Development of cell lines that provide tightly controlled temporal translation of the human cytomegalovirus IE2 proteins for complementation and functional analyses of growth-impaired and nonviable IE2 mutant viruses.
The human cytomegalovirus (HCMV) IE2 86 protein is essential for viral replication. Two other proteins, IE2 60 and IE2 40, which arise from the C-terminal half of IE2 86, are important for later stages of the infection. Functional analyses of IE2 86 in the context of the infection have utilized bacterial artificial chromosomes as vectors to generate mutant viruses. One limitation is that many mutations result in debilitated or nonviable viruses. Here, we describe a novel system that allows tightly controlled temporal expression of the IE2 proteins and provides complementation of both growth-impaired and nonviable IE2 mutant viruses. The strategy involves creation of cell lines with separate lentiviruses expressing a bicistronic RNA with a selectable marker as the first open reading frame (ORF) and IE2 86, IE2 60, or IE2 40 as the second ORF. Induction of expression of the IE2 proteins occurs only following DNA recombination events mediated by Cre and FLP recombinases that delete the first ORF. HCMV encodes Cre and FLP, which are expressed at immediate-early (for IE2 86) and early-late (for IE2 40 and IE2 60) times, respectively. We show that the presence of full-length IE2 86 alone provides some complementation for virus production, but the correct temporal expression of IE2 86 and IE2 40 together has the most beneficial effect for early-late gene expression and synthesis of infectious virus. This approach for inducible protein translation can be used for complementation of other mutations as well as controlled expression of toxic cellular and microbial proteins.
Sanders
Rebecca L
RL
Department of Cellular and Molecular Medicine and Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0712, USA.
Clark
Charles L
CL
Morello
Christopher S
CS
Spector
Deborah H
DH
D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics / Q000254:growth & development*; D017353:Gene Deletion; D015870:Gene Expression; D005816:Genetic Complementation Test / Q000379:methods*; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics* / Q000378:metabolism; D014176:Protein Biosynthesis; D011995:Recombination, Genetic; D013997:Time Factors; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D014773:Virology / Q000379:methods*; D014779:Virus Replication / Q000235:genetics*
C069186:IE2 protein, Cytomegalovirus; D017874:Immediate-Early Proteins; D015534:Trans-Activators