Abstract
Functional analysis of the essential proteins encoded by human cytomegalovirus (HCMV) is hindered by the lack of complementing systems. To overcome this difficulty, we have established a novel approach, termed the intein-mediated modulation of protein stability (imPS), in which a destabilizing domain and part of a split intein are fused to the essential protein. The growth of the mutant virus can then be regulated by the degradation and splicing of the protein. We found that an ultrafast gp41-1 split intein was able to rescue or degrade the protein of interest (POI) by removing or adding a strong degron through protein splicing. As a result, the function of the POI was turned on or off during the process. Using HCMV essential gene IE1/IE2, we confirmed that imPS worked remarkably well in conditionally regulating protein stability during viral infection. This conditional approach is likely to be applicable for dissecting the gene functions of HCMV or other viruses.
Highlights
Functional analysis of the essential proteins encoded by human cytomegalovirus (HCMV) is hindered by the lack of complementing systems
It is sometimes difficult to achieve complementation by expressing the essential genes in trans, most likely because the expression patterns of the genes are important for their function and because exogenous promoters often fail to reproduce the tight regulation of endogenous promoters
To determine whether this intein could be used in the intein-mediated modulation of protein stability (imPS) system, a destabilizing domain derived from a mutant variant of the human FKBP12 protein[11] was fused to the N-terminus of the C intein (IntC) and monitored using enhanced green fluorescent protein (GFP; Fig. 1B)
Summary
Functional analysis of the essential proteins encoded by human cytomegalovirus (HCMV) is hindered by the lack of complementing systems To overcome this difficulty, we have established a novel approach, termed the intein-mediated modulation of protein stability (imPS), in which a destabilizing domain and part of a split intein are fused to the essential protein. A conditional approach has been developed to analyze the proteins essential to viral growth and pathogenesis[5,6,7,8,9,10] This approach utilizes a mutant variant of the human FKBP12 protein (ddFKBP), which is intrinsically unstable and degrades rapidly when expressed in mammalian cells. The intact intein mediates protein splicing in trans[14,15,16]
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