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Generation of a novel human cytomegalovirus bacterial artificial chromosome tailored for transduction of exogenous sequences.
Virus Research 2017 October 16
The study of herpesviruses, including human cytomegalovirus (HCMV), is complicated by viral genome complexity and inefficient methods for genetic manipulation in tissue culture. Reverse genetics of herpesviruses has been facilitated by propagating their genomes in E. coli as bacterial artificial chromosomes (BACs), which enables complex and precise genetic manipulation using bacterial recombinational systems. Internal capsid volume imposes a strict limit on the length of genome that can be packaged efficiently. This necessitates deletion of presumably nonessential segments of the viral genome to allow for incorporation of the E. coli mini-F plasmid propagation sequence. To avoid deleting viral genes, several BACs utilize a Cre/LoxP system to self-excise the mini-F sequence upon reconstitution of virus in tissue culture. Here, we describe the adaptation of Cre/LoxP to modify the mini-F sequence of the HCMV TB40/E BAC, thus generating a new self-excisable BAC, TB40/E/Cre. After excision of the E. coli propagation sequence, a 2.7 kbp genome length deficit is created due to a preexisting deletion within the US2-US6 coding region. We exploited this deficit and an FKBP12 protein destabilization domain (ddFKBP) to create a novel gene transduction system for studying exogenous proteins during HCMV infection. Using TB40/E/Cre, we: i) found genome length-associated differences in growth and ii) demonstrated its utility as a system capable of efficient transduction of exogenous proteins and regulation of their accumulation over periods as short as 2h. TB40/E/Cre is a powerful tool of broad applicability that can be adapted to study HCMV replication and cell biology in a variety of contexts.
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