Spatial patterns of protein expression in focal infections of human cytomegalovirus

Abstract

Human cytomegalovirus (HCMV) is a medically significant human pathogen that infects a wide range of cell and tissue types. During infection, HCMV activates a variety of signal transduction pathways that induce profound changes in cellular processes and dramatically affect cellular gene expression patterns. To better define how these virus-host interactions affect the local microenvironment and influence the spatial and temporal spread of HCMV, we initiated HCMV focal infections on normal human dermal fibroblast monolayers and monitored viral gene expression patterns and infection spread over 45 days. To establish baseline temporal measurements of HCMV infection and spread in cell monolayers, we characterized the influence of three experimental variables on viral gene expression: cell plating density, the presence of serum, and neutralization of cellular antiviral responses with an antibody against interferon-beta. We found that high cell plating density or the inclusion of serum correlated with enhanced HCMV infection spread. Dramatic differences in the expression pattern of the viral immediate early 2 (IE2) gene were observed under these conditions as compared to low plating density or the absence of serum. In the latter case round, uniform foci were observed with a clear wave of IE2 expression visible in advance of a late stage viral protein, envelope glycoprotein B. By contrast, larger irregular foci with arms of IE2 expression were observed in the presence of serum. Addition of the antibody had little effect on the rate of spread, which is consistent with the knowledge that HCMV represses antiviral responses during infection. This experimental system provides a useful means to visualize and quantify complex virus-host interactions.

Visualizing infection spread: Dual‐color fluorescent reporting of virus–host interactions

“…The engineering of recombinant virus strains to express fluorescent proteins provides a facile method to identify infected cells and track the spread of infection in cells and tissues (Duprex et al, , ; Jöns and Mettenleiter, ; Lam et al, ; Payne et al, ). For example, we employed a recombinant strain of human cytomegalovirus (HCMV), engineered to express a GFP‐fusion with immediate early (IE2) protein of HCMV, to track the spread of focal infections in monolayers of normal human dermal fibroblasts (Lam et al, ). In a 45‐day culture, the expression of viral proteins in the cell monolayer corresponded with their temporal order; specifically, the viral immediate early protein (IE2) was detected at larger radii of the expanding infection than the late expressed viral glycoprotein B, which was detected by immunolabeling.…”

Chemical engineering and virology: Challenges and opportunities at the interface

“…We showed how the changes in the infection velocity correlated with processes of virus evolution,11 and activation of cell‐cell communication between infected and healthy cells 12. Interestingly, different temporal stages of the infection process appear to spatially segregate across vast expanses of the infected cells 13. This observation suggests prospects for elucidating the dynamics of virus‐cell interactions by labeling and imaging spatial patterns of gene expression as infections spread.…”

Spatial-Temporal Patterns of Viral Amplification and Interference Initiated by a Single Infected Cell

“…The process becomes still more complex when defective interfering particles enter the mix, coinfecting susceptible cells, perturbing normal viral replication, triggering cellular responses and signaling, and spreading to near or distant naïve cells and tissues. Here we have taken initial steps to dissect and reconstruct parts of virus-DIP interactions within cells and the subsequent impacts on infection spread in a plaque growth system, neglecting, for now, contributions from host immunity or physical transport by processes other than free diffusion, factors that have been considered elsewhere ( 10 , 50 , 55 , 56 , 63 – 67 ).…”