Infectious Full-Length Clones of Calibrachoa Mottle Virus (CbMV)
Full-length cDNA clones derived from genomic RNA (gRNA) and subgenomic RNAs (sgRNAs) of Calibrachoa mottle virus (CbMV) were constructed under the control of the T7 RNA promoter and ligated into plasmid pUC-18. The capped and uncapped in vitro transcripts, synthesized from full length genomic cDNA clone pUC-CbMV-FL generated chlorotic local lesions on mechanical inoculated Chenopodium quinoa within 10 to 15 days post-inoculation. The progeny virions recovered from inoculated symptomatic leaves had the same morphological properties as the parental virions. These virions are also reacted positively with CbMV antiserum in double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and the viral coat protein was detected by Western blotting. The presence of five open reading frames (ORFs) on the CbMV genome and synthesis of corresponding proteins by gRNA and sgRNAs were confirmed by Northern blotting and in vitro translation. The transcription start site of three sgRNAs of CbMV was also determined. Successful construction of full-length infectious cDNA clone of CbMV makes it possible to develop molecular tools that can be used to understand the gene functions of this virus.
Calibrachoa mottle virus (CbMV) infects Calibrachoa, an important new horticultural plant in Europe and in the United States. It belongs to the genus Carmovirus of family Tombusviridae and has a single stranded, positive-sense RNA genome of 3.919 kb (Genbank accession number GQ244431). Molecular analysis of the CbMV genomic sequence revealed the presence of at least five open reading frames (ORFs) flanked by a short (34 nt) 5’ untranslated region (UTR) and 234 nt non polyadenylated 3’ UTR. The first 5’ proximal ORF (ORF1) encodes a protein of 28 kDa (p28) and terminates with an amber stop codon, whose readthrough would result in the synthesis of a protein of 87 kDa (ORF2; p87) that encloses the motifs of the viral RNA dependent-RNA polymerase (RdRp). A comparison of amino acid sequences of CbMV with other carmoviruses has shown that both these proteins are involved with expression and/or regulation of the RdRp domain in viruses. In vitro translation studies of Carnation mottle virus (CarMV) showed that these two ORFs were expressed from the viral genomic RNA. Two small centrally located ORFs encode overlapping proteins of approximately 8 kDa (p8) and 9 kDa (p9), respectively. These proteins have been shown in Turnip crinkle virus (TCV) to be involved with viral cell-to-cell movement (movement proteins or MPs). The 3’ proximal ORF5 encodes viral coat protein (CP) of 37 kDa (p37). There is no evidence for the presence of cap at 5’ end of carmovirus genomes. Construction of infectious clones corresponding to the genomes of RNA viruses, obtained either as cDNA clones or as in vitro-transcribed RNA copies, has allowed the study of virus-host interactions such as cell to cell movement. Important information is obtained about the expression and replication of RNA viruses by using in vitro mutagenesis (deletions, insertions, substitutions) and complementation tests. In the study of natural or induced RNA recombination, mechanisms of defect interfering RNA and satellite RNA genesis can also be obtained through infectious clones. Infectious clones, considered as the pools of viral genes for designing antiviral strategies, are an essential source of material for the preparation of new viral vectors
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