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...