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

Many signaling circuits face a fundamental tradeoff between accelerating their response speed while maintaining final levels below a cytotoxic threshold. Here, we describe a transcriptional circuitry that dynamically converts signaling inputs into faster rates without amplifying final equilibrium levels. Using time-lapse microscopy, we find that transcriptional activators accelerate human cytomegalovirus (CMV) gene expression in single cells without amplifying steady-state expression levels, and this acceleration generates a significant replication advantage. We map the accelerator to a highly self-cooperative transcriptional negative-feedback loop (Hill coefficient ∼7) generated by homomultimerization of the virus's essential transactivator protein IE2 at nuclear PML bodies. Eliminating the IE2-accelerator circuit reduces transcriptional strength through mislocalization of incoming viral genomes away from PML bodies and carries a heavy fitness cost. In general, accelerators may provide a mechanism for signal-transduction circuits to respond quickly to external signals without increasing steady-state levels of potentially cytotoxic molecules.

Infection by human cytomegalovirus (CMV) elicits a strong humoral immune response and robust anti-CMV antibody production. Diagnosis of virus infection can be carried out by using a variety of serological assays; however, quantification of serum antibodies against CMV may not present an accurate measure of a patient's ability to control a virus infection. CMV strains that express green fluorescent protein (GFP) fusion proteins can be used as screening tools for evaluating characteristics of CMV infection in vitro. In this study, we employed a CMV virus strain, AD169, that ectopically expresses a yellow fluorescent protein (YFP) fused to the immediate-early 2 (IE2) protein product (AD169IE2-YFP) to quantify a CMV infection in human cells. We created a high-throughput cell-based assay that requires minimal amounts of material and provides a platform for rapid analysis of the initial phase of virus infection, including virus attachment, fusion, and immediate-early viral gene expression. The AD169IE2-YFP cell infection system was utilized to develop a neutralization assay with a monoclonal antibody against the viral surface glycoprotein gH. The high-throughput assay was extended to measure the neutralization capacity of serum from CMV-positive subjects. These findings describe a sensitive and specific assay for the quantification of a key immunological response that plays a role in limiting CMV dissemination and transmission. Collectively, we have demonstrated that a robust high-throughput infection assay can analyze the early steps of the CMV life cycle and quantify the potency of biological reagents to attenuate a virus infection.