High-Throughput Characterization of Viral and Cellular Protein Expression Patterns During JC Polyomavirus Infection.

Jeanne K Dushane,Melissa S Maginnis,Mason A Crocker,Michael P Wilczek

doi:10.3389/fmicb.2019.00783

Abstract

JC polyomavirus (JCPyV) is a ubiquitous human pathogen and the causative agent of a fatal demyelinating disease in severely immunocompromised individuals. Due to the lack of successful pharmacological interventions, the study of JCPyV infection strategies in a rapid and highly sensitive manner is critical for the characterization of potential antiviral therapeutics. Conventional methodologies for studying viral infectivity often utilize the detection of viral proteins through immunofluorescence microscopy-based techniques. While these methodologies are well established in the field, they require significant time investments and lack a high-throughput modality. Scanning imager-based detection methods like the In-cell Western (ICW)TM have been previously utilized to overcome these challenges incurred by traditional microscopy-based infectivity assays. This automated technique provides not only rapid detection of viral infection status, but can also be optimized to detect changes in host-cell protein expression during JCPyV challenge. Compared to traditional manual determinations of infectivity through microscopy-based techniques, the ICW provides an expeditious and robust determination of JCPyV infection. The optimization of the ICW for the detection of viral and cellular proteins during JCPyV infection provides significant time and cost savings by diminishing sample preparation time and increasing resource utilization. While the ICW cannot provide single-cell analysis information and is limited in the detection of quantitation of low-expressing proteins, this assay provides a high-throughput system to study JCPyV, previously unavailable to the field. Thus, the high-throughput nature and dynamic experimental range of the ICW can be applied to the study of JCPyV infection.

Highlights

JC polyomavirus (JCPyV), the etiological agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML), infects between 50–80% of the human population (Egli et al, 2009; Kean et al, 2009)
The In-cell Western (ICW) has been shown to accurately characterize JCPyV infectivity at variable levels of infection, including viral inhibition through chemical and siRNA treatments, and for the quantification of host-cell protein expression during viral challenge. These findings demonstrate that the ICW assay provides an effective measure of viral infection and can be utilized as a platform for high-throughput screening of JCPyV infectivity
To determine whether the ICW can be used to accurately quantitate JCPyV infection, SVG-A cells were infected with varying JCPyV MOIs and analyzed for infectivity using manual fluorescent focus unit (FFU) quantitation and ICW analyses performed in parallel (Figures 1A,B)

Summary

Introduction

JC polyomavirus (JCPyV), the etiological agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML), infects between 50–80% of the human population (Egli et al, 2009; Kean et al, 2009). The lytic infection of these glial cell types leads to CNS demyelination and the onset of PML, for which there are currently no effective therapies (Ferenczy et al, 2012). Polyomaviruses have a double-stranded DNA genome enclosed within a proteinaceous capsid comprised of viral proteins 1 (VP1), 2, and 3 (Ferenczy et al, 2012). While the initial stages of SV40 infection vary slightly from JCPyV, all polyomaviruses traffic into the ER prior to genomic deposition in the nucleus. Viral genes, such as the T-antigens, are transcribed first followed by DNA replication and the transcription of viral late genes that encode the capsid proteins. New viral progeny are encapsidated and eventually egress from the host cell (Loeber and Dorries, 1988; Ault and Stoner, 1993; Agostini et al, 1997; Bhattacharjee and Chattaraj, 2017)

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