Defective RNAs of Citrus tristeza virus analogous to Crinivirus genomic RNAs

AbstractThe 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 ∼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

Accumulation of a 5′ proximal subgenomic RNA of \u3ci\u3eCitrus tristeza virus\u3c/i\u3e is correlated with encapsidation by the minor coat protein

During replication, Citrus tristeza virus (CTV) produces large amounts of two unusual subgenomic (sg) RNAs that are positive-stranded and 5′ coterminal. Although these RNAs are produced in similar amounts and are similar in size, with LMT1 (~750 nt) only slightly larger than LMT2 (~650), we found that the similar sgRNAs are produced differently. We previously showed that the LMT1 RNA is produced by premature termination during genomic RNA synthesis. However, LMT2 production was found to correlate with virion assembly instead of RNA replication. The time course of accumulation of the LMT2 RNA occurred late, coinciding with virion accumulation. The long flexuous virions of CTV contain two coat proteins that encapsidate the virions in a polar manner. The major coat protein encapsidates ~97% of the virion, while the minor capsid protein encapsidates the remainder of the genome beginning in the 5′ non-translated region with the transition zone at ~630 nucleotides from the 5′ end. The section of the virion RNA that was encapsidated by CPm was identical in size to the LMT2 RNA, suggesting that the LMT2 RNA represented a portion of the viral RNA protected by CPm encapsidation. Mutations that abrogated encapsidation by CPm also abolished the accumulation of LMT2 RNA. Thus, these two unusual but similar RNAs are produced via different pathways, one from RNA replication and one processed by the virion assembly process. To our knowledge, this represents the first evidence of a viral RNA processed by the assembly mechanism

Molecular Characterization of \u3ci\u3eCitrus tatter leaf virus\u3c/i\u3e Historically Associated with Meyer Lemon Trees: Complete Genome Sequence and Development of Biologically Active In Vitro Transcripts

Citrus tatter leaf virus isolated from Meyer lemon trees (CTLV-ML) from California and Florida induces bud union incompatibility of citrus trees grafted on the widely used trifoliate and trifoliate hybrid rootstocks. The complete genome sequence of CTLV-ML was determined to be 6,495 nucleotides (nts), with two overlapping open reading frames (ORFs) and a poly (A) tail at the 3′ end. The genome organization is similar to other capilloviruses, with ORF1 (nts 37 to 6,354) encoding a putative 242-kDa polyprotein which contains replication-associated domains plus a coat protein (CP), and ORF2 (nts 4,788 to 5,750), which is located within ORF1 in a different reading frame and encodes a putative movement protein. Although the proteins encoded by CTLV-ML possesses 84 to 96% amino acid sequence identity with strains of Apple stem grooving virus (ASGV), we observed two strikingly different regions in ORF1: variable region I (amino acids 532 to 570) and variable region II (amino acids 1,583 to 1,868), with only 15 to 18 and 56 to 62% identities, respectively, with the corresponding regions of ASGV strains. Conditions for a herbaceous systemic assay host were optimized in which the wildtype virus induced systemic infection in Phaseolus vulgaris cv. Light Red Kidney (LRK) bean plants at 19 or 22°C but not at higher temperatures. In vitro transcripts generated from full-length cDNA clones induced systemic symptoms on LRK bean plants similar to that of the wild-type virus. Replication of the recombinant virus was confirmed by hybridization of a 5′ positive-stranded RNA-specific probe to a genome-sized RNA and by reverse-transcription polymerase chain reaction

Characterization of the 5′- and 3′-Terminal Subgenomic RNAs Produced by a Capillovirus: Evidence for a CP Subgenomic RNA

The members of Capillovirus genus encode two overlapping open reading frames (ORFs): ORF1 encodes a large polyprotein containing the replication-associated proteins plus a coat protein (CP), and ORF2 encodes a movement protein (MP), located within ORF1 in a different reading frame. Organization of the CP sequence as part of the replicase ORF is unusual in capilloviruses. In this study, we examined the capillovirus genome expression strategy by characterizing viral RNAs produced by Citrus tatter leaf virus (CTLV), isolate ML, a Capillovirus. CTLV-ML produced a genome-length RNA of ∼6.5-kb and two 3′-terminal sgRNAs in infected tissue that contain the MP and CP coding sequences (3′-sgRNA1), and the CP coding sequence (3′-sgRNA2), respectively. Both 3′-sgRNAs initiate at a conserved octanucleotide (UUGAAAGA), and are 1826 (3′-sgRNA1) and 869 (3′-sgRNA2) nts with 119 and 15 nt leader sequences, respectively, suggesting that these two 3′- sgRNAs could serve to express the MP and CP. Additionally, accumulation of two 5′-terminal sgRNAs of 5586 (5′-sgRNA1) and 4625 (5′-sgRNA2) nts was observed, and their 3′-termini mapped to 38–44 nts upstream of the transcription start sites of 3′-sgRNAs. The presence of a separate 3′-sgRNA corresponding to the CP coding sequence and its cognate 5′-terminal sgRNA (5′-sgRNA1) suggests that CTLV-ML produces a dedicated sg mRNA for the expression of its CP

Metabolic Changes in Skin Caused by Scd1 Deficiency: A Focus on Retinol Metabolism

We previously reported that mice with skin-specific deletion of stearoyl-CoA desaturase-1 (Scd1) recapitulated the skin phenotype and hypermetabolism observed in mice with a whole-body deletion of Scd1. In this study, we first performed a diet-induced obesity experiment at thermoneutral temperature (33°C) and found that skin-specific Scd1 knockout (SKO) mice still remain resistant to obesity. To elucidate the metabolic changes in the skin that contribute to the obesity resistance and skin phenotype, we performed microarray analysis of skin gene expression in male SKO and control mice fed a standard rodent diet. We identified an extraordinary number of differentially expressed genes that support the previously documented histological observations of sebaceous gland hypoplasia, inflammation and epidermal hyperplasia in SKO mice. Additionally, transcript levels were reduced in skin of SKO mice for genes involved in fatty acid synthesis, elongation and desaturation, which may be attributed to decreased abundance of key transcription factors including SREBP1c, ChREBP and LXRα. Conversely, genes involved in cholesterol synthesis were increased, suggesting an imbalance between skin fatty acid and cholesterol synthesis. Unexpectedly, we observed a robust elevation in skin retinol, retinoic acid and retinoic acid-induced genes in SKO mice. Furthermore, SEB-1 sebocytes treated with retinol and SCD inhibitor also display an elevation in retinoic acid-induced genes. These results highlight the importance of monounsaturated fatty acid synthesis for maintaining retinol homeostasis and point to disturbed retinol metabolism as a novel contributor to the Scd1 deficiency-induced skin phenotype

e-Book on Closteroviridae

Flores Pedauye, R.; Moreno, P.; Falk, B.; Martelli, GP.; Dawson, WO. (2013). e-Book on Closteroviridae. Frontiers in Microbiology. 4:411-1-411-3. doi:10.3389/fmicb.2013.00411S411-1411-3