Green fluorescent protein expression from recombinant lettuce infectious yellows virus-defective RNAs originating from RNA 2.
(1/33)
Lettuce infectious yellows virus (LIYV) RNA 2 defective RNAs (D RNAs) were compared in protoplasts for their ability to replicate and to express the green fluorescent protein (GFP) from recombinant D RNA constructs. Initially four LIYV D RNAs of different genetic composition were compared, but only two (LIYV D RNA M5 and M18) replicated to high levels. Both of these contained at least two complete ORFs, one being the 3'-terminal ORF encoding P26. Northern hybridization analysis using probes corresponding to 3' regions of LIYV RNA 2 detected the P26 subgenomic RNA from protoplasts infected with LIYV RNAs 1 and 2 or protoplasts inoculated only with RNA 1 plus either the LIYV D RNA M5 or M18, suggesting that these LIYV D RNAs served as templates to generate the P26 subgenomic RNA. The GFP coding region was inserted as an in-frame insertion into the P26 coding region of the LIYV M5 and M18 D RNAs, yielding M5gfp and M18gfp. When transcripts of M5gfp and M18gfp were used to inoculate protoplasts, bright fluorescence was seen only when they were co-inoculated with LIYV RNA 1. The percentage of fluorescent protoplasts ranged from experiment to experiment, but was as high as 5.8%. Time course analyses showed that fluorescence was not detected before 48 h pi, and this correlated with the timing of LIYV RNA 2 and RNA 2 D RNA accumulation, but not with that of LIYV RNA 1. (+info)
Genome structure and phylogenetic analysis of lettuce infectious yellows virus, a whitefly-transmitted, bipartite closterovirus.
(2/33)
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. (+info)
Complete genome sequence and analyses of the subgenomic RNAs of sweet potato chlorotic stunt virus reveal several new features for the genus Crinivirus.
(3/33)
The complete nucleotide sequences of genomic RNA1 (9,407 nucleotides [nt]) and RNA2 (8,223 nt) of Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae) were determined, revealing that SPCSV possesses the second largest identified positive-strand single-stranded RNA genome among plant viruses after Citrus tristeza virus. RNA1 contains two overlapping open reading frames (ORFs) that encode the replication module, consisting of the putative papain-like cysteine proteinase, methyltransferase, helicase, and polymerase domains. RNA2 contains the Closteroviridae hallmark gene array represented by a heat shock protein homologue (Hsp70h), a protein of 50 to 60 kDa depending on the virus, the major coat protein, and a divergent copy of the coat protein. This grouping resembles the genome organization of Lettuce infectious yellows virus (LIYV), the only other crinivirus for which the whole genomic sequence is available. However, in striking contrast to LIYV, the two genomic RNAs of SPCSV contained nearly identical 208-nt-long 3' terminal sequences, and the ORF for a putative small hydrophobic protein present in LIYV RNA2 was found at a novel position in SPCSV RNA1. Furthermore, unlike any other plant or animal virus, SPCSV carried an ORF for a putative RNase III-like protein (ORF2 on RNA1). Several subgenomic RNAs (sgRNAs) were detected in SPCSV-infected plants, indicating that the sgRNAs formed from RNA1 accumulated earlier in infection than those of RNA2. The 5' ends of seven sgRNAs were cloned and sequenced by an approach that provided compelling evidence that the sgRNAs are capped in infected plants, a novel finding for members of the Closteroviridae. (+info)
Nucleotide sequence and genome organization of Cucumber yellows virus, a member of the genus Crinivirus.
(4/33)
The genome of Cucumber yellows virus (CuYV), isolated in Japan from cucumber (Cucumis sativus L.), was completely sequenced and shown to be bipartite. CuYV RNA1 consisted of 7889 nucleotides and encompassed seven open reading frames (ORFs), which is typical of the Closteroviridae, including a heat-shock protein 70 homologue, a coat protein and a diverged coat protein (CPd). CuYV RNA2 consisted of 7607 nucleotides and included two ORFs: ORF1a potentially encoded a polyprotein containing putative papain-like protease, methyltransferase and helicase domains, and ORF 1b potentially encoded an RNA-dependent RNA polymerase, which is probably expressed via a +1 ribosomal frameshift. The size and organization of the CuYV genome are similar to those of Lettuce infectious yellows virus (LIYV), the type member of the genus Crinivirus in the family Closteroviridae, indicating that CuYV is a member of that genus, although CuYV differed in several points from LIYV. (+info)
Defective RNAs of Citrus tristeza virus analogous to Crinivirus genomic RNAs.
(5/33)
The family Closteroviridae includes the genera Closterovirus and Ampelovirus with monopartite genomes and the genus Crinivirus with bipartite genomes. Plants infected with the Closterovirus, Citrus tristeza virus (CTV), often contain one or more populations of defective RNAs (dRNAs). Although most dRNAs are comparatively small (2-5 kb) consisting of the genomic RNA termini with large internal deletions, we recently characterized large dRNAs of approximately 12 kb that retained the open reading frames (ORFs) 1a plus 1b. These were self-replicating RNAs and appeared to be analogous to the genomic RNA 1 of the bipartite criniviruses. The present report describes the finding of an additional group of large dRNAs (LdRNAs) that retained all or most of the 10 3' ORFs and appeared to be analogous to genomic RNA 2 of criniviruses. Isolates associated with LdRNAs were found associated with double-recombinant dRNAs (DR-dRNAs) of various sizes (1.7 to 5.1 kb) that comprised the two termini and a noncontiguous internal sequence from ORF2. The genetic and epidemiological implications of the architectural identities of LdRNAs and DR dRNAs and their apparent analogy with the genomic RNA 2 of criniviruses are discussed. (+info)
Further variability within the genus Crinivirus, as revealed by determination of the complete RNA genome sequence of Cucurbit yellow stunting disorder virus.
(6/33)
The complete nucleotide (nt) sequences of genomic RNAs 1 and 2 of Cucurbit yellow stunting disorder virus (CYSDV) were determined for the Spanish isolate CYSDV-AlLM. RNA1 is 9123 nt long and contains at least five open reading frames (ORFs). Computer-assisted analyses identified papain-like protease, methyltransferase, RNA helicase and RNA-dependent RNA polymerase domains in the first two ORFs of RNA1. This is the first study on the sequences of RNA1 from CYSDV. RNA2 is 7976 nt long and contains the hallmark gene array of the family Closteroviridae, characterized by ORFs encoding a heat shock protein 70 homologue, a 59 kDa protein, the major coat protein and a divergent copy of the coat protein. This genome organization resembles that of Sweet potato chlorotic stunt virus (SPCSV), Cucumber yellows virus (CuYV) and Lettuce infectious yellows virus (LIYV), the other three criniviruses sequenced completely to date. However, several differences were observed. The most striking novel features of CYSDV compared to SPCSV, CuYV and LIYV are a unique gene arrangement in the 3'-terminal region of RNA1, the identification in this region of an ORF potentially encoding a protein which has no homologues in any databases, and the prediction of an unusually long 5' non-coding region in RNA2. Additionally, the CYSDV genome resembles that of SPCSV in having very similar 3' regions in RNAs 1 and 2, although for CYSDV similarity in primary structures did not result in predictions of equivalent secondary structures. Overall, these data reinforce the view that the genus Crinivirus contains considerable genetic variation. Additionally, several subgenomic RNAs (sgRNAs) were detected in CYSDV-infected plants, suggesting that generation of sgRNAs is a strategy used by CYSDV for the expression of internal ORFs. (+info)
Analysis of the RNA of Potato yellow vein virus: evidence for a tripartite genome and conserved 3'-terminal structures among members of the genus Crinivirus.
(7/33)
Double-stranded RNA preparations produced from potato plants graft-inoculated with a Peruvian isolate of Potato yellow vein virus (PYVV; genus Crinivirus, family Closteroviridae) contain five RNA species denoted RNA 1, RNA 2, RNA 3, x and y of approximately 8, 5.3, 3.8, 2.0 and 1.8 kbp, respectively. The complete nucleotide sequences of PYVV RNAs 1, 2 and 3 and Northern hybridization analysis showed that PYVV RNA 1 contained the replication module and an additional open reading frame (p7), while two distinct species, RNAs 2 and 3, contain the Closteroviridae hallmark gene array. Pairwise comparisons and phylogeny of genome-encoded proteins showed that PYVV shares significant homology with other criniviruses but is most closely related to the Trialeurodes vaporariorum-vectored Cucumber yellows virus. Secondary structure prediction of the 3'-untranslated regions of all three PYVV RNAs revealed four conserved stem-loop structures and a 3'-terminal pseudoknot structure, also predicted for all fully characterized members of the genus Crinivirus and some members of the genera Closterovirus and Ampelovirus. (+info)
Quantitative parameters determining whitefly (Bemisia tabaci) transmission of Lettuce infectious yellows virus and an engineered defective RNA.
(8/33)
In this study, quantitative parameters affecting in vitro acquisition and whitefly (Bemisia tabaci) transmission of Lettuce infectious yellows virus (LIYV) were examined and B. tabaci transmission of an engineered defective RNA (D-RNA) was demonstrated. Virions purified from virus- and virion RNA-inoculated Chenopodium murale plants and protoplasts of Nicotiana tabacum, respectively, were consistently transmitted to plants by B. tabaci when virion concentrations were 0.1 ng microl(-1) or greater. Transmission efficiency increased with increasing virion concentration and number of whiteflies used for inoculation. When in vitro-derived transcripts of the M5gfp D-RNA (engineered to express the green fluorescent protein, GFP) were co-inoculated to protoplasts with wild-type LIYV virion RNAs, the resulting virions were transmissible to plants. LIYV and the M5gfp D-RNA systemically invaded inoculated plants; however, GFP expression was not detected in these plants. Unlike LIYV, the M5gfp D-RNA was not subsequently transmitted by B. tabaci from the initially infected plants, but, when high concentrations of virions from plants infected by LIYV and the M5gfp D-RNA were used for in vitro acquisition by whiteflies, both were transmitted to plants. Quantitative and qualitative analyses showed that, although the M5gfp D-RNA replicated within and systemically invaded plants along with LIYV, compared with LIYV RNA 2 it was not as abundant in plants or in the resulting virions, and concentration of encapsidated RNAs is an important factor affecting transmission efficiency. (+info)