Cloning, sequencing, and expression in Escherichia coli of the coat protein gene of a new potyvirus infection South African Passiflora

Abstract

Complementary DNA representing 1418 nucleotides (nt) of the 3'-poly(A)-proximal tract of the genomic RNA of a potyvirus causing woodiness disease in South African passion fruit, was cloned and sequenced. The sequence contained a single long open reading frame (ORF) of 1188 nt with no initiation codon, and a 3'-non-coding region (3'-NCR) of 230 nt followed by a poly-adenylate tract. Comparison of the ORF with other potyviral coat protein (CP) sequences led to the prediction that a 279 residue CP of MW 31722 is encoded by 836 nt at the 3'-terminus of the ORF. This virus is not merely a South African strain of passion fruit woodiness virus (PWV): the deduced CP sequence is only distantly related to CPs of other sequenced strains of PWV, although it is part of a distinct subgroup of potyviruses related to PWV. The virus was therefore designated as South African passiflora virus (SAPV). The DNA containing the putative CP was cloned into the pUEX2 bacterial expression vector and expressed in Escherichia coli as a beta-gal-CP fusion protein. The fusion protein reacted with polyclonal antisera raised against the native virus, and antisera raised against partially purified fusion protein reacted with viral CP in Western blots.