Effects of Modification of the Transcription Initiation Site Context on Citrus Tristeza Virus Subgenomic RNA Synthesis

Abstract

Citrus tristeza virus (CTV), a member of the Closteroviridae, has a positive-sense RNA genome of about 20 kb organized into 12 open reading frames (ORFs). The last 10 ORFs are expressed through 3-coterminal subgenomic RNAs (sgRNAs) regulated in both amounts and timing. Additionally, relatively large amounts of complementary sgRNAs are produced. We have been unable to determine whether these sgRNAs are produced by internal promotion from the full-length template minus strand or by transcription from the minus-stranded sgRNAs. Understanding the regulation of 10 sgRNAs is a conceptual challenge. In analyzing commonalities of a replicase complex in producing so many sgRNAs, we examined initiating nucleotides of the sgRNAs. We mapped the 5 termini of intermediate-(CP and p13) and low-(p18) produced sgRNAs that, like the two highly abundant sgRNAs (p20 and p23) previously mapped, all initiate with an adenylate. We then examined modifications of the initiation site, which has been shown to be useful in defining mechanisms of sgRNA synthesis. Surprisingly, mutation of the initiating nucleotide of the CTV sgRNAs did not prevent sgRNA accumulation. Based on our results, the CTV replication complex appears to initiate sgRNA synthesis with purines, preferably with adenylates, and is able to initiate synthesis using a nucleotide a few positions 5 or 3 of the native initiation nucleotide. Furthermore, the context of the initiation site appears to be a regulatory mechanism for levels of sgRNA production. These data do not support either of the established mechanisms for synthesis of sgRNAs, suggesting that CTV sgRNA production utilizes a different mechanism.

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

“…Although a precise role for any of the identified CTV suppressors in host infection remains to be established, it is interesting to note that all are encoded by the most abundantly transcribed subgenomic mRNAs of CTV (24,25). 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).…”

“…The 3Ј half of the genome encodes 10 genes that are not required for replication in protoplasts and are each expressed through a nested set of 3Ј-coterminal subgenomic mRNAs (24,25). This large genome size, as well as the genome organization and expression strategy, is very similar to that of the members of the animal Nidovirales, of which the human severe acute respiratory syndrome Coronavirus is a member (24,26). Our results show that the large CTV genome encodes at least three suppressors of RNA silencing for protection against the host RNA silencing defense.…”

El virus del manchado foliar de los cítricos: caracterización del promotor del RNA subgenómico del gen de la proteína de la cápsida y del supresor del silenciamiento de RNA

“…La inoculación de protoplastos con transcritos de RNA sintetizados in vitro a partir de clones infecciosos de cDNA ha sido una herramienta muy valiosa para estudiar in vivo las secuencias asociadas con la promoción y síntesis de los sgRNAs de muchos virus de plantas (Grdzelishvili et al, 2000;Ayllón et al, 2003Ayllón et al, ,2004Li y Wong, 2006;. Sin embargo, en protoplatos de N. occidentalis, N. benthamiana y cidro Etrog inoculados con transcritos infecciosos de CLBV, sólo se detectaron pequeñas cantidades de RNA viral (gRNA y sgRNAs), por lo que fue imposible realizar estos estudios utilizando este sistema genético.…”

“…Los promotores que son reconocidos por la RdRp para iniciar la síntesis de los sgRNAs pueden ser tanto estructuras primarias Johnson et al, 2003) como secundarias (Grdzelishvili et al, 2000;Li y Wong, 2006) y se localizan en la cadena negativa del gRNA del virus. Hay virus que presentan diferentes promotores para los diferentes sgRNAs, lo que permite regular la transcripción de los distintos genes codificados por el mismo, como ocurre con el virus de la tristeza de cítricos (CTV) (Ayllón et al, 2003). Por otro lado, algunos virus como BMV (Marsh y Hall., 1987;Marsh et al, 1988), CMV (Boccard y Baulcombe, 1993), el virus del mosaico de la alfalfa (Alfalfa mosaic virus, AMV) (van der Kuyl et al, 1990), el virus del mosaico del trébol rojo (Red clover mottle virus, RCMV) (Zavriev et al, 1996), el virus del moteado clorótico del maíz (Maize chlorotic mottle virus, MCMV) (Lommel et al, 1991), TCV (Wang y Simon, 1997), el virus baciliforme del champiñón (Mushroom bacilliform virus, MBV) (Revill et al, 1999), el virus del enanismo amarillo de la cebada (Barley yellow dwarf virus, BYDV) (Kelly et al, 1994) y el virus del manchado foliar de los cítricos (Citrus leaf blotch virus, CLBV) , presentan secuencias conservadas en el extremo 5´ tanto del gRNA como de sus sgRNAs.…”

“…Hasta la fecha, todos los estudios realizados para caracterizar in vivo las secuencias implicadas en la promoción de los sgRNAs de virus de plantas, se han realizado mediante la inoculación de transcritos del virus en protoplastos Ayllón et al, 2003Ayllón et al, , 2004Sivakumaran et al, 2004;Li y Wong, 2006). Sin embargo, como se ha visto en el capítulo anterior, en protoplastos de N. occidentalis, N. benthamiana y cidro Etrog transfectados con viriones o con transcritos infecciosos de CLBV generados in vitro, sólo se detectaron pequeñas cantidades de RNA viral (gRNA y sgRNAs), por lo que es imposible utilizar este sistema genético para caracterizar el promotor del CP-sgRNA.…”