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...

The use of internal ribosome entry sites (IRESs) is one of the unorthodox mechanisms exploited by viruses to initiate the translation of internal genes. Herein, we report a plant virus exploiting an IRES and its 3-untranslated region (UTR) to express its internal genes, notably the 3-proximal viral coat protein gene. Hibiscus chlorotic ringspot virus (HCRSV), a positive-strand nonpolyadenylated RNA virus, was demonstrated to harbor a unique 100-nucleotide (nt) IRES, located 124 nt upstream of the coat protein gene, that could function in wheat germ extract, rabbit reticulocyte lysate, and mammalian cells. In comparison with other known IRESs of picornaviruses and eukaryotic mRNAs, this 100-nt IRES is distinctively short and simple. The IRES activity was tested in homologous and heterologous bicistronic constructs, and the expression of the 3-proximal gene was enhanced when the 3-UTR was present. When the IRES element was bisected, each half still possessed IRES activity and could initiate internal translation on its own. Site-directed mutagenesis and deletion analyses revealed that the primary sequence within the 5 half was crucial for IRES activity, whereas the primary sequence of the second half and a GNRA motif were non-essential. To our knowledge, this is the first report describing a mechanism whereby an IRES, located in the 3 portion of the virus genome, co-operates with the 3-UTR to enhance gene expression differentially.Viruses, being obligate parasites, depend heavily on their host for replication. To compete successfully with cellular mRNAs for translation and to fully utilize their compact genomes, viruses have evolved various mechanisms either to redirect the translational machinery to favor viral transcripts or to regulate the expression of internal genes. Genome partitioning and the use of subgenomic RNAs (sgRNAs) 1 are common mechanisms used by many plant viruses to make their internal genes accessible for ribosomes (1). In addition, several non-orthodox strategies, such as the use of internal ribosome entry sites (IRES) have been exploited by viruses to express multiple genes from a single RNA species. Hibiscus chlorotic ringspot virus (HCRSV), a member of the genus Carmovirus, is an isometric monopartite plant virus. HCRSV possesses a single-stranded, positive-sense RNA that is non-polyadenylated at the 3Ј terminus and may not contain a 5Ј cap. The genomic RNA (gRNA) is 3911 nt long with the potential to encode seven open reading frames (ORFs), including two novel ORFs, ORF(p23) and ORF(p25) (2). Two 3Ј-coterminated sgRNA species have been identified. The 3Ј-UTR of HCRSV was previously reported to differentially enhance the translation of various ORFs on the gRNA and sgRNA, such as ORF(p25) and ORF (p38) (3).In this study, we report the identification and characterization of a 100-nt IRES that could co-operate with the 3Ј-UTR of HCRSV to differentially enhance the translation of ORFs on gRNA and sgRNA and a heterologous gene in a bicistronic construct. Located in the 3Ј portion of the gRNA and 1...

Osmotic priming in a polyethylene glycol solution (300 g/kg water) for 48 h resulted in a partial loss of desiccation tolerance for seeds of Vigna radiata (L.) Wilczek (mung bean). The percentage of germination began to decrease after primed seeds were dried down to water contents less than 0.06 g/g DW. As compared with control seeds, primed mung bean seeds also had poorer storage stability. The decline of storage stability after osmotic priming was correlated with the modifications of seed water sorption properties. Priming significantly increased the amount of water associated with the weak water-binding sites, and reduced the amount of water associated with the strong binding sites and multi-molecular binding sites in seed tissues. The enhancement of molecular mobility in seeds, as a result of such water redistribution, probably accelerates seed deterioration and decreases storage stability.

RNA plant viruses use various translational regulatory mechanisms to control their gene expression.Translational enhancement of viral mRNAs that leads to higher levels of protein synthesis from specific genes may be essential for the virus to successfully compete for cellular translational machinery. The control elements have yet to be analyzed for members of the genus Carmovirus, a small group of plant viruses with positive-sense RNA genomes. In this study, we examined the 3 untranslated region (UTR) of hibiscus chlorotic ringspot virus (HCRSV) genomic RNA (gRNA) and subgenomic RNA (sgRNA) for its role in the translational regulation of viral gene expression. The results showed that the 3 UTR of HCRSV significantly enhanced the translation of several open reading frames on gRNA and sgRNA and a viral gene in a bicistronic construct with an inserted internal ribosome entry site. Through deletion and mutagenesis studies of both the bicistronic construct and full-length gRNA, we demonstrated that a six-nucleotide sequence, GGGCAG, that is complementary to the 3 region of the 18S rRNA and a minimal length of 180 nucleotides are required for the enhancement of translation induced by the 3 UTR.The 3Ј untranslated regions (UTRs) of plant virus RNAs are likely to function in a manner similar to that of the corresponding regions of cellular mRNA in regulating translation and maintaining RNA stability. Due to their great variety in terminal structures, the 3Ј UTRs of plus-strand viral RNAs play varied roles in replication and translation (5). For example, the turnip yellow mosaic virus RNA contains a 3Ј tRNA mimic believed to regulate minus-strand RNA synthesis (6). For brome mosaic virus, the 3Ј UTR harbors a unique promoter element that directs minus-strand RNA synthesis (23). The 3Ј UTR of tobacco mosaic virus contains a translation enhancer (8).Hibiscus chlorotic ringspot virus (HCRSV), a member of the genus Carmovirus, is an isometric monopartite plant virus which measures 28 nm in diameter. The virus is found worldwide where hibiscus is cultivated (16,41,42). The symptoms on HCRSV-infected plants range from generalized mottle to chlorotic ringspots and vein-banding patterns, severe stunting, and flower distortion (42,45). HCRSV possesses a singlestranded positive-sense RNA that is not polyadenylated at the 3Ј terminus. The genomic RNA (gRNA) is 3,911 nucleotides (nt) long and has the potential to encode seven open reading frames (ORFs), including two novel ORFs, p23 and p25 (12). Two 3Ј-coterminated subgenomic RNA (sgRNA) species have been identified.In this study, we examined the 3Ј UTR of HCRSV gRNA and sgRNA for its role in the regulation of translation. Through deletion studies and elimination of putative translation enhancer elements, we were able to identify the prerequisites required for the translational enhancement induced by the 3Ј UTR of HCRSV. The results showed that the 3Ј UTR of HCRSV differentially enhanced the translation of ORFs on gRNA and sgRNA and an HCRSV gene in a bicistronic construct by 1.5-t...

We previously showed that translation from the rat BACE1 5' leader is cap-dependent and that four AUG codons (AUG1-4) in the 5' leader were bypassed, partially or completely, depending on the cell line. Two other groups reported comparable results with human BACE1 sequences in different cell lines, although different mechanisms were postulated. In contrast, a third group working with the human sequence reported that most translation events are initiated at AUG2. Using reporter constructs with the rat BACE1 5' leader in rat cells, we now show that this apparent discrepancy between studies can be explained by the use of different expression systems and differences in interpretation. When reporter constructs were transcribed in the nucleus, the upstream AUG codons did not affect translation, but when mRNAs were transcribed in the cytoplasm or when in vitro transcripts were transfected into cells, the upstream AUG codons inhibited translation. These findings suggest that when transcription occurs in the nucleus, the BACE1 mRNA initiates translation by a shunting mechanism. The results are less consistent with either leaky scanning or reinitiation and provide a caveat against the use of cytoplasmic expression systems or RNA transfection for analyses of translation initiation.