Abstract
Here, we present the complete genome sequence of a cytomegalovirus Towne-BAC (bacterial artificial chromosome) isolate that we first genetically engineered to mutate the UL96 gene and then serially passaged in human fibroblasts to allow for the accumulation of compensatory mutations. A total of 17 single-base substitutions were discovered in the passaged genome compared to the reference sequence (KF493877).
Highlights
Replicating mutant viruses can be passaged in cell culture to allow for the accumulation of compensatory mutations
We engineered a cluster mutation in the human cytomegalovirus Towne-BAC genome [6], replacing the UL96 C-terminal 122EVDDAV127 residues with the neutral charged alanine residues. This mutant virus (DUL96C) manifested a smaller plaque size and significantly reduced growth rate compared to the wild-type virus (TowneBAC)
The mutant virus was serially passaged in fibroblasts until a bigger plaque size and an approximately 10,000fold increased virus growth rate was achieved
Summary
Replicating mutant viruses can be passaged in cell culture to allow for the accumulation of compensatory mutations. This passaging often results in increased virus growth rates over a period of time [1,2,3,4,5]. We engineered a cluster mutation in the human cytomegalovirus Towne-BAC (bacterial artificial chromosome) genome [6], replacing the UL96 C-terminal 122EVDDAV127 residues with the neutral charged alanine residues. This mutant virus (DUL96C) manifested a smaller plaque size and significantly reduced growth rate compared to the wild-type virus (TowneBAC) (data not shown).
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