Complete Nucleotide Sequence and Genome Organization of Hibiscus Chlorotic Ringspot Virus, a New Member of the Genus Carmovirus : Evidence for the Presence and Expression of Two Novel Open Reading Frames

Abstract

The complete nucleotide sequence of hibiscus chlorotic ringspot virus (HCRSV) was determined. The genomic RNA (gRNA) is 3,911 nucleotides long and has the potential to encode seven viral proteins in the order of 28 (p28), 23 (p23), 81 (p81), 8 (p8), 9 (p9), 38 (p38), and 25 (p25) kDa. Excluding two unique open reading frames (ORFs) encoding p23 and p25, the ORFs encode proteins with high amino acid similarity to those of carmoviruses. In addition to gRNA, two 3-coterminated subgenomic RNA (sgRNA) species were identified. Full-length cDNA clones derived from gRNA and sgRNA were constructed under the control of a T7 promoter. Both capped and uncapped transcripts derived from the full-length genomic cDNA clone were infectious. In vitro translation and mutagenesis assays confirmed that all the predicted ORFs except the ORF encoding p8 are translatable, and the two novel ORFs (those encoding p23 and p25) may be functionally indispensable for the viral infection cycle. Based on virion morphology and genome organization, we propose that HCRSV be classified as a new member of the genus Carmovirus in family Tombusviridae.Hibiscus chlorotic ringspot virus (HCRSV) is an isometric monopartite plant virus which measures 28 nm in diameter. It was first identified in a hibiscus cultivar imported to the United States from El Salvador. The virus is found worldwide where hibiscus is cultivated (15,36,37). The symptoms on HCRSVinfected plants range from a generalized mottle to chlorotic ring spots and vein-banding patterns (37). Many hibiscus hybrids grown in the tropics as ornamental plants showed severe stunting and flower distortion when infected by HCRSV (41).Serological evidence has demonstrated that HCRSV belongs to the genus Carmovirus (13). So far, the complete nucleotide sequences for seven carmoviruses have been determined. These include the type member carnation mottle virus (CarMV) (9), turnip crinkle virus (TCV) (3), melon necrotic spot virus (MNSV) (27), cardamine chlorotic fleck virus (CCFV) (33), cowpea mottle virus (CPMoV) (42), saguaro cactus virus (SCV) (38), and galinsoga mosaic virus (GaMV) (6).All carmoviruses have a genome organization similar to that of CarMV (Fig. 1). These viruses possess two open reading frames (ORFs), one of which results from an in-frame readthrough mechanism, with the potential to encode two polypeptides starting from the first AUG (5,8,22). Both proteins are putative subunits of the viral replicase. Two centrally located small ORFs encode proteins p8 and p9, which are required for virus movement (10, 17). The coat protein gene is located in the 3Ј region of the genome. CPMoV also has a unique ORF that encodes a 28-kDa protein by in-frame readthrough of the ORF encoding p9 [ORF(p9)] (42). The function of p28 of CPMoV is still unknown.Here we report the complete nucleotide sequence, genome organization, construction of a cDNA clone from which infectious transcripts can be derived, and expression of HCRSV in vitro. In addition to the five ORFs found in other carmoviruses, the HCRSV geno...

Hibiscus chlorotic ringspot virus coat protein inhibits trans-acting small interfering RNA biogenesis in Arabidopsis

“…Generation of CP-and DCP-transgenic Arabidopsis. The CP coding sequence of HCRSV (Huang et al, 2000) was PCR amplified with a 59 leader (59-AAGGAGATATAACA-39) and cloned into pBI121 by replacing the GUS sequence between the 35S promoter and the nopaline synthase terminator to yield pBICP (Fig. 1a).…”

“…Hibiscus chlorotic ringspot virus (HCRSV) is a positivesense, single-stranded RNA virus in the genus Carmovirus (Huang et al, 2000). The coat protein (CP) of HCRSV has been identified as a strong gene-silencing suppressor, which suppresses transiently expressed sense-transgeneinduced PTGS, and a complete CP is required for the effective suppression function (Meng et al, 2006).…”

Análisis de relaciones estructura-función en el virus del arabesco del Pelargonium.

“…El mayor de ellos (sgRNA1), presenta un tamaño aproximado de 1.7 kb, es bicistrónico y está implicado en la traducción de las dos proteínas de movimiento, mientras que el otro (sgRNA2), de tamaño aproximado 1.5 kb, es responsable de la traducción de la CP (revisado en Lommel et al, 2005). Ensayos de traducción in vitro han permitido confirmar la producción de todas las proteínas codificadas tanto por los gRNAs como por los sgRNAs del CarMV, del TCV y del HCRSV (Carrington y Morris, 1985; Harbison et al, 1985; Carrington et al, 1987; Hacker et al, 1992;Huang et al, 2000).…”

“…Actualmente se conoce la secuencia completa de una especie provisional del género, el virus de la necrosis del tallo del guisante (Pea stem necrosis virus, PSNV) (Suzuki et al, 2002), y de diez especies definitivas: CarMV (Guilley et al, 1985), TCV (Carrington et al, 1989), MNSV (Riviere y Rochon, 1990), virus del salpicado clorótico del cardamomo (Cardamine chlorotic fleck virus, CCFV) (Skotnicki et al, 1993), CPMoV (You et al, 1995), virus del cactus Saguaro (Saguaro cactus virus, SCV) (Weng y Xiong, 1997), virus del mosaico del Galisonga (Galisonga mosaic virus, GaMV) (Ciuffreda et al, 1998), HCRSV (Huang et al, 2000), virus de los anillos necróticos del iris japonés (Japanese iris necrotic ring virus, JINRV) (Takemoto et al, 2000) y PFBV (Rico y Hernández, 2004). Además, también se conoce la secuencia completa del virus del aclaramiento de las venas del lupino Nootka (Nootka lupine vein-clearing virus, NLVCV), y del virus de la rotura del color de la flor de la angelonia (Angelonia flower break virus, AnFBV) que han sido propuestos como nuevas especies de este género (Adkins et al, 2006;Robertson et al, 2007).…”

“…El mayor de ellos (sgRNA1), presenta un tamaño aproximado de 1.7 kb, es bicistrónico y está implicado en la traducción de las dos proteínas de movimiento, mientras que el otro (sgRNA2), de tamaño aproximado 1.5 kb, es responsable de la traducción de la CP (revisado en Lommel et al, 2005). Ensayos de traducción in vitro han permitido confirmar la producción de todas las proteínas codificadas tanto por los gRNAs como por los sgRNAs del CarMV, del TCV y del HCRSV (Carrington y Morris, 1985; Harbison et al, 1985; Carrington et al, 1987; Hacker et al, 1992;Huang et al, 2000).Los mecanismos de traducción empleados por los virus de este género para la expresión de sus distintas ORFs son diversos. Como ya se ha indicado antes, las dos primeras ORFs de carmovirus se traducen a partir del gRNA, la segunda de ellas mediante un mecanismo de lectura a través de un codón de terminación débil.…”