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Bryce W. Falk - One of the best experts on this subject based on the ideXlab platform.
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using partially purified virions and the whitefly Bemisia tabaci
2016Co-Authors: Tongyan Tian, Luis Rubio, Hsin-hung Yeh, Brett Crawford, Bryce W. FalkAbstract:Virions of lettuce infectious yellows virus (LIYV; genus Crinivirus) were purified from LIYV-infected plants and their protein composition was analysed by SDS–PAGE and immunoblotting. Virion preparations contained the major capsid protein (CP), but the minor capsid protein (CPm), p59 and the HSP70 homologue were also identified by immunoblot analysis. Immunogold labelling analysis showed that CP constituted the majority of the LIYV virion capsid, but CPm was also part of the capsid and localized to one end of the virion, similar to the polar morphology seen for viruses in the genus Closterovirus. p59 and the HSP70 homologue were not detected on virions by immunogold labelling, but were always detected in virion preparations by immunoblot analysis. Purified LIYV virions were used for in vitro acquisition analysis with Bemisia tabaci whiteflies and were efficiently transmitted to plants. Infectivity neutralization analyses were done using antisera to the LIYV-encoded CP, CPm, p59 and HSP70 homologue. Only antiserum to the CPm effectively neutralized LIYV transmission by B. tabaci. These data suggest that the LIYV–B. tabaci transmission determinants are associated with purified virions, and that the LIYV virion structural protein CPm is involved in transmission by B. tabaci
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Population Structure and Genetic Diversity within California Citrus tristeza virus (CTV) Isolates
Virus Genes, 2000Co-Authors: Ping Kong, Luis Rubio, Marylou Polek, Bryce W. FalkAbstract:The Closterovirus, Citrus tristeza virus (CTV) is an aphid-borne RNA virus that is the causal agent of important worldwide economic losses in citrus. Biological and molecular variation has been observed for many CTV isolates. In this work we detected and analyzed sequence variants (haplotypes) within individual CTV isolates. We studied the population structure of five California CTV isolates by single strand conformation polymorphism (SSCP) analysis of four CTV genomic regions. Also, we estimated the genetic diversity within and between isolates by analysis of haplotype nucleotide sequences. Most CTV isolates were composed of a population of genetically related variants (haplotypes), one being predominant. However in one case, we found a high nucleotide divergence between haplotypes of the same isolate. Comparison of these haplotypes with those from other isolates suggests that some CTV isolates could have arisen as result of a mixed infection of two divergent isolates.
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specific inclusion bodies are associated with replication of lettuce infectious yellows virus rnas in nicotiana benthamiana protoplasts
Journal of General Virology, 1998Co-Authors: Vicente Medina, Tongyan Tian, Jacek Wierzchos, Bryce W. FalkAbstract:Nicotiana benthamiana mesophyll protoplasts, either mock-inoculated or inoculated using in vitro transcripts derived from lettuce infectious yellows virus (LIYV) RNA 1- and/or RNA 2-cloned cDNAs were analysed by transmission electron microscopy (TEM) and, in some cases, also by immunogold labelling. TEM revealed the main cytopathological effects of LIYV infections in N. benthamiana protoplasts infected with RNAs 1 and 2: (a) typical Closterovirus-induced (beet yellows virus-type) accumulations of vesiculated cytoplasmic membranes as inclusion bodies, sometimes with associated virions; (b) scattered aggregations of virions within the cytoplasm; and (c) electron-dense plasmalemma deposits. These were not seen in mock-inoculated protoplasts. Protoplasts inoculated only with LIYV RNA 1 contained vesiculated cytoplasmic inclusion bodies, but not virions or plasmalemma deposits. Thus, infection by only LIYV RNA 1 is sufficient to induce characteristic Closterovirus vesiculated cytoplasmic inclusion bodies. However, both LIYV RNAs 1 and 2 are needed for production of virions and plasmalemma deposits.
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tomato infectious chlorosis virus has a bipartite genome and induces phloem limited inclusions characteristic of the Closteroviruses
Phytopathology, 1996Co-Authors: Gail C Wisler, H.-y. Liu, James E Duffus, V A Klaassen, Bryce W. FalkAbstract:Tomato infectious chlorosis virus (TICV) is a newly described Closterovirus. Virions purified from TICV-infected plants contained two single-stranded (ss) RNAs, one of approximately 7,800 (RNA 1) and the other 7,400 (RNA 2) nucleotides. Double-stranded (ds) RNA analysis showed two prominent dsRNAs of approximately 7,800 and 7,400 bp, as well as several smaller dsRNAs. The TICV virion ssRNAs were used for cDNA cloning. Of 200 cDNA clones analyzed, 10 clones containing cDNAs ranging in size from about 900 to 1,500 nucleotides were used to generate digoxigenin-UTP-labeled transcripts. These transcripts hybridized with the TICV ssRNAs in Northern blot hybridization analyses and were used in dot-blot analyses to confirm TICV infection in several host plants including tomato, potato, Physalis wrightii, Nicotiana clevelandii, and artichoke. None of the probes reacted with any uninfected host plant tested or with plants infected with four other clostero- or clostero-like viruses including lettuce infectious yellows Closterovirus, lettuce chlorosis virus, cucurbit yellow stunting disorder virus, and beet pseudo yellows virus. Northern blot hybridization analyses using selected riboprobes showed no detectable homology between TICV dsRNA 1 and 2, or between subsets of smaller dsRNAs. Inclusion bodies, characteristic of the Closteroviruses, were consistently associated with the phloem of TICV-infected N. clevelandii.
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genome structure and phylogenetic analysis of lettuce infectious yellows virus a whitefly transmitted bipartite Closterovirus
Virology, 1995Co-Authors: Vicki Klaassen, Tongyan Tian, Eugene V. Koonin, Maury L Boeshore, Bryce W. FalkAbstract:We report the complete nucleotide sequences of lettuce infectious yellows virus (LIYV) RNAs 1 and 2. LIYV RNA 1 is 8118 nucleotides and includes three open reading frames (ORFs). Computer-assisted analysis of LIYV RNA 1 ORFs identified domains for a papain-like protease, methyltransferase (MTR), RNA helicase (HEL), and RNA-dependent RNA polymerase (RdRp). We suggest that the RdRp domain is expressed independently of the other replication-associated domains via a + 1 ribosomal frameshift. Amino acid sequences of the MTR, HEL, and RdRp show highly significant similarity to the homologous sequences from other Closteroviruses and lower similarity to the respective proteins of tobamoviruses, tobraviruses, hordeiviruses, bromoviruses, and furoviruses. LIYV RNA 2 is 7193 nucleotides and includes six ORFs. These ORFs include a gene array that is characteristic of the Closteroviruses: ORFs encoding a small membrane protein, a homologue of the HSP70 family of chaperone proteins, a protein whose function is unknown, the coat protein, and a diverged duplicate of the coat protein. LIYV is distinguished from the monopartite Closteroviruses in the following ways: its genome consists of two RNAs, the positions of the coat protein gene and its diverged duplicate are reversed, and LIYV includes ORFs that are unrelated to ORFs found in other Closteroviruses.
Valerian V. Dolja - One of the best experts on this subject based on the ideXlab platform.
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The Closterovirus-derived gene expression and RNA interference vectors as tools for research and plant biotechnology
Frontiers in Microbiology, 2013Co-Authors: Valerian V. Dolja, Eugene V. KooninAbstract:Important progress in understanding replication, interactions with host plants, and evolution of Closteroviruses enabled engineering of several vectors for gene expression and virus-induced gene silencing (VIGS). Due to the broad host range of Closteroviruses, these vectors expanded vector applicability to include important woody plants such as citrus and grapevine. Furthermore, large Closterovirus genomes offer genetic capacity and stability unrivaled by other plant viral vectors. These features provided immense opportunities for using Closterovirus vectors for the functional genomics studies and pathogen control in economically valuable crops. This review briefly summarizes advances in Closterovirus research during the last decade, explores the relationships between virus biology and vector design, and outlines the most promising directions for future application of Closterovirus vectors.
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genomic and biological analysis of grapevine leafroll associated virus 7 reveals a possible new genus within the family closteroviridae
Virus Research, 2012Co-Authors: Maher Al Rwahnih, Valerian V. Dolja, Eugene V. Koonin, Steve Daubert, Adib RowhaniAbstract:Deep sequencing analysis of an asymptomatic grapevine revealed a virome containing five RNA viruses and a viroid. Of these, Grapevine leafroll-associated virus 7 (GLRaV-7), an unassigned Closterovirus, was by far the most prominently represented sequence in the analysis. Graft-inoculation of the infection to another grape variety confirmed the lack of the leafroll disease symptoms, even though GLRaV-7 could be detected in the inoculated indicator plants. A 16,496 nucleotide-long genomic sequence of this virus was determined from the deep sequencing data. Its genome architecture and the sequences encoding its nine predicted proteins were compared with those of other Closteroviruses. The comparison revealed that two other viruses, Little cherry virus-1 and Cordyline virus-1 formed a well supported phylogenetic cluster with GLRaV-7.
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class viii myosins are required for plasmodesmatal localization of a Closterovirus hsp70 homolog
Journal of Virology, 2008Co-Authors: Dror Avisar, Alexey I Prokhnevsky, Valerian V. DoljaAbstract:The Hsp70 homolog (Hsp70h) of Beet yellows virus (BYV) functions in virion assembly and cell-to-cell movement and is autonomously targeted to plasmodesmata in association with the actomyosin motility system (A. I. Prokhnevsky, V. V. Peremyslov, and V. V. Dolja, J. Virol. 79:14421-14428, 2005). Myosins are a diverse category of molecular motors that possess a motor domain and a tail domain involved in cargo binding. Plants have two classes of myosins, VIII and XI, whose specific functions are poorly understood. We used dominant negative inhibition to identify myosins required for Hsp70h localization to plasmodesmata. Six full-length myosin cDNAs from the BYV host plant Nicotiana benthamiana were sequenced and shown to encode apparent orthologs of the Arabidopsis thaliana myosins VIII-1, VIII-2, VIII-B, XI-2, XI-F, and XI-K. We found that the ectopic expression of the tail domains of each of the class VIII, but not the class XI, myosins inhibited the plasmodesmatal localization of Hsp70h. In contrast, the overexpression of the motor domains or the entire molecules of the class VIII myosins did not affect Hsp70h targeting. Further mapping revealed that the minimal cargo-binding part of the myosin VIII tails was both essential and sufficient for the inhibition of the proper Hsp70h localization. Interestingly, plasmodesmatal localization of the Tobacco mosaic virus movement protein and Arabidopsis protein RGP2 was not affected by myosin VIII tail overexpression. Collectively, our data implicate class VIII myosins in protein delivery to plasmodesmata and suggest that more than one mechanism of such delivery exist in plants.
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Virion Tails of Beet Yellows Virus: Coordinated Assembly by Three Structural Proteins
Virology, 2006Co-Authors: Dina V. Alzhanova, Alexey I Prokhnevsky, Valera V. Peremyslov, Valerian V. DoljaAbstract:Abstract Filamentous virions of Beet yellows virus contain a long body formed by a major capsid protein and a short tail that is assembled by a minor capsid protein (CPm), an Hsp70-homolog (Hsp70h), a 64-kDa protein (p64), and a 20-kDa protein (p20). Using mutation analysis and newly developed in planta assays, here we investigate the genetic requirements for the tail assembly. We show that the inactivation of CPm dramatically reduces incorporation of both Hsp70h and p64. Furthermore, inactivation of Hsp70h prevents incorporation of p64 into virions and vice versa. Hsp70h and p64 are each required for efficient incorporation of CPm. We also show that the tails possessing normal relative amounts of CPm, Hsp70h, and p64 can be formed in the absence of the major capsid protein and p20. Similar to the tails isolated from the wild-type virions, these mutant tails encapsidate the ∼ 700 nt-long, 5′-terminal segments of the viral RNA. Taken together, our results imply that CPm, Hsp70h and p64 act cooperatively to encapsidate a defined region of the Closterovirus genome.
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comparative and functional genomics of Closteroviruses
Virus Research, 2006Co-Authors: Valerian V. Dolja, Jan KreuzeAbstract:The largest extant RNA genomes are found in two diverse families of positive-strand RNA viruses, the animal Coronaviridae and the plant Closteroviridae. Comparative analysis of the viruses from the latter family reveals three levels of gene conservation. The most conserved gene module defines RNA replication and is shared with plant and animal viruses in the alphavirus-like superfamily. A module of five genes that function in particle assembly and transport is a hallmark of the family Closteroviridae and was likely present in the ancestor of all three Closterovirus genera. This module includes a homologue of Hsp70 molecular chaperones and three diverged copies of the capsid protein gene. The remaining genes show dramatic variation in their numbers, functions, and origins among Closteroviruses within and between the genera. Proteins encoded by these genes include suppressors of RNA silencing, RNAse III, papain-like proteases, the AlkB domain implicated in RNA repair, Zn-ribbon-containing protein, and a variety of proteins with no detectable homologues in the current databases. The evolutionary processes that have shaped the complex and fluid genomes of the large RNA viruses might be similar to those that have been involved in evolution of genomic complexity in other divisions of life.
Gail C Wisler - One of the best experts on this subject based on the ideXlab platform.
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tomato chlorosis virus a new whitefly transmitted phloem limited bipartite Closterovirus of tomato
Phytopathology, 1998Co-Authors: Gail C Wisler, H.-y. Liu, D S Lowry, James E DuffusAbstract:ABSTRACT Tomato chlorosis virus (ToCV) is the second whitefly-transmitted, phloem-limited, bipartite Closterovirus described infecting tomato. ToCV is distinct from tomato infectious chlorosis virus (TICV), based on lack of serological and nucleic acid cross-reactions and differences in vector specificity. TICV is transmitted only by the greenhouse whitefly (Trialeurodes vaporariorum), whereas ToCV is transmitted by the greenhouse whitefly, the banded-wing whitefly (T. abutilonea), and Bemisia tabaci biotypes A and B (B. argentifolii). Double-stranded (ds) RNA analyses of ToCV show two prominent dsRNAs of approximately 7,800 and 8,200 bp, with several small dsRNAs. Digoxigenin-11-UTP-labeled riboprobes derived from cDNA clones representing portions of RNAs 1 and 2 were used in Northern blot hybridizations to detect two large nonhomologous dsRNAs and a subset of smaller dsRNAs. These probes were used in dot blot hybridizations to detect ToCV in infected tomato. Inclusion bodies and cytoplasmic vesicles were consistently observed in phloem tissues of ToCV-infected Nicotiana clevelandii. Computer-assisted sequence analysis showed significant homology between ToCV clones that hybridize specifically with RNAs 1 and 2 and the lettuce infectious yellows virus methyltransferase of RNA 1 and the HSP70 heat shock protein homolog of RNA 2, respectively. Thus, ToCV is another member of the growing subgroup of bipartite Closteroviruses transmitted by whiteflies.
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tomato infectious chlorosis virus has a bipartite genome and induces phloem limited inclusions characteristic of the Closteroviruses
Phytopathology, 1996Co-Authors: Gail C Wisler, H.-y. Liu, James E Duffus, V A Klaassen, Bryce W. FalkAbstract:Tomato infectious chlorosis virus (TICV) is a newly described Closterovirus. Virions purified from TICV-infected plants contained two single-stranded (ss) RNAs, one of approximately 7,800 (RNA 1) and the other 7,400 (RNA 2) nucleotides. Double-stranded (ds) RNA analysis showed two prominent dsRNAs of approximately 7,800 and 7,400 bp, as well as several smaller dsRNAs. The TICV virion ssRNAs were used for cDNA cloning. Of 200 cDNA clones analyzed, 10 clones containing cDNAs ranging in size from about 900 to 1,500 nucleotides were used to generate digoxigenin-UTP-labeled transcripts. These transcripts hybridized with the TICV ssRNAs in Northern blot hybridization analyses and were used in dot-blot analyses to confirm TICV infection in several host plants including tomato, potato, Physalis wrightii, Nicotiana clevelandii, and artichoke. None of the probes reacted with any uninfected host plant tested or with plants infected with four other clostero- or clostero-like viruses including lettuce infectious yellows Closterovirus, lettuce chlorosis virus, cucurbit yellow stunting disorder virus, and beet pseudo yellows virus. Northern blot hybridization analyses using selected riboprobes showed no detectable homology between TICV dsRNA 1 and 2, or between subsets of smaller dsRNAs. Inclusion bodies, characteristic of the Closteroviruses, were consistently associated with the phloem of TICV-infected N. clevelandii.
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tomato infectious chlorosis virus a new clostero like virus transmitted by trialeurodes vaporariorum
European Journal of Plant Pathology, 1996Co-Authors: James E Duffus, Hsing Yeh Liu, Gail C WislerAbstract:A previously undescribed virus disease of tomato, other crops and weed hosts was found in California. Affected tomato plants exhibited interveinal yellowing, necrosis and severe yield losses. Leaf dips and purified preparations contained Closterovirus-like long flexuous, filamentous particles approximately 12×850–900 nm. The virus, designated as tomato infectious chlorosis virus (TICV), is transmitted in a semipersistent manner by the greenhouse whitefly,Trialeurodes vaporariorum. The host range of the virus is moderate (26 species in 8 plant families) but includes some important crops and ornamental species including tomato, (Lycopersicon esculentum), tomatillo (Physalis ixocarpa), potato (Solanum tuberosum), artichoke (Cynara scolymus), lettuce (Lactuca sativa) and petunia (Petunia hybrida). The virus has been found in a number of different locations in California and has a number of potential vehicles of movement including greenhouse grown ornamentals, tomato transplants, artichoke cuttings and potato seed. The virus has the potential to spread to other growing regions with resident populations of the greenhouse whitefly. The host range, particle size, insect transmission, and serology clearly distinguish TICV from previously described viruses.
James E Duffus - One of the best experts on this subject based on the ideXlab platform.
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tomato chlorosis virus a new whitefly transmitted phloem limited bipartite Closterovirus of tomato
Phytopathology, 1998Co-Authors: Gail C Wisler, H.-y. Liu, D S Lowry, James E DuffusAbstract:ABSTRACT Tomato chlorosis virus (ToCV) is the second whitefly-transmitted, phloem-limited, bipartite Closterovirus described infecting tomato. ToCV is distinct from tomato infectious chlorosis virus (TICV), based on lack of serological and nucleic acid cross-reactions and differences in vector specificity. TICV is transmitted only by the greenhouse whitefly (Trialeurodes vaporariorum), whereas ToCV is transmitted by the greenhouse whitefly, the banded-wing whitefly (T. abutilonea), and Bemisia tabaci biotypes A and B (B. argentifolii). Double-stranded (ds) RNA analyses of ToCV show two prominent dsRNAs of approximately 7,800 and 8,200 bp, with several small dsRNAs. Digoxigenin-11-UTP-labeled riboprobes derived from cDNA clones representing portions of RNAs 1 and 2 were used in Northern blot hybridizations to detect two large nonhomologous dsRNAs and a subset of smaller dsRNAs. These probes were used in dot blot hybridizations to detect ToCV in infected tomato. Inclusion bodies and cytoplasmic vesicles were consistently observed in phloem tissues of ToCV-infected Nicotiana clevelandii. Computer-assisted sequence analysis showed significant homology between ToCV clones that hybridize specifically with RNAs 1 and 2 and the lettuce infectious yellows virus methyltransferase of RNA 1 and the HSP70 heat shock protein homolog of RNA 2, respectively. Thus, ToCV is another member of the growing subgroup of bipartite Closteroviruses transmitted by whiteflies.
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tomato infectious chlorosis virus has a bipartite genome and induces phloem limited inclusions characteristic of the Closteroviruses
Phytopathology, 1996Co-Authors: Gail C Wisler, H.-y. Liu, James E Duffus, V A Klaassen, Bryce W. FalkAbstract:Tomato infectious chlorosis virus (TICV) is a newly described Closterovirus. Virions purified from TICV-infected plants contained two single-stranded (ss) RNAs, one of approximately 7,800 (RNA 1) and the other 7,400 (RNA 2) nucleotides. Double-stranded (ds) RNA analysis showed two prominent dsRNAs of approximately 7,800 and 7,400 bp, as well as several smaller dsRNAs. The TICV virion ssRNAs were used for cDNA cloning. Of 200 cDNA clones analyzed, 10 clones containing cDNAs ranging in size from about 900 to 1,500 nucleotides were used to generate digoxigenin-UTP-labeled transcripts. These transcripts hybridized with the TICV ssRNAs in Northern blot hybridization analyses and were used in dot-blot analyses to confirm TICV infection in several host plants including tomato, potato, Physalis wrightii, Nicotiana clevelandii, and artichoke. None of the probes reacted with any uninfected host plant tested or with plants infected with four other clostero- or clostero-like viruses including lettuce infectious yellows Closterovirus, lettuce chlorosis virus, cucurbit yellow stunting disorder virus, and beet pseudo yellows virus. Northern blot hybridization analyses using selected riboprobes showed no detectable homology between TICV dsRNA 1 and 2, or between subsets of smaller dsRNAs. Inclusion bodies, characteristic of the Closteroviruses, were consistently associated with the phloem of TICV-infected N. clevelandii.
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tomato infectious chlorosis virus a new clostero like virus transmitted by trialeurodes vaporariorum
European Journal of Plant Pathology, 1996Co-Authors: James E Duffus, Hsing Yeh Liu, Gail C WislerAbstract:A previously undescribed virus disease of tomato, other crops and weed hosts was found in California. Affected tomato plants exhibited interveinal yellowing, necrosis and severe yield losses. Leaf dips and purified preparations contained Closterovirus-like long flexuous, filamentous particles approximately 12×850–900 nm. The virus, designated as tomato infectious chlorosis virus (TICV), is transmitted in a semipersistent manner by the greenhouse whitefly,Trialeurodes vaporariorum. The host range of the virus is moderate (26 species in 8 plant families) but includes some important crops and ornamental species including tomato, (Lycopersicon esculentum), tomatillo (Physalis ixocarpa), potato (Solanum tuberosum), artichoke (Cynara scolymus), lettuce (Lactuca sativa) and petunia (Petunia hybrida). The virus has been found in a number of different locations in California and has a number of potential vehicles of movement including greenhouse grown ornamentals, tomato transplants, artichoke cuttings and potato seed. The virus has the potential to spread to other growing regions with resident populations of the greenhouse whitefly. The host range, particle size, insect transmission, and serology clearly distinguish TICV from previously described viruses.
R F Lee - One of the best experts on this subject based on the ideXlab platform.
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genetic transformation of citrus paradisi with antisense and untranslatable rna dependent rna polymerase genes of citrus tristeza Closterovirus
Turkish Journal of Agriculture and Forestry, 2006Co-Authors: Bayram Cevik, R F Lee, C L NiblettAbstract:Protein and RNA-mediated forms of pathogen-derived resistance (PDR) have been developed against many viruses in different plants. However, no resistance has been reported against Citrus tristeza virus (CTV), a Closterovirus, in Citrus species transformed with coat protein genes or other sequences of CTV. The successful use of replication-associated genes in RNA-mediated resistance in other crops prompted the use of the RNA-dependent RNA polymerase (RdRp) gene of CTV for the development of RNA-mediated PDR in Citrus. The RdRP gene was amplified from CTV isolate DPI3800 from Florida and used to generate antisense (RdRp-AS) and untranslatable (RdRp-UT) constructs with point mutation consecutive stop codons in the 5’ end of the RdRp gene for use in plant transformation. A total of 3120 etiolated epicotyl segments of Duncan grapefruit (Citrus paradisi Macf. cv. Duncan) were transformed with these constructs using Agrobacterium tumefaciens-mediated transformation. From these segments 1040 kanamycin-resistant shoots were regenerated, and a total of 131 putative transgenic shoots were identified by fluorescent microscopy and histochemical b-glucuronidase (GUS) assays. One hundred GUS positive plants were rooted and 66 plants survived and were established on soil. A total of 41 plants were tested by polymerase chain reaction (PCR) for the presence of the GUS gene and for the transgenes. Eighteen GUS-positive and transgene-positive plants (8 with RdRp-AS, and 10 with RdRp-UT) were identified.
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complete sequence of the citrus tristeza virus rna genome
Virology, 1995Co-Authors: Alexander V Karasev, Siddarame Gowda, Eugene V. Koonin, C L Niblett, D J Gumpf, V P Boyko, Olga V Nikolaeva, M E Hilf, K Cline, R F LeeAbstract:Abstract The sequence of the entire genome of citrus tristeza virus (CTV), Florida isolate T36, was completed. The 19,296-nt CTV genome encodes 12 open reading frames (ORFs) potentially coding for at least 17 protein products. The 5′-proximal ORF 1a starts at nucleotide 108 and encodes a large polyprotein with calculated MW of 349 kDa containing domains characteristic of (from 5′ to 3′) two papain-like proteases (P-PRO), a methyltransferase (MT), and a helicase (HEL). Alignment of the putative P-PRO sequences of CTV with the related proteases of beet yellows Closterovirus (BYV) and potyviruses allowed the prediction of catalytic cysteine and histidine residues as well as two cleavage sites, namely Val-Gly/Gly for the 5′ proximal P-PRO domain and Met-Gly/Gly for the 5′ distal P-PRO domain. The autoproteolytic cleavage of the polyprotein at these sites would release two N-terminal leader proteins of 54 and 55 kDa, respectively, and a 240-kDa C-terminal fragment containing MT and HEL domains. The apparent duplication of the leader domain distinguishes CTV from BYV and accounts for most of the size increase in the ORF 1a product of CTV. The downstream ORF 1b encodes a 57-kDa putative RNA-dependent RNA polymerase (RdRp), which is probably expressed via a + 1 ribosomal frameshift. Sequence analysis of the frameshift region suggests that this + 1 frameshift probably occurs at a rare arginine codon CGG and that elements of the RNA secondary structure are unlikely to be involved in this process. The complete polyprotein resulting from this frameshift event has a calculated MW of 401 kDa and after cleavage of the two N-terminal leaders would yield a 292-kDa protein containing the MT, HEL, and RdRp domains. Phylogenetic analysis of the three replication-associated domains, MT, HEL, and RdRp, indicates that CTV and BYV form a separate Closterovirus lineage within the alpha-like supergroup of positive-strand RNA viruses. Two gene blocks or modules can be easily identified in the CTV genome. The first includes the replicative MT, HEL, and RdRp genes and is conserved throughout the entire alpha-like superfamily. The second block consists of five ORFs, 3 to 7, conserved among Closteroviruses, including genes for the CTV homolog of HSP70 proteins and a duplicate of the coat protein gene. The 3′-terminal ORFs 8 to 11 encode a putative RNA-binding protein (ORF 11), and three proteins with unknown functions; this gene array is poorly conserved among Closteroviruses. The genomic doublestranded CTV RNA had an extra G at the 3′ terminus of the minus strand and an extra U at the 3′ terminus of the plus strand.
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defective rna molecules associated with citrus tristeza virus
Virology, 1995Co-Authors: Munir Mawassi, Ron Gafny, Alexander V Karasev, R F Lee, Elzbieta Mietkiewska, Moshe BarjosephAbstract:Abstract Preparations of single-stranded (ss) RNA extracted from particles of the Israeli VT strain of citrus tristeza virus (CTV-VT), and ss- and double-stranded (ds) RNA preparations extracted from infected Alemow (Citrus macrophylla) plants, contained a population of molecules with features that suggest that they are defective RNAs. The prototype of 2424 nt was cloned and sequenced and was found to be composed of two genomic regions corresponding to the 5′ (1151 nt) and the 3′ (1259 nt) termini of the genomic CTV-RNA, with two perfect direct repeats of eight nucleotides of unknown origin at the junction site. Northern hybridization analysis demonstrated that this 2.4-kb defective RNA is an abundant species among the other CTV-specific ss- and ds-RNAs in infected plants. The 2.4-kb RNA was found encapsidated by the CTV coat protein indicating that the CTV origin of assembly is located close to the 5′ or 3′ terminus. This is the first defective RNA to be reported for a member of the Closterovirus group.