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...

Wound-induced protein kinase (WIPK) is a tobacco (Nicotiana tabacum) mitogen-activated protein kinase known to play an essential role in defense against wounding and pathogens, although its downstream targets have yet to be clarified. This study identified a gene encoding a protein of 648 amino acids, which directly interacts with WIPK, designated as N. tabacum WIPK-interacting factor (NtWIF). The N-terminal region with approximately 250 amino acids showed a high similarity to the plant-specific DNA binding domain, B3, but no other similarity with known proteins. The C terminus of approximately 200 amino acids appeared to be essential for the interaction with WIPK, and a Luciferase-reporter gene assay using Bright Yellow 2 cells indicated the full-length protein to possess trans-activation activity, located to the middle region of approximately 200 amino acids. In vitro phosphorylation assays indicated that WIPK efficiently phosphorylates the full-length protein and the N terminus but not the C terminus. When full-length NtWIF was coexpressed with WIPK in Bright Yellow 2 cells, the Luciferase transcriptional activity increased up to 5-fold that of NtWIF alone, whereas no effect was observed with a kinase-deficient WIPK mutant. Transcripts of NtWIF began to simultaneously accumulate with those of WIPK 30 min after wounding and 1 h after the onset of hypersensitive response upon tobacco mosaic virus infection. These results suggest that NtWIF is a transcription factor that is directly phosphorylated by WIPK, thereby being activated for transcription of target gene(s) involved in wound and pathogen responses.

Despite significant efforts in many countries, there is still no commercially viable dengue vaccine. Currently, attention is focused on the development of either live attenuated vaccines or live attenuated chimaeric vaccines using a variety of backbones. Alternate vaccine approaches, such as whole inactivated virus and subunit vaccines are in the early stages of development, and are each associated with different problems. Subunit vaccines offer the advantage of providing a uniform antigen of well-defined nature, without the added risk of introducing any genetic material into the person being inoculated. Preliminary trials of subunit vaccines (using dengue E protein) in rhesus monkeys have shown promising results. However, the primary disadvantages of dengue subunit vaccines are the low levels of expression of dengue proteins in mammalian or insect cells, as well as the added unknown risks of antigens produced from mammalian cells containing other potential sources of contamination. In the past two decades, plants have emerged as an alternative platform for expression of biopharmaceutical products, including antigens of bacterial, fungal or viral origin. In the present minireview, we highlight the current plant expression technologies used for expression of biopharmaceutical products, with an emphasis on plants as a production system for dengue subunit vaccines.

NtWIF is a transcription factor activated upon phosphorylation by wound-induced protein kinase (WIPK) in tobacco plants. Transgenic tobacco plants overexpressing NtWIF exhibited constitutive accumulation of transcripts for pathogenesis-related genes, PR-1a and PR-2. Salicylic acid levels were 50-fold higher than those in wild-type plants. The levels of jasmonic acid and IAA did not significantly differ, while an increase of ABA upon wounding was delayed by 3 h in the transgenics. When challenged with tobacco mosaic virus, lesions developed faster and were smaller in the transgenic plants. The results suggest that NtWIF is likely to influence salicylic acid biosynthesis, being located downstream of WIPK.

P3 protein is one of the least characterized potyviral proteins in both functions and sub-cellular localization. In this study, we examined the sub-cellular localization of PRSV P3 and its intermediate, P3-6K1 by expressing their GFP fusion proteins in onion epidermal cells. Our results showed that both P3- and P3-6K1 GFP fusion proteins were localized at the endoplasmic reticulum. Deletion analysis indicated that C-terminal of P3 protein contained localization signal, and a 19 amino acids hydrophobic domain from this region was able to target the GFP fusion protein to endoplasmic reticulum. C-terminal of P3 proteins has been suggested to be involved in both viability and pathogenicity of the potyvirus. Therefore, our result suggests that localization of P3 protein at endoplasmic reticulum is essential for functionality of P3 protein.