A translation strategy and gene map for pea seedborne mosaic virus
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
A method was developed to purify pea seed borne mosaic virus (PSbMV) and its genomic RNA from peas. The RNA of PSbMV was found to be polyadenylated and approximately 10,000 nucleotides long. A cDNA library of 360 clones was made. Of these, 80 clones were screened to determine the size of the inserts. One or more Rsal fragments were sequenced from each of three clones, pPSb54, pPSb66 and pPSb401. A fourth clone, pPSb70, was used as a probe in Northern blots. The cylindrical inclusions of PSbMV were found to have several different morphologies. Pinwheels, scrolls and short laminate aggregates were all observed to be induced by PSbMV. The presence of all three types of inclusions places PSbMV in subdivision-IV according to the classification of Edwardson et al. (1984). The molecular weights of 3 virus-encoded proteins were established by Western blots. The molecular weights are: coat protein 33 kDa, nuclear inclusion b protein 48 kDa, and the cylindrical inclusion protein 70 kDa. The molecular weight of the Nla protein (45 kDa) was established by immunoprecipitation of in vitro translation products. A gene map for PSbMV was proposed, using information from serologically cross-reacting products of the in vitro translations of PSbMV-RNA together with information from Western blots. The proposed map places the coat protein gene at the 3' end of the genome. Upstream of this is the putative replicase gene (Nib), then a putative protease (Nla), followed by the cylindrical inclusion gene. The gene encoding the amorphous inclusion protein is nearer the 5' end. The translation strategy of PSbMV was determined using in vitro translation of PSbMV-RNA in rabbit reticulocyte lysate. The virus produces a polyprotein that is proteolytically cleaved to produce 7 or more mature products. Full length PSbMV-RNA was isolated from the polyribosomes of infected plant material. The amount of PSbMV-RNA electroporated into the protoplasts of Nicotiana species was found to increase very rapidly for the first 4 hours of incubation, then the RNA level was maintained for a further 8 hours. After 12 hours incubation, the amount of RNA was observed to decrease. Western blots using antiserum to the coat protein of PSbMV indicated that PSbMVRNA was translated in Nicotiana tabacum protoplasts.