UC San Diego

Systems biology is an emergent field in the biological sciences that has enabled biologists to quantitatively study gene circuits and networks by integrating mathematical modeling, bioinformatics, and biological approaches. Here, we use mathematical modeling and single- cell fluorescence microscopy to reveal a novel gene circuitry in CMV, a global pathogen and major health concern in infants and immunocompromised individuals. This gene circuitry produces acceleration while limiting amplification of output protein levels in the presence of transactivators. IE2 (Immediate-Early 2), CMV's essential viral transactivator, drives this accelerator circuit by autoregulating itself via cooperative binding to the cis repression sequence (crs), a 12 base-pair (bp) sequence located just upstream of the Major Immediate-Early promoter (MIEP)'s Initiator site. IE2 acceleration produces a viral replication fitness advantage, and mutation of the crs sequence eliminates IE2 acceleration and produces amplification of IE2 protein in response to transactivators, severely limiting CMV's replication ability. The [Delta]crs mutant virus exhibits lowered transcriptional strength due to its inability to efficiently localize to sub-nuclear PML bodies, where CMV immediate-early transcription typically occurs. The low transcriptional strength in the [Delta]crs mutant virus results in slower IE2 expression and a severe fitness cost. To further understand the mechanism behind the IE2 accelerator circuit, IE2's interaction with the crs sequence was studied using gel filtration chromatography and electron microscopy. IE2 self-multimerizes into a ring -like structure in the presence of crs DNA, using multiple IE2 subunits. IE2 multimerization only occurs in the presence of the crs sequence and does not occur in the presence of a mutated crs sequence