Identification of an epitope on the P and V proteins of simian virus 5 that distinguishes between two isolates with different biological characteristics

Abstract

Two canine isolates of simian virus 5 (SV5), termed CPI+ and CPI-, were examined for their ability to react with a bank of monoclonal antibodies (MAbs) that had been previously raised against a human isolate of SV5. CPI-virus was originally isolated from the brain of a gnotobiotic dog infected with CPI+ virus and establishes persistent infections more readily than CPI+ in vitro. Of more than 50 MAbs tested, only one (P-k) reacted with CPI+ but not CPI-, enabling distinction between the two canine isolates. It had been shown previously that MAb P-k reacts with an epitope common to both the P and V proteins. In order to characterize further the epitope binding site of this MAb the P/V genes of CPI+ and CPI-were sequenced.There were four nucleotide differences between CPI+ and CPI-, three of which resulted in predicted amino acid substitutions. Synthetic peptides corresponding to regions encompassing these changes were made and radioimmune competition assays were used to identify the epitope binding site of MAb P-k. Sequence comparison of the P/V gene of CPI+ with the published sequence of a monkey isolate of SV5 (W3) revealed 14 nucleotide differences with five amino acid substitutions. The only amino acid substitution observed between CPI+, CPI-and W3 which altered the predicted secondary structures of the P and V proteins was a leueine to proline change that induced a predicted /I-turn and resulted in the loss of binding of MAb P-k.

Two Related Kinesins,klp5+andklp6+, Foster Microtubule Disassembly and Are Required for Meiosis in Fission Yeast

“…A plasmid vector was constructed containing a [Glycine-Alanine] 2 spacer, three tandem copies of the Pk1 epitope (Southern et al, 1991), the green fluorescent protein (GFP) (F64L, S65T allele) (Cormack et al, 1996), the nmt1 ϩ terminator sequence (Maundrell, 1993), and the ura4 ϩ gene. A DNA molecule containing this construction of (GA) 2 -(Pk1) 3 -GFP-ura4 ϩ was PCR amplified with the use of a 5Ј primer containing 75 bases identical to the 3Ј end of each respective Klp open-reading frame and 33 bases, in-frame, containing the Glycine-Alanine spacer and the [Pk1] 3 sequence.…”

cut11+: A Gene Required for Cell Cycle-dependent Spindle Pole Body Anchoring in the Nuclear Envelope and Bipolar Spindle Formation inSchizosaccharomyces pombe

“…A plasmid vector containing the green fluorescent protein (S65T allele [Heim et al, 1995]), followed in frame by three tandem copies of the Pk1 epitope tag (Southern et al, 1991), and the ura4 ϩ gene was generously provided by B. Carson and C. Troxell (University of Colorado). PCR was used to amplify a continuous ϳ3.5 kb fragment containing the Pk1 and GFP tags together with the ura4 ϩ gene.…”

Construction of solid matrix-antibody-antigen complexes containing simian immunodeficiency virus p27 using tag-specific monoclonal antibody and tag-linked antigen

“…14 amino acids of SV5 P and V proteins (Fig. 2) recognized by MAb SV5-P-k (Southern et al, 1991), was inserted at the 3' terminus of p27 gene, yielding plasmid pSV27T. In order to insert the hybrid gene into pGEX-2T, the BclI site at the 5' end of the p27-TAG gene had to be regenerated by PCR amplification from pSV27T using p27BACK primer and the negative strand of the tag DNA linker as primers.…”