Synthesis of figwort mosaic virus (FMV) coat protein-reverse transcriptase fusion protein in reticulocyte lysates from a full-length transcript
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abstract
Genetic organization of figwort mosaic virus (FMV) genome (circular, double stranded DNA of about 8 kbp) reveals six major open reading frames (I-Vl) and a small (VII) open reading frame (ORF) which are expressed from two different transcripts. One of the transcripts spans the entire length of the viral genome and may function as a messenger RNA for the translation of 5-6 closely spaced genes appearing on this RNA; the other spans the gene VI. In the present study, synthesis of a native viral polypeptide(s) was investigated from a full-length and a partial transcript of a naturally occurring deletion mutant of FMV. The deletion in the mutant results in the fusion of the coat protein (IV) and the reverse transcriptase (V) cistrons, consequently, a fusion cistron (IV/V ORF) is generated. In constructs pH52 and pH55 used in the present study, chloramphenicol acetyltransferase (CAT) gene in the deletion mutant is positioned in cistron II and VI, respectively. Translation of full-length transcripts generated from these constructs in reticulocyte lysates results in simultaneous synthesis of CAT polypeptide with a predicted size of the fusion (IV/V) polypeptide of 86,000 daltons. These polypeptides are immunoselected using anti-CAT and antiviral coat protein antibodies. When IV/V ORF is deleted from these constructs by digestion with Sac I before a transcript is prepared, with pH55, the in vitro expression of the fusion polypeptide (IV/V) and the CAT polypeptide in lysates are both abolished. With construct pH52, on the other hand, the synthesis of CAT polypeptide is still observed. These studies are consistant with the full-length viral RNA functioning as a messenger RNA.
