Synthesis of an infectious full-length cDNA clone of rice yellow mottle virus andmutagenesis of the coat protein

Abstract

A full-length cDNA clone of rice yellow mottle sobemovirus (RYMV) was synthesized and placed adjacent to a bacteriophage T7 RNA polymerase promoter sequence. Capped-RNA transcripts produced in vitro were infectious when mechanically inoculated onto rice plants (Oryza sativa L). Individual full-length clones varied in their degree of infectivity but all were less infectious than native viral RNA. A representative clone, designated RYMV-FL5, caused a disease phenotype identical to that produced by viral RNA except that symptoms were somewhat slower to appear than those induced by viral RNA. The infectivity of RYMV-FL5 was verified by ELISA, Western blot analysis, Northern blot hybridization, RT-PCR, and Southern blot hybridization. Frameshift and deletion mutations introduced into the coat protein cistron demonstrated that the coat protein was dispensable for RNA replication in rice protoplasts. However, the coat protein was required for full infectivity in rice plants, presumably by playing a role in phloem-mediated long-distance movement and possibly in cell-to-cell movement.

Internal Point Mutations of the Capsid Modify the Serotype of Rice Yellow Mottle Virus

“…The CP gene sequence of a second sample of 152 isolates was determined as described by Pinel (21). Briefly, genome fragments with the CP gene and part of the 3Ј untranslated region were transcribed and amplified by reverse transcription-PCR after extraction of total RNA from leaves as described previously (5,21). Sequencing was performed by using the Taq terminator sequencing kit (Applied Biosystems) with primers described previously (21) and analyzed on an Applied Biosystems 373A sequencer.…”

“…Mutations were introduced into an infectious RYMV full-length cDNA (clone FL5 originated from isolate CIa cloned downstream of the T7 promoter) (5). Two nonsynonymous point substitutions in the RYMV coat protein cistron at nucleotides 3982 and 3988 (amino acids 178 and 180, respectively) were generated in two mutated clones named FL5-CPT180K and FL5-CPG178D, respectively.…”

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

“…El virus se detectó a los 4 dpt, un periodo cercano al límite de supervivencia de los protoplastos. Este periodo de incubación es superior al necesario cuando se inoculan protoplastos con otros virus como CTV (Navas-Castillo, 1997), el virus del moteado amarillo del arroz (RYMV) (Brugidou et al, 1995), el virus del amarilleo de las cucurbitáceas (Cucurbit aphid-borne yellows virus, CABYV) (Prüfer et al, 1995), el virus del amarilleo infeccioso de la lechuga (Lettuce infectious yellows virus, LIYV) (Klassen et al, 1996), el virus del rizado moteado de la alcachofa (Artichoke mottle crinkle virus, AMCV) (Tavazza et al, 1994) y el virus de los anillos cloróticos del hibiscus (hibiscus chlorotic ringspot virus, HCRSV) (Liang et al, 2002), donde la infección viral se detecta a los 2 dpt. Esto puede ser debido a que CLBV tenga una tasa de multiplicación menor que otros virus de plantas o a que el método de transfección empleado en este trabajo sea menos eficiente.…”

“…Este resultado sugiere que la estructura CAP contribuye a la infectividad aumentando la traducción inicial del transcrito (Shih et al, 1976;Contreras et al, 1982), y/o aumentando su estabilidad al limitar la degradación mediada por las nucleasas del hospedador (Furuichi et al, 1977;Shimotohno et al, 1977;Green et al, 1983). De la misma manera, en CTV y LIYV sólo los transcritos virales a los que se les añadió una estructura CAP resultaron infecciosos (Satyanarayana et al, 1999;Klaassen et al, 1996), mientras que en el caso de RYMV y AMCV, ambos tipos de transcritos (con y sin CAP), resultaron infecciosos (Tavazza et al, 1994;Brugidou et al, 1995).…”

“…Esta diferencia de acumulación también se observó con CTV, donde la multiplicación viral se detectó a los 2 dpt al inocular viriones y a los 3 dpt al inocular transcritos (Satyanarayana et al, 1999), pero no con la mayoría de virus, que se acumulan de forma similar en protoplastos transfectados con viriones o transcritos (Tavazza et al, 1994;Brugidou et al, 1995;Klassen et al, 1996). Los aislados de CLBV, al igual que otros virus de genoma de RNA, están compuestos por una población de variantes de secuencia.…”