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

Abstract

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 Ϸ20 kilobases that expresses the replicase-associated genes as an Ϸ400-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.

Three distinct suppressors of RNA silencing encoded by a 20-kb viral RNA genome

“…Thus, it is likely that abundant expression of three distinct suppressors would potently suppress the siRNA-mediated antiviral pathway (11)(12)(13)(14)(15)(16) and possibly also interfere with the host development pathway controlled by micro-RNAs (9,10,(36)(37)(38), at multiple points in a manner similar to virus synergy in mixed infections with viruses carrying distinct suppressors (22,39). Indeed, this may in part explain why CTV causes the most destructive viral disease in citrus worldwide (27). 1-4, 8, 10, and 12) and from the T19 scions 6 weeks after grafting on rootstocks made from various genotypes (lanes 5-7, 9, 11, and 13).…”

“…Importantly, p23 and coat protein (CP) target the intracellular and intercellular silencing, respectively, whereas the third, p20, inhibits silencing at both levels. We suggest that the simultaneous suppression of intracellular and intercellular silencing antiviral defense by CTV proteins may explain, in part, why CTV causes the most destructive viral disease in citrus worldwide (27).…”

Evidence of Recombinant Citrus tristeza virus Isolate Occurring in Acid Lime cv. Pant Lemon Orchard in Uttarakhand Terai Region of Northern Himalaya in India

“…The CTV genome contains single stranded RNA of about 19.3 kb with 12 open reading frames (ORFs) encoding at least 19 proteins [12,21]. The ORFs 1a and 1b encode replication related proteins, whereas, the 3 0 end ten ORFs (ORFs 2-ORF11) encodes proteins p33, p6, p65, p61, p27, p25, p18, p13, p20 and p23 [11,22].…”

The Complex Genetics of Citrus tristeza virus

“…In fact, CTV evolved ending up with three proteins, CP, p20 and p23, which are suppressors of the plant RNA silencing mechanism in N. benthamiana and N. tabacum plants [18]. Unexpectedly, the ORFs that encode proteins p33, p18 and p13 are not required either for replication or assembly [6,13] or for systemic infection of Mexican lime [C. aurantifolia (Christm.) Swing.]…”

“…However, in a decade, a remarkable progress has been achieved in developing the genetic engineering tools to overcome the challenges of examining CTV genetics. A cDNA clone (T36-CTV9) of the Florida isolate T36 was generated and an in vitro genetic system was developed to analyze CTV genotypes, D-RNAs, mutants and self-replicating constructs in Nicotiana benthamiana protoplasts or indexing plants [6,7,8,9,10]. The last advances in CTV genetics and the different biotechnological approaches used to study CTV are discussed in this chapter.…”