grown and greenhouse-grown grapevines. Conversely, evidence was found for ubiquitous presence of Grapevine Rupestris Stem Pitting-associated Virus (GRSPaV), Hop Stunt Viroid (HSVd), and Grapevine Yellow Speckle Viroid 1 (GYSVd-1) in both plant groups. Moreover, in every field-grown grapevine, GPGV was detected by real-time RT-PCR. Ultrastructural observations and immunogold labelling assays showed filamentous flexuous viruses in the bundle sheath cells, often located inside membrane-bound organelles. No cytological differences were observed among field-grown grapevine samples showing different symptom severity. GPGV localization and associated ultrastructural modifications are reported and discussed, in the perspective of assisting management and control of the disease.

Combining Thermotherapy with Cryotherapy for Efficient Eradication of Apple stem grooving virus from Infected In-vitro-cultured Apple Shoots.

Apple stem grooving virus (ASGV), a difficult-to-eradicate virus from apple propagative materials, causes serious damage to apple production. The use of virus-free plants has been and is an effective strategy for control of plant viral diseases. This study aimed to eradicate ASGV from virus-infected in-vitro-cultured shoots of four apple cultivars and one rootstock by combining thermotherapy with cryotherapy. In vitro stock shoots infected with ASGV were thermo-treated using an alternating temperature of 36°C (day) and 32°C (night). Shoot tips were excised from the treated stock shoots and subjected to cryotherapy. Results showed that, although thermotherapy did not influence shoot survival rates, it reduced shoot growth and proliferation of in vitro shoots. Shoot regrowth rates decreased while virus eradication frequencies increased in cryo-treated shoot tips as time durations of thermotherapy increased from 0 to 6 weeks. Shoot regrowth and frequency of virus eradication were positively and negatively correlated, respectively, with the size of shoot tips. The protocol established here yielded shoot regrowth rates and virus eradication frequencies of 33 to 76% and 30 to 100%, respectively, in the four apple cultivars and one rootstock. Thermotherapy altered virus distribution patterns, subsequently resulting in production of a larger virus-free area in the thermo-treated shoot tips. Many cells in the top layers of apical dome and some cells in the youngest leaf primordia survived in cryo-treated shoot tips; these cells were most likely free of virus infection. Thus, plants regenerated from the procedure of combining thermotherapy with cryotherapy were free of ASGV, as judged by reverse-transcription polymerase chain reaction. To the best of our knowledge, this is the widest-spectrum technique reported thus far for the production of ASGV-free plants and provides a novel biotechnology for the production of virus-free plants in Malus spp.

Effect of repeated Ribavirin treatment on grapevine viruses.

The effect of Ribavirin treatment for the chemotherapy of several grapevine viruses was evaluated. Four grapevine cultivars were repeatedly treated with Ribavirin in two different concentrations and with three different lengths of treatment. Repeating the Ribavirin treatment always had a significant effect on the number of healthy grapevine plants obtained. Ribavirin concentration and length of exposure showed a significant difference in sanitation of the Grapevine rupestris stem pitting-associated virus. During sanitation of the Grapevine Pinot gris virus and Grapevine fleck virus, those two factors did not show significant differences in the elimination of grapevine viruses.

Transmission of Grapevine virus A and Grapevine leafroll-associated virus 1 and 3 by Heliococcus bohemicus (Hemiptera: Pseudococcidae) Nymphs From Plants With Mixed Infections.

Mealybugs (Hemiptera: Pseudococcidae) represent a serious threat for viticulture as vectors of phloem-restricted viruses associated with the grapevine rugose wood and leafroll diseases. Heliococcus bohemicus (Sulc) is known to be involved in the spread of these two viral diseases, being a vector of the Grapevine virus A (GVA) and the Grapevine leafroll-associated virus 1 and 3 (GLRaV-1 and GLRaV-3). This study investigated the acquisition and transmission efficiency of H. bohemicus fed on mixed-infected plants. Nymphs were field-collected onto GVA, GLRaV-1, and GLRaV-3 multiple-infected grapevines in two vineyards in North-Western Italy, and were used in transmission experiments under controlled conditions. Even if most of the collected nymphs were positive to at least one virus, transmission occurred only to a low number of test grapevines. The transmission frequency of GLRaV-3 was the highest, whereas GVA was transmitted to few test plants. The transmission of multiple viruses occurred at low rates, and nymphs that acquired all the three viruses then failed to transmit them together. Statistical analyses showed that the three viruses were independently acquired and transmitted by H. bohemicus and neither synergistic nor antagonistic interactions occurred among them. GVA and GLRaVs transmission efficiencies by H. bohemicus were lower than those reported for other mealybug vectors. This finding is consistent with the slow spread of leafroll and rugose wood diseases observed in Northern Italy, where H. bohemicus is the predominant vector species.

Identification and characterization of microRNAs from in vitro-grown pear shoots infected with Apple stem grooving virus in response to high temperature using small RNA sequencing.

BACKGROUND: MicroRNAs (miRNAs) have functions in diverse biological processes such as growth, signal transduction, disease resistance, and stress responses in plants. Thermotherapy is an effective approach for elimination of viruses from fruit trees. However, the role of miRNAs in this process remains elusive. Previously, we showed that high temperature treatment reduces the titers of Apple stem grooving virus (ASGV) from the tips of in vitro-grown Pyrus pyrifolia plants. In this study, we identified high temperature-altered pear miRNAs using the next generation sequencing technology, and futher molecularly characterized miRNA-mediated regulaton of target gene expression in the meristem tip and base tissues of in vitro-grown, ASGV-infected pear shoots under different temperatures. RESULTS: Using in vitro-grown P. pyrifolia shoot meristem tips infected with ASGV, a total of 22,592,997 and 20,411,254 clean reads were obtained from Illumina high-throughput sequencing of small RNA libraries at 24 °C and 37 °C, respectively. We identified 149 conserved and 141 novel miRNAs. Seven conserved miRNAs and 77 novel miRNAs were differentially expressed at different temperatures. Target genes for differentially expressed known and novel miRNAs were predicted and functionally annotated. Gene Ontology (GO) analysis showed that high-ranking miRNA target genes were involved in metabolic processes, responses to stress, and signaling, indicating that these high temperature-responsive miRNAs have functions in diverse gene regulatory networks. Spatial expression patterns of the miRNAs and their target genes were found to be expressed in shoot tip and base tissues by qRT-PCR. In addition, high temperature reduced viral titers in the shoot meristem tip, while negatively regulated miRNA-mediated target genes related to resistance disease defense and hormone signal transduction pathway were up-regulated in the P. pyrifolia shoot tip in response to high temperature. These results suggested that miRNAs may have important functions in the high temperature-dependent decrease of ASGV titer in in vitro-grown pear shoots. CONCLUSIONS: This is the first report of miRNAs differentially expressed at 24 °C and 37 °C in the meristem tip of pear shoots infected with ASGV. The results of this study provide valuable information for further exploration of the function of high temperature-altered miRNAs in suppressing viral infections in pear and other fruit trees.

Subcellular localization and membrane association of the replicase protein of grapevine rupestris stem pitting-associated virus, family Betaflexiviridae.

As a member of the newly established Betaflexiviridae family, grapevine rupestris stem pitting-associated virus (GRSPaV) has an RNA genome containing five ORFs. ORF1 encodes a putative replicase polyprotein typical of the alphavirus superfamily of positive-strand ssRNA viruses. Several viruses of this superfamily have been demonstrated to replicate in structures designated viral replication complexes associated with intracellular membranes. However, structure and cellular localization of the replicase complex have not been studied for members of Betaflexiviridae, a family of mostly woody plant viruses. As a first step towards the elucidation of the replication complex of GRSPaV, we investigated the subcellular localization of full-length and truncated versions of its replicase polyprotein via fluorescent tagging, followed by fluorescence microscopy. We found that the replicase polyprotein formed distinctive punctate bodies in both Nicotiana benthamiana leaf cells and tobacco protoplasts. We further mapped a region of 76 amino acids in the methyl-transferase domain responsible for the formation of these punctate structures. The punctate structures are distributed in close proximity to the endoplasmic reticulum network. Membrane flotation and biochemical analyses demonstrate that the N-terminal region responsible for punctate structure formation associated with cellular membrane is likely through an amphipathic α helix serving as an in-plane anchor. The identity of this membrane is yet to be determined. This is, to our knowledge, the first report on the localization and membrane association of the replicase proteins of a member of the family Betaflexiviridae.

The global trade in fresh produce and the vagility of plant viruses: a case study in garlic.

As cuisine becomes globalized, large volumes of fresh produce are traded internationally. The potential exists for pathogens infecting fresh produce to hitchhike to new locations and perhaps to establish there. It is difficult to identify them using traditional methods if pathogens are novel, scarce, and/or unexpected. In an attempt to overcome this limitation, we used high-throughput sequencing technology as a means of detecting all RNA viruses infecting garlic (Allium sativum L.) bulbs imported into Australia from China, the USA, Mexico, Argentina and Spain, and those growing in Australia. Bulbs tested were grown over multiple vegetative generations and all were stably infected with one or more viruses, including two species not previously recorded in Australia. Present in various combinations from 10 garlic bulbs were 41 virus isolates representing potyviruses (Onion yellow dwarf virus, Leek yellow stripe virus), carlaviruses (Shallot latent virus, Garlic common latent virus) and allexiviruses (Garlic virus A, B, C, D, and X), for which 19 complete and 22 partial genome sequences were obtained, including the first complete genome sequences of two isolates of GarVD. The most genetically distinct isolates of GarVA and GarVX described so far were identified from Mexico and Argentina, and possible scenarios explaining this are presented. The complete genome sequence of an isolate of the potexvirus Asparagus virus 3 (AV3) was obtained in Australia from wild garlic (A. vineale L.), a naturalized weed. This is first time AV3 has been identified from wild garlic and the first time it has been identified beyond China and Japan. The need for routine generic diagnosis and appropriate legislation to address the risks to primary production and wild plant communities from pathogens spread through the international trade in fresh produce is discussed.

Co-evolution between Grapevine rupestris stem pitting-associated virus and Vitis vinifera L. leads to decreased defence responses and increased transcription of genes related to photosynthesis.

Grapevine rupestris stem pitting-associated virus (GRSPaV) is a widespread virus infecting Vitis spp. Although it has established a compatible viral interaction in Vitis vinifera without the development of phenotypic alterations, it can occur as distinct variants that show different symptoms in diverse Vitis species. The changes induced by GRSPaV in V. vinifera cv 'Bosco', an Italian white grape variety, were investigated by combining agronomic, physiological, and molecular approaches, in order to provide comprehensive information about the global effects of GRSPaV. In two years, this virus caused a moderate decrease in physiological efficiency, yield performance, and sugar content in berries associated with several transcriptomic alterations. Transcript profiles were analysed by a microarray technique in petiole, leaf, and berry samples collected at véraison and by real-time RT-PCR in a time course carried out at five grapevine developmental stages. Global gene expression analyses showed that transcriptomic changes were highly variable among the different organs and the different phenological phases. GRSPaV triggers some unique responses in the grapevine at véraison, never reported before for other plant-virus interactions. These responses include an increase in transcripts involved in photosynthesis and CO(2) fixation, a moderate reduction in the photosynthesis rate and some defence mechanisms, and an overlap with responses to water and salinity stresses. It is hypothesized that the long co-existence of grapevine and GRSPaV has resulted in the evolution of a form of mutual adaptation between the virus and its host. This study contributes to elucidating alternative mechanisms used by infected plants to contend with viruses.

Infection of capilloviruses requires subgenomic RNAs whose transcription is controlled by promoter-like sequences conserved among flexiviruses.

The first open-reading frame (ORF) of apple stem grooving virus (ASGV), of the genus Capillovirus, encodes an apparently chimeric polyprotein containing conserved regions for replicase (Rep) and coat protein (CP). However, our previous study revealed that ASGV mutants with distinct and discontinuous Rep- and CP-coding regions successfully infect plants, indicating that CP expressed via a subgenomic RNA (sgRNA) is sufficient for viability of the virus. Here we identified a transcription start site of the CP sgRNA and revealed that CP translated from the sgRNA is essential for ASGV infection. We mapped the transcription start sites of both the CP and the movement protein (MP) sgRNAs of ASGV and found a hexanucleotide motif, UUAGGU, conserved upstream from both sgRNA transcription start sites. Mutational analysis of the putative CP initiation codon and of the UUAGGU sequence upstream from the transcription start site of CP sgRNA demonstrated their importance for ASGV accumulation. Our results also demonstrated that potato virus T (PVT), an unassigned species closely related to ASGV, produces two sgRNAs putatively deployed for the CP and MP expression and that the same hexanucleotide motif as found in ASGV is located upstream from the transcription start sites of both sgRNAs. This motif, which constituted putative core elements of the sgRNA promoter, is broadly conserved among viruses in the families Alphaflexiviridae and Betaflexiviridae, suggesting that the gene expression strategy of the viruses in both families has been conserved throughout evolution.

The ORF3-encoded proteins of vitiviruses GVA and GVB induce tubule-like and punctate structures during virus infection and localize to the plasmodesmata.

The genomic RNA of vitiviruses contains 5 open reading frames (ORF). ORF3 encodes a protein to which the function of a movement protein (MP) was assigned, based on sequence homology with other viral proteins. The aim of the research described in this paper was to gain further insight in distribution profile of the ORF3 product encoded by the vitiviruses Grapevine virus A (GVA) and Grapevine virus B (GVB). Expression of the GVA MP-GFP fusion protein via the virus genome in Nicotiana benthamiana leaves resulted in the formation of irregular spots and fibrous network structures on the outermost periphery of epidermal cells. Expression of GVA MP-GFP and GVB MP-GFP was involved in the formation of the tubule-like and punctate structures on the periphery of N. benthamiana and Vitis vinifera protoplasts. Co-expression of the GVA MP-GFP and GVA MP-RFP in protoplasts resulted in co-localization of these proteins into the same punctate structures, indicating that the MP is not accumulated randomly onto the cell surface, but targeted to particular sites at the cell periphery, where punctate and tubule-like structures are likely formed. With the use of cytoskeleton and secretory pathway inhibitors, we showed that the cytoskeletal elements are not likely to be involved in targeting of the MP-GFP to the punctate cellular structures. In addition to MP, a functional coat protein was found to be essential for virus spread within inoculated leaves.

The grapevine-infecting vitiviruses, with particular reference to grapevine virus A.

A number of vitiviruses infect grapevine, arguably the most important fruit crop, and the host from which they derive their genus name. In contrast to most grapevine viruses, the etiological role of these viruses is unclear, albeit that they are associated with several well-known--and a number of emerging--diseases of grapevine. Here, we review the genus Vitivirus, with special reference to its most omnipresent member, grapevine virus A. We discuss the latest taxonomic status of the genus, as well as the genome and genomic organisation, replication mechanism, and genetic variability of GVA, and we also present the latest research progress with vitivirus-based vectors; the identification of a new vitivirus, GVE, the discovery of VIGG, a unique GVA-induced host protein, the molecular characterisation of hitherto unknown or puzzling genetic elements in the GVA genome, and the latest developments in vitivirus diagnostics.

The capsid protein of Grapevine rupestris stem pitting-associated virus contains a typical nuclear localization signal and targets to the nucleus.

Grapevine rupestris stem pitting-associated virus (GRSPaV) is a positive strand, ssRNA virus of the genus Foveavirus (family Betaflexiviridae; order Tymovirales). GRSPaV is distributed in table and wine grapes worldwide and comprises a large family of sequence variants. As a newly discovered virus, mechanisms of virus replication and movement of GRSPaV have not been elucidated. We recently revealed the subcellular localization of the proteins encoded by the triple gene block of GRSPaV (Rebelo et al., 2008). However, the subcellular localization and interaction of its capsid protein (CP) have not been explored. We report here that GRSPaV CP contains a nuclear localization signal "KRKR" near its N-terminus, which is conserved among all five strains whose genomes are completely sequenced. Similar sequences were also detected in the CP of two other viruses of the same family: African oil palm ringspot virus and Cherry green ring mottle virus. Using fluorescent protein tagging, we demonstrate that the CP targets to the nucleus in tobacco protoplasts. Mutation to this nuclear localization signal abolished the nuclear localization. Using bi-molecular fluorescence complementation, we show that the capsid protein of GRSPaV engages in homologous interaction. To our knowledge, this is the first report on the nuclear localization of a CP encoded by a RNA plant virus.

Rugose wood-associated viruses do not appear to be involved in Shiraz (Syrah) decline in South Africa.

The presence of rugose-wood-associated viruses of the genera Foveavirus and Vitivirus in the family Betaflexiviridae was investigated in various clones of own-rooted and grafted Vitis vinifera cv. Shiraz that were affected, or not, by Shiraz decline, and in rootstocks. RT nested-PCR amplification of double-stranded RNA using degenerate primers for the simultaneous detection of foveaviruses and vitiviruses (Dovas CI, Katis NI in J Virol Meth 170:99-106, 2003), cloning of DNA amplicons, SSCP analysis of clones, sequencing and computer-assisted analysis of sequences was used to characterize viral genetic variability. A total of 1,137 clones were analysed by SSCP, and, of those, 371 clones were sequenced. The results revealed that variants of five molecular groups belonging to the species Grapevine rupestris stem pitting-associated virus (GRSPaV), including highly divergent variants related to strain SY (Lima MF et al. in Arch Virol 151:1889-1894, 2006) were present in plants of various clones of Shiraz regardless of their Shiraz decline status, and in rootstocks. Grapevine virus A (GVA) and grapevine virus B (GVB) were detected in a relatively small number of plants. This study suggested no involvement of GRSPaV, GVA or GVB in Shiraz decline.

Pseudo-polyprotein translated from the full-length ORF1 of capillovirus is important for pathogenicity, but a truncated ORF1 protein without variable and CP regions is sufficient for replication.

The first open-reading frame (ORF) of the genus Capillovirus encodes an apparently chimeric polyprotein containing conserved regions for replicase (Rep) and coat protein (CP), while other viruses in the family Flexiviridae have separate ORFs encoding these proteins. To investigate the role of the full-length ORF1 polyprotein of capillovirus, we generated truncation mutants of ORF1 of apple stem grooving virus by inserting a termination codon into the variable region located between the putative Rep- and CP-coding regions. These mutants were capable of systemic infection, although their pathogenicity was attenuated. In vitro translation of ORF1 produced both the full-length polyprotein and the smaller Rep protein. The results of in vivo reporter assays suggested that the mechanism of this early termination is a ribosomal -1 frame-shift occurring downstream from the conserved Rep domains. The mechanism of capillovirus gene expression and the very close evolutionary relationship between the genera Capillovirus and Trichovirus are discussed.