Biomedical subjects

W O Dawson

Publications and source records attributed to W O Dawson.

At least 19 recordsLinked to original sources

PubMed2001

Characterization of the cis-acting elements controlling subgenomic mRNAs of citrus tristeza virus: production of positive- and negative-stranded 3'-terminal and positive-stranded 5'-terminal RNAs.

Citrus tristeza virus (CTV), a member of the Closteroviridae, has an approximately 20-kb positive-sense RNA genome with two 5' ORFs translated from the genomic RNA and 10 3' genes expressed via nine or ten 3'-terminal subgenomic (sg) RNAs. The expression of the 3' genes appears to have properties intermediate between the smaller viruses of the "alphavirus supergroup" and the larger viruses of the Coronaviridae. The sgRNAs are contiguous with the genome, without a common 5' leader, and are associated with large amounts of complementary sgRNAs. Production of the different sgRNAs is regulated temporally and quantitatively, with the highly expressed genes having noncoding regions (NCR) 5' of the ORFs. The cis-acting elements that control the highly expressed major coat protein (CP) gene and the intermediately expressed minor coat protein (CPm) gene were mapped and compared. Mutational analysis showed that the CP sgRNA controller element mapped within nts -47 to -5 upstream of the transcription start site, entirely within the NCR, while the CPm control region mapped within a 57 nt sequence within the upstream ORF. Although both regions were predicted to fold into two stem-loop structures, mutagenesis suggested that primary structure might be more important than the secondary structure. Because each controller element produced large amounts of 3'-terminal positive- and negative-stranded sgRNAs, we could not differentiate whether the cis-acting element functioned as a promoter or terminator, or both. Reversal of the control element unexpectedly produced large amounts of a negative-stranded sgRNA apparently by termination of negative-stranded genomic RNA synthesis. Further examination of controller elements in their native orientation showed normal production of abundant amounts of positive-stranded sgRNAs extending to near the 5'-terminus, corresponding to termination at each controller element. Thus, each controller element produced three sgRNAs, a 5'-terminal positive strand and both positive- and negative-stranded 3'-terminal RNAs. Therefore, theoretically CTV could produce 30-33 species of RNAs in infected cells.

Base Sequence↗

PubMed2001

Assessment of recombinants that arise from the use of a TMV-based transient expression vector.

A potential use of virus-based transient expression vectors is the large-scale production of commercial specialty products, which would require the inoculation of many acres of plants with the viral vector. However, there are several concerns about the widespread use of virus-based vectors. Among these are the spread of the engineered virus to susceptible plants and the generation and persistence of recombinant viruses in the environment. Using a Tobacco mosaic virus (TMV)-based transient gene expression vector, 30B, which expresses the jellyfish green fluorescent protein (30B-GFP), we describe the predominant types of hybrid viruses that developed in plants. In general, the recombinants deleted the foreign gene and repeated sequences, retaining only those sequences required for optimal replication and movement. In pathogenicity studies and challenge experiments designed to make a comparative assessment of the competitiveness of the recombinants with the parent virus, the recombinants had reduced vigor and were less competitive and pathogenic than TMV, a virus which is already present in the areas where tobacco is grown.

Animals↗

PubMed2001

5'-coterminal subgenomic RNAs in citrus tristeza virus-infected cells.

Three unusual 5' coterminal positive-stranded subgenomic (sg) RNAs, two of about 0.8 kb and one of 10 kb (designated LMT1, LMT2, and LaMT, respectively), from Citrus spp. plants and Nicotiana benthamiana protoplasts infected with Citrus tristeza virus (CTV) were characterized. The 5' termini of the LMT RNAs were mapped by runoff reverse transcription and found to correspond with the 5' terminus of the genomic RNA. The LMT 5'-coterminal sgRNAs consisted of two modal lengths of 744--746 and 842--854 nts. The 3' of the LaMT RNAs terminated near the junction of ORF 1b and ORF 2 (p33). None of the 5' sgRNAs had detectable amounts of corresponding negative-sense RNAs, as occurs with the genomic and 3' coterminal subgenomic RNAs of CTV. The abundance of the short and long 5' sgRNAs differed considerably in infected cells. The LMT RNAs were considerably more abundant than the genomic RNAs, while the larger LaMT RNA accumulated to much lower levels. The kinetics of accumulation of LMT1 and LMT2 in synchronously infected protoplasts differed. The larger RNA, LMT1, accumulated earlier with a strong hybridization signal at 2 days postinfection, a time when only traces of genomic and 3' sgRNAs were detected. The lack of corresponding RNAs, that could be 3' cleavage products corresponding to the 5' coterminal sgRNAs and the lack of complementary negative strands, suggest that these sgRNAs were produced by termination during the synthesis of the genomic positive strands.

Base Sequence↗

PubMed2001

Amplification of Citrus tristeza virus from a cDNA clone and infection of citrus trees.

Isolates of the Closterovirus, Citrus tristeza virus (CTV), are populations of disparate genotypes and defective RNAs developed during long periods of vegetative propagation of citrus trees. Because it has not been possible to obtain pure cultures of the virus, it is not known what components of the population are primarily responsible for induction of diseases. We previously developed an infectious cDNA clone from which in vitro-produced RNA transcripts could infect protoplasts (Satyanarayana et al., 1999, Proc. Natl. Acad. Sci. USA 96, 7433-7438). However, neither the RNA transcripts nor virions from transcript-infected protoplasts were competent for infection of citrus trees. Using a green fluorescent protein-marked virus as inoculum, we found that the approximately 20-kb RNA from virions or transcripts of cDNA infected only a small percentage of protoplasts ( approximately 0.01%), but virions could infect more than 80% of the protoplasts. Based on this information, we amplified the virus from the cDNA clone (recombinant virus) by successive passages in protoplasts using virions in crude sap as inoculum. By the third to seventh passages in protoplasts, maximal amounts of recombinant progeny virus were produced, which were used for inoculation of small citrus trees by slashing stems in the presence of virion preparations. A relatively high percentage of plants became infected with the recombinant virus from protoplasts, resulting in the first defined pure culture of CTV in plants. The comparative biology of the pure culture of recombinant CTV with that of the parental population in planta demonstrated that the recombinant virus retained through all of the recombinant DNA manipulations the normal functions of replication, movement, and aphid transmissibility, and had a symptom phenotype indistinguishable from that of the parental population. Additionally, fulfilling Koch's postulates of the first pure culture of CTV in plants suggested that the major genotype of the CTV T36 population is the primary determinant of the symptom phenotype. We could distinguish no biological contributions resulting from the minor genotypes and defective RNAs of the parental population.

Animals↗

PubMed2001

Conundrum of the lack of defective RNAs (dRNAs) associated with tobamovirus Infections: dRNAs that can move are not replicated by the wild-type virus; dRNAs that are replicated by the wild-type virus do not move.

Two classes of artificially constructed defective RNAs (dRNAs) of Tobacco mosaic virus (TMV) were examined in planta with helper viruses that expressed one (183 kDa) or both (126 and 183 kDa) of the replicase-associated proteins. The first class of artificially constructed dRNAs had the helicase and polymerase (POL) domains deleted; the second had an intact 126-kDa protein open reading frame (ORF). Despite extremely high levels of replication in protoplasts, the first class of dRNAs did not accumulate in plants. The dRNAs with an intact 126-kDa protein ORF were replicated at moderate levels in protoplasts and in planta when supported by a TMV mutant that expressed the 183-kDa protein but not the 126-kDa protein (183F). These dRNAs were not supported by helper viruses expressing both replicase-associated proteins. De novo dRNAs were generated in plants infected by 183F but not in plants infected with virus with the wild-type replicase. These novel dRNAs each contained a new stop codon near the location of the wild-type stop codon for the 126-kDa protein and had most of the POL domain deleted. The fact that only dRNAs that contained a complete 126-kDa protein ORF moved systemically suggests that expression of a functional 126-kDa protein or the presence of certain sequences and/or structures within this ORF is required for movement of dRNAs. At least two factors may contribute to the lack of naturally occurring dRNAs in association with wild-type TMV infections: an inability of TMV to support dRNAs that can move in plants and the inability of dRNAs that can be replicated by TMV to move in plants.

Defective Viruses↗

PubMed2001

Functional specialization and evolution of leader proteinases in the family Closteroviridae.

Members of the Closteroviridae and Potyviridae families of the plant positive-strand RNA viruses encode one or two papain-like leader proteinases. In addition to a C-terminal proteolytic domain, each of these proteinases possesses a nonproteolytic N-terminal domain. We compared functions of the several leader proteinases using a gene swapping approach. The leader proteinase (L-Pro) of Beet yellows virus (BYV; a closterovirus) was replaced with L1 or L2 proteinases of Citrus tristeza virus (CTV; another closterovirus), P-Pro proteinase of Lettuce infectious yellows virus (LIYV; a crinivirus), and HC-Pro proteinase of Tobacco etch virus (a potyvirus). Each foreign proteinase efficiently processed the chimeric BYV polyprotein in vitro. However, only L1 and P-Pro, not L2 and HC-Pro, were able to rescue the amplification of the chimeric BYV variants. The combined expression of L1 and L2 resulted in an increased RNA accumulation compared to that of the parental BYV. Remarkably, this L1-L2 chimera exhibited reduced invasiveness and inability to move from cell to cell. Similar analyses of the BYV hybrids, in which only the papain-like domain of L-Pro was replaced with those derived from L1, L2, P-Pro, and HC-Pro, also revealed functional specialization of these domains. In subcellular-localization experiments, distinct patterns were observed for the leader proteinases of BYV, CTV, and LIYV. Taken together, these results demonstrated that, in addition to a common proteolytic activity, the leader proteinases of closteroviruses possess specialized functions in virus RNA amplification, virus invasion, and cell-to-cell movement. The phylogenetic analysis suggested that functionally distinct L1 and L2 of CTV originated by a gene duplication event.

Amino Acid Sequence↗

PubMed2000

Closterovirus encoded HSP70 homolog and p61 in addition to both coat proteins function in efficient virion assembly.

Assembly of the viral genome into virions is a critical process of the virus life cycle often defining the ability of the virus to move within the plant and to be transmitted horizontally to other plants. Closteroviridae virions are polar helical rods assembled primarily by a major coat protein, but with a related minor coat protein at one end. The Closteroviridae is the only virus family that encodes a protein with similarity to cellular chaperones, a 70-kDa heat-shock protein homolog (HSP70h). We examined the involvement of gene products of Citrus tristeza virus (CTV) in virion formation and found that the chaperone-like protein plus the p61 and both coat proteins were required for efficient virion assembly. Competency of virion assembly of different CTV mutants was assayed by their ability to be serially passaged in Nicotiana benthamiana protoplasts using crude sap as inoculum, and complete and partial virus particles were analyzed by serologically specific electron microscopy. Deletion mutagenesis revealed that p33, p6, p18, p13, p20, and p23 genes were not needed for virion formation. However, deletion of either minor- or major-coat protein resulted in formation of short particles which failed to be serially transferred in protoplasts, suggesting that both coat proteins are required for efficient virion assembly. Deletion or mutation of HSP70h and/or p61 dramatically reduced passage and formation of full-length virions. Frameshift mutations suggested that the HSP70h and p61 proteins, not the RNA sequences, were needed for virion assembly. Substitution of the key amino acid residues in the ATPase domain of HSP70h, Asp(7) to Lys or Glu(180) to Arg, reduced assembly, suggesting that the chaperone-like ATPase activity is involved in assembly. Both HSP70h and p61 proteins appeared to contribute equally to assembly, consistent with coordinate functions of these proteins in closterovirus virion formation. The requirement of two accessory proteins in addition to both coat proteins for efficient assembly is uniquely complex for helical virions.

Amino Acid Substitution↗

PubMed2000

The fitness of citrus tristeza virus defective RNAs is affected by the lengths of their 5'- and 3'-termini and by the coding capacity.

Populations of the Closterovirus Citrus tristeza virus (CTV) generally contain defective RNAs (dRNAs) that vary in size, abundance, and sequence. The variation in abundance of the different dRNAs in a population suggests selection for those of higher fitness. To examine factors affecting fitness of dRNAs, we investigated a series of in vitro constructed dRNAs for their ability to be amplified in protoplasts by an efficiently replicated CTV deletion mutant. The minimal sequences required for accumulation of the dRNAs were within the genomic 5' proximal approximately 1 kb and the 3' 270 nucleotides. However, other factors were involved, because a dRNA with only the minimal sequences failed to be replicated. Rescue of a nonviable dRNA by insertion of nonviral sequences between the termini suggested that "spacing" between terminal cis-acting signals influenced fitness. A continuous open reading frame (ORF) through most of the sequences derived from the 5' of the genome was a requirement for dRNA amplification. In general, insertions, deletions, or nucleotide substitutions were tolerated in the dRNAs as long as an ORF was retained, whereas dRNAs with mutations that prematurely terminated the ORF were not viable. To discriminate between a requirement for an essential protein and ribosomal travel, perhaps to present replication signals to the replicase complex, mutations were made to modify the potential protein but still maintain an ORF. Deletions, insertions of nonviral sequences, or switching of reading frames that altered the amino acid sequence of the protein, except the N-terminal 161 amino acids, did not destroy the fitness of the dRNAs. Yet termination of the ORF in the middle of nonviral sequences did destroy the ability of the dRNAs to be amplified. These results suggest that even though a continuous ORF was needed for fitness, its protein product did not affect the amplification of the dRNAs.

Closterovirus↗

PubMed2000

Mapping of the Tobacco mosaic virus movement protein and coat protein subgenomic RNA promoters in vivo.

The Tobacco mosaic virus movement protein (MP) and coat protein (CP) are expressed from 3'-coterminal subgenomic RNAs (sgRNAs). The transcription start site of the MP sgRNA, previously mapped to positions 4838 (Y. Watanabe, T. Meshi, and Y. Okada (1984), FEBS Lett. 173, 247-250) and 4828 (K. Lehto, G. L. Grantham, and W. O. Dawson (1990), Virology 174, 145-157) for the TMV OM and U1 strains, respectively, has been reexamined and mapped to position 4838 for strain U1. Sequences of the MP and CP sgRNA promoters were delineated by deletion analysis. The boundaries for minimal and full MP sgRNA promoter activity were localized between -35 and +10 and -95 and +40, respectively, relative to the transcription start site. The minimal CP sgRNA promoter was mapped between -69 and +12, whereas the boundaries of the fully active promoter were between -157 and +54. Computer analysis predicted two stem-loop structures (SL1 and SL2) upstream of the MP sgRNA transcription start site. Deletion analysis and site-directed mutagenesis suggested that SL1 secondary structure, but not its sequence, was required for MP sgRNA promoter activity, whereas a 39-nt deletion removing most of the SL2 region increased MP sgRNA accumulation fourfold. Computer-predicted folding of the fully active CP sgRNA promoter revealed one long stem-loop structure. Deletion analysis suggested that the upper part of this stem-loop, located upstream of the transcription start site, was essential for transcription and that the lower part of the stem had an enhancing role.

Base Sequence↗

PubMed2000

The p20 gene product of Citrus tristeza virus accumulates in the amorphous inclusion bodies.

Citrus tristeza virus (CTV) has 10 3' open reading frames (ORFs) of unknown function except for the two coat proteins. The highest produced subgenomic RNAs are those of the major coat protein gene (p25) and the 3' most genes, p20 and p23. The proteins from three ORFs, p25, p27, and p20, were examined in the yeast two-hybrid assay for the interactions between themselves and to one another. The p20 protein exhibited a high affinity for itself, suggesting that it might aggregate in infected cells. The cytopathology of CTV infections includes characteristic paracrystalline and amorphous inclusions in the phloem elements of infected citrus. Polyclonal antiserum raised against the bacterial expressed p20 gene product detected a protein of approximately 22-23 kDa, which accumulated to relatively high levels in CTV-infected citrus, but not in healthy citrus. Immunogold localization using antibodies to p20 protein showed strong and specific labeling of the amorphous inclusion bodies present in CTV-infected cells. Mesophyll protoplasts of Nicotiana benthamiana transfected with a CTV mutant containing the green fluorescent protein (GFP) ORF fused in-frame to the 3' end of p20 protein ORF expressed high levels of GFP. The fusion protein was concentrated in one specific area in the cytoplasm and lacked an organized shape. Accumulation of high levels of p20 protein in infected tissue, specific localization of the p20-GFP fusion protein, immunolocalization of p20 protein into amorphous inclusions, and strong homologous p20 protein-p20 protein interactions in the yeast-two-hybrid assay suggest that the p20 protein of CTV is a major component of the amorphous inclusion bodies present in CTV-infected cells.

Blotting, Western↗

PubMed2000

Full-length tobacco mosaic virus RNAs and defective RNAs have different 3' replication signals.

The viral replicase complex of positive-stranded RNA viruses interacts with cis-acting elements that are usually located at the termini of the viral RNAs. On comparison of the replication requirement of a tobacco mosaic virus (TMV)-based defective RNA (dRNA) and its helper virus, we found different requirements for replication of TMV RNAs in cis and in trans. The level of replication of full-length TMV RNA decreased substantially in the absence of pseudoknot (pk) 1 and/or 2, whereas identical deletions in dRNAs did not affect their replication. However, pk3 was required for replication of both full-length TMV RNAs and dRNAs. The requirements for homologous sequences were greater for dRNA replication than for replication of full-length TMV RNAs. Defective RNAs with heterologous 3' nontranslated regions (NTRs) failed to be replicated or replicated minimally, whereas replication of similarly mutated full-length RNAs was much less affected. Increasing amounts of contiguous heterologous sequences in the dRNAs compensated for the impaired interactions between the replicase and 3' NTR. The precision requirement appeared to involve the terminal 28 nucleotides, specifically the pseudoknot in the aminoacyl acceptor arm of the tRNA like structure, which was important in replication of both dRNAs and full-length TMV RNAs.

Base Sequence↗

PubMed2000

Functions of the 126- and 183-kDa proteins of tobacco mosaic virus.

Tobacco mosaic virus produces two proteins that contain domains similar to the methyltransferase (MT) and helicase (HEL)-like domains of the replicase-associated proteins of other RNA viruses. The more abundant 126-kDa protein contains only the MT and HEL-like domains, whereas the 183-kDa readthrough protein additionally contains the polymerase domain. We examined the functions of these proteins by constructing a bipartite system to express the 126- and 183-kDa proteins from separate RNAs. Mutants expressing the 183-kDa protein recognized promoters for negative- and positive-stranded RNA synthesis, transcribed subgenomic mRNAs, capped RNAs, synthesized proteins, moved cell to cell within the plant, and replicated defective RNAs (dRNAs). The principal function of the 126-kDa protein was to increase the rate of replication approximately tenfold. The 126-kDa protein appeared to function primarily in cis, and production of the 126-kDa protein in trans did not enhance replication of the helper virus. dRNAs producing a functional 126-kDa protein were replicated efficiently by helper viruses that produced only the 183-kDa protein but not by wild-type virus, suggesting that efficient replication required the 183-kDa protein to form a heterodimer with the 126-kDa protein already bound to the target dRNA.

Frameshift Mutation↗

PubMed2000

Replication of heterologous combinations of helper and defective RNA of citrus tristeza virus.

Citrus tristeza virus (CTV) populations are among the more complex of plant RNA viruses with unusual mixtures of strains and defective RNAs (dRNAs). Citrus plants infected with different CTV isolates contain multiple dRNA molecules that differ in size and relative abundance within and between isolates. Additionally, we found mixtures of heterologous dRNAs in populations. To examine the replication of CTV dRNAs, the protoplast system had to be extended to support helper-assisted amplification of input dRNAs. The use of freshly extracted sap of CTV-infected tissue as inoculum increased the infection of Nicotiana benthamiana protoplasts sufficiently to result in accumulation of high levels of CTV RNAs as well as dRNAs within 2 or 3 days postinoculation. A series of dRNA-like molecules, each with a single large internal deletion, were created from an infectious cDNA clone of the CTV T36 isolate and examined for amplification in N. benthamiana protoplasts using a CTV deletion mutant as the helper virus. Of 12 synthetic dRNAs, only three with sizes of 3650, 3819, and 4460 nucleotides were efficiently replicated. CTV dRNA replication did not appreciably affect levels of accumulation of the genomic or the subgenomic RNAs of the helper virus. To investigate the maintenance of dRNAs in CTV populations, we examined heterologous interactions between dRNAs and helper viruses. Wild-type populations of heterologous strains T68 and T3, as well as the homologous T36, supported replication of synthetic T36 dRNAs. Replacement in the T36 dRNA of the 5' region, which is most variable among CTV strains, with the corresponding sequences from VT, T68, T3, or T30 resulted in chimeric dRNAs that failed to be replicated by the T36 helpers but were replicated to detectable levels by the T68 helper. The differential specificities of different CTV replicase complexes with dRNA replication signals is one possible factor that affects the maintenance of dRNA population structures.

Citrus↗

PubMed2000

Sequences of Citrus tristeza virus separated in time and space are essentially identical.

The first Citrus tristeza virus (CTV) genomes completely sequenced (19.3-kb positive-sense RNA), from four biologically distinct isolates, are unexpectedly divergent in nucleotide sequence (up to 60% divergence). Understanding of whether these large sequence differences resulted from recent evolution is important for the design of disease management strategies, particularly the use of genetically engineered mild (essentially symptomless)-strain cross protection and RNA-mediated transgenic resistance. The complete sequence of a mild isolate (T30) which has been endemic in Florida for about a century was found to be nearly identical to the genomic sequence of a mild isolate (T385) from Spain. Moreover, samples of sequences of other isolates from distinct geographic locations, maintained in different citrus hosts and also separated in time (B252 from Taiwan, B272 from Colombia, and B354 from California), were nearly identical to the T30 sequence. The sequence differences between these isolates were within or near the range of variability of the T30 population. A possible explanation for these results is that the parents of isolates T30, T385, B252, B272, and B354 have a common origin, probably Asia, and have changed little since they were dispersed throughout the world by the movement of citrus. Considering that the nucleotide divergence among the other known CTV genomes is much greater than those expected for strains of the same virus, the remarkable similarity of these five isolates indicates a high degree of evolutionary stasis in some CTV populations.

Base Sequence↗

PubMed1999

An engineered closterovirus RNA replicon and analysis of heterologous terminal sequences for replication.

Citrus tristeza virus (CTV) populations in citrus trees are unusually complex mixtures of viral genotypes and defective RNAs developed during the long-term vegetative propagation of the virus and by additional mixing by aphid transmission. The viral replication process allows the maintenance of minor amounts of disparate genotypes and defective RNAs in these populations. CTV is a member of the Closteroviridae possessing a positive-stranded RNA genome of approximately 20 kilobases that expresses the replicase-associated genes as an approximately 400-kDa polyprotein and the remaining 10 3' genes through subgenomic mRNAs. A full-length cDNA clone of CTV was generated from which RNA transcripts capable of replication in protoplasts were derived. The large size of cDNA hampered its use as a genetic system. Deletion of 10 3' genes resulted in an efficient RNA replicon that was easy to manipulate. To investigate the origin and maintenance of the genotypes in CTV populations, we tested the CTV replicase for its acceptance of divergent sequences by creating chimeric replicons with heterologous termini and examining their ability to replicate. Exchange of the similar 3' termini resulted in efficient replication whereas substitution of the divergent (up to 58% difference in sequence) 5' termini resulted in reduced but significant replication, generally in proportion to the extent of sequence divergence.

Closterovirus↗

PubMed1999

Tobacco mosaic virus virulence and avirulence.

In celebration of a century of research on tobacco mosaic virus that initiated the science of virology, I review recent progress relative to earlier contributions concerning how viruses cause diseases of plants and how plants defend themselves from viruses.

Immunity, Innate↗

PubMed1999

Heterologous sequences greatly affect foreign gene expression in tobacco mosaic virus-based vectors.

A series of tobacco mosaic virus (TMV)-based hybrid vectors for transient gene expression were constructed with similar designs but differing in the source of heterologous tobamovirus sequence: Odontoglossum ringspot virus, tobacco mild green mosaic virus variants U2 and U5, tomato mosaic virus, and sunn-hemp mosaic virus. These vectors contained a heterologous coat protein subgenomic mRNA promoter and coat protein open reading frame (ORF) and either TMV or heterologous 3' nontranslated region. The foreign ORF, from the jellyfish green fluorescent protein (GFP) gene, was transcribed from the native TMV coat protein subgenomic mRNA promoter, which extended into the coat protein ORF. The presence of an in-frame stop codon within the GFP mRNA leader and the choice of sequence of GFP ORFs substantially affected translational efficiency. However, the major regulatory component of gene expression in these vectors appeared to be transcriptional rather than translational. There was an inverse relationship between expression of GFP and the heterologous coat protein genes that was reflected in accumulation of the respective mRNAs and proteins. The most effective vector in this series (30B) contained sequences encoding the coat protein subgenomic mRNA promoter, coat protein ORF, and 3' nontranslated region from tobacco mild green mosaic virus U5. Expressed from 30B, GFP accumulated up to 10% of total soluble protein in leaves.

3' Untranslated Regions↗

PubMed1999

The complete genome sequence of the major component of a mild citrus tristeza virus isolate.

The genome of the Spanish mild isolate T385 of citrus tristeza virus (CTV) was completely sequenced and compared with the genomes of the severe isolates T36 (Florida), VT (Israel) and SY568 (California). The genome of T385 was 19,259 nt in length, 37 nt shorter than the genome of T36, and 33 and 10 nt longer than those of VT and SY568, respectively, but their organization was identical. T385 had mean nucleotide identities of 81.3, 89.3 and 94% with T36, VT and SY568, respectively. The 3' UTR had over 97% identity in all isolates, whereas the 5' UTR of T385 had 67% identity with VT, 66.3% with SY568 and only 42.5% with T36. In the coding regions, the nucleotide differences between T385 and VT were evenly distributed along the genome (around 90% identity); this was not observed between T385 and the other isolates. T385 and T36 had nucleotide identities around 90% in the eight 3'-terminal ORFs of the genome, but only 72.3% in ORF 1a, a divergence pattern similar to that reported previously for T36 and VT. T385 and SY568 had nucleotide identities close to 90% in the 5'- and 3'-terminal regions of the genome, whereas the central region had over 99% identity. Our data suggest that the central region in the SY568 genome results from RNA recombination between two CTV genomes, one of which was almost identical to T385.

Base Sequence↗