Human cytomegalovirus strain Towne pp65 gene: nucleotide sequence and expression in Escherichia coli

Abstract

The human cytomegalovirus (HCMV) encodes a 65-kDa tegument protein (pp65), which has been reported to be a target of immune response during natural infection. We have cloned and sequenced the gene encoding pp65 of HCMV Towne strain (pp65Towne), and have expressed this gene in Escherichia coli in order to study certain antigenic and structural properties of this polypeptide. The pp65Towne gene had a 99% nucleotide similarity and 99.7% amino acid similarity to pp65 of HCMV AD169 strain (pp65AD169). However, unlike the pp65AD169 gene, the pp65Towne gene was found to be incapable of undergoing RNA splicing due to a base substitution in the critical 3' splice-acceptor site. Insertion of this protein coding sequence into the bacterial expression plasmids enabled synthesis in E. coli of an immunoreactive pp65-related polypeptide. The recombinant pp65 (rpp65) reacted strongly in immunoblot analysis with pp65-specific murine and human monoclonal antibodies as well as with anti-pp65 rabbit antiserum. In immunoblot analysis, the reactivity of rpp65 with a panel of human HCMV-immune sera indicated that some sera were reactive while other HCMV seropositive sera were nonreactive, a finding similar to that for native pp65.