Development of Tools to Study how Virus-host Cell Interactions Influence Infection Spread PDF Download

Author: Bahar Inankur
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Languages : en
Pages : 255

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Book Description
Viruses cause a variety of human diseases including AIDS, the common cold, hepatitis, and cancer. During a virus infection, the various processes and interactions of the virus, host, and environment form a complex and dynamic system with a heterogeneous range of outcomes. While there has been a strong focus on investigation of molecular mechanisms of virus-host cell interactions within a single-infection cycle and clinical studies that entail multiple infection cycles, relatively less has been done to link how the interactions within a single-infection cycle are reflected over multiple rounds of infection. In this context, the work in this thesis focuses on the development of experimental and computational quantitative tools to study the factors that influence infection spread from cell to cell in vitro. The potential of an infection to spread is determined mainly by the ability of the infecting virus to effectively enter the host cell, replicate efficiently, and evade or suppress any host restrictions. Conversely, host cell inhibition of the infection relies on the ability to detect the invading virus, mount a response that typically includes the activation of genes that have either direct antiviral roles or signal to warn other cells of the infection, and avoid the suppressive activity of the virus that seeks to limit these responses. In order to investigate this dynamic interaction between virus and host, we used vesicular stomatitis virus (VSV), a well characterized model virus. We first designed microfluidic channels to study virus spread under stagnant fluid environments. In parallel, to quantify the effects of antiviral responses we developed an antiviral activity assay that measures the overall functional ability of secreted antiviral molecules to inhibit infections. Moreover, using a novel dual color fluorescent reporter system to detect viral replication and cellular antiviral activity; we performed real-time fluorescent microscopy imaging studies of infection spread within cell culture monolayers in conventional well plates and microchannels. Quantitative analysis of spatial and temporal features of spreading infections in these two different cultures, together with the use of activity assay revealed the dose and duration dependency of antiviral activation prior to infection that ultimately determined the spread or arrest of the infection. On a separate project, using the image processing and analysis tools, we quantified the infection spread behavior of VSV in the presence of a virus mutant that lacked essential genes for growth, but was able to divert the viral proteins produced by an infectious virus to its own replication. The detailed analysis and modeling of the diverse interfered patterns revealed the increase in the diversity of spread patterns with increasing mutant virus input. The tools developed here can aid the in vitro testing of antiviral drugs by helping assess how the treatments influence the interactions between virus and the host cells over multiple rounds of infection. Further improving the quantitative methods developed using VSV, finally we focused on a more clinically relevant virus, rhinovirus (RV), which contributes to a variety of respiratory illnesses. Aiming to link the genetic diversity observed in different RV strains with differences in the severity of infections observed clinically, we developed single-cell image analysis workflows. The workflows enabled tracking of individual cells and extraction of fluorescent intensities from different cellular compartments in a dual color fluorescent transfection assay. Using these tools we quantified the RVs’ ability to disrupt nuclear transport pathways over time. Comparison of different strains revealed the differential disruption of nucleocytoplasmic transport, a key mechanism for intracellular antiviral signaling. Extending these studies to probe spatial and temporal dynamics of spreading RV infections, we quantified the spread patterns of a recombinant fluorescent RV strain and extracted parameters that can be further used for characterizing the various features of the spread phenotypes of different RV strains. The multi-cycle infection analysis of various RV strains may help gain insights into varying degrees of illness severities observed for infections of different RV strains. Overall, this work demonstrates the potential of these new experimental and computational tools for enabling a more quantitative approach to elucidating the dynamic and complex mechanisms of virus-host interactions.