Journal of General Virology

Fig. 1. Genetic manipulation of the infectious clone, pGGVs. Regions of the virus genome (5´®3´; thick arrows) and vector plasmids (thin curved lines) are shown. Vertical bars indicate the Vs virus genome nucleotide position with unique restriction sites above. Short DNA linkers (dotted lines) were inserted between the unique restriction sites AgeI and Sse8387I to construct plasmid pGGVs_660–1982, and between NotI and AgeI and Sse8387I and PspAI to construct plasmid pGGVs_{660–1982del}, both from pGGVs. An additional NotI restriction site was introduced into pGGVs_{660–1982del} with a linker. pGEMT-E and pGEMT-3´UTR were made by cloning cDNA (produced by RT–PCR) from the original Vs virus into the pGEMT vector. Asterisks indicate two sites on the infectious clone, pGGVs, one within the E protein (nucleotide G₁₆₁₉ with corresponding amino acid R₄₉₆ in the polyprotein) and the other within the 3´UTR (nucleotide C₁₀₈₈₄), which were substituted for A₁₆₁₉ and T₁₀₈₈₄, respectively, to reproduce the wild-type genotypes of Vs virus genome. The DNA fragments excised from pGEMT-E and pGEMT-3´UTR (dotted vertical arrows) to replace corresponding fragments in plasmids pGGVs_660–1982 and pGGVs_{660–1982del}, respectively, to introduce mutations are shown.

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