A TB40/E-Derived Human Cytomegalovirus Genome with an Intact US-Gene Region and a Self-Excisable BAC Cassette for Immunological Research

For immunological research on the human cytomegalovirus (HCMV), a virus that combines the broad cell tropism of clinical isolates, efficient replication in cell culture, the complete set of MHC-I modulator genes, and suitability for genetic engineering is desired. Here, we aimed to generate a genetically complete derivative of HCMV strain TB40/E as a bacterial artificial chromosome (BAC) with a self-excisable BAC cassette. The BAC cassette was inserted into the US2–US6 gene region (yielding TB40-BAC KL7 ), relocated into the UL73/UL74 region with modifications that favor excision of the BAC cassette during replication in fibroblasts, and finally the US2–US6 region was restored, resulting in BAC clone TB40-BAC KL7 -SE When this BAC clone was transfected into fibroblasts at efficiencies >0.1%, replicating virus that had lost the BAC cassette appeared within 2 weeks after transfection, grew to high titers, and displayed the broad tropism of the parental virus. The degree of MHC-I down-regulation by this virus was consistent with functional restoration of US2–US6. To enable detection of infected cells by flow cytometry, an enhanced green fluorescent protein (EGFP)-expression cassette was inserted downstream of US34A, yielding the fluorescent virus RV-TB40-BAC KL7 -SE-EGFP.