A versatile reporter system to monitor virus infected cells and its application to dengue virus and SARS-CoV-2

Abstract

Positive-strand RNA viruses have been the etiological agents in several major disease outbreaks over the last few decades. Examples of that are flaviviruses, such as dengue virus and Zika virus that cause millions of yearly infections and spread around the globe, and coronaviruses, such as SARS-CoV-2, which is the cause of the current pandemic. The severity of outbreaks caused by these viruses stresses the importance of virology research in determining mechanisms to limit virus spread and to curb disease severity. Such studies require molecular tools to decipher virus-host interactions and to develop effective interventions. Here, we describe the generation and characterization of a reporter system to visualize dengue virus and SARS-CoV-2 replication in live cells. The system is based on viral protease activity causing cleavage and nuclear translocation of an engineered fluorescent protein that is expressed in the infected cells. We show the suitability of the system for live cell imaging and visualization of single infected cells as well as for screening and testing of antiviral compounds. Given the modular building blocks, the system is easy to manipulate and can be adapted to any virus encoding a protease, thus offering a high degree of flexibility.IMPORTANCEReporter systems are useful tools for fast and quantitative visualization of viral replication and spread within a host cell population. Here we describe a reporter system that takes advantage of virus-encoded proteases that are expressed in infected cells to cleave an ER-anchored fluorescent protein fused to a nuclear localization sequence. Upon cleavage, the fluorescent protein translocates to the nucleus, allowing for rapid detection of the infected cells. Using this system, we demonstrate reliable reporting activity for two major human pathogens from the Flaviviridae and the Coronaviridae families: dengue virus and SARS-CoV-2. We apply this reporter system to live cell imaging and use it for proof-of-concept to validate antiviral activity of a nucleoside analogue. This reporter system is not only an invaluable tool for the characterization of viral replication, but also for the discovery and development of antivirals that are urgently needed to halt the spread of these viruses.

Integrative Imaging Reveals SARS-CoV-2-Induced Reshaping of Subcellular Morphologies

“…To analyze the dynamics of intermediate filament remodeling in real time, we established a SARS-CoV-2 reporter system (Pahmeier et al, 2020) in which a green fluorescent protein (GFP), tagged with a nuclear localization sequence (NLS), is anchored to the cytosolic side of the ER by the transmembrane domain of Sec61b through a linker containing a recognition sequence for the SARS-CoV-2 main protease 3-chymotrypsin-like (3CL) (Figure 5C, top). Upon infection by SARS-CoV-2, the reporter protein is cleaved by 3CL, releasing the GFP-NLS moiety, which then translocates into the nucleus.…”

“…The SARS-CoV-2 reporter construct used for live cell imaging will be described in detail elsewhere (Pahmeier et al, 2020). In brief, it is an engineered fusion protein composed of (from N-to C terminus) green fluorescence protein, the nuclear localization sequence from simian virus 40, a linker sequence containing the consensus cleavage site of the 3CL protease of SARS-CoV-1, and the transmembrane region of Sec61b.…”

Identification of host dependency factors involved in SARS-CoV-2 replication organelle formation through proteomics and ultrastructural analysis

“…To this end, we employed a previously described reporter cell line that utilizes nuclear translocation of a fluorescent protein to identify virus-infected cells ( Fig. 4A ) ( 41 ). Forty-eight hours after reverse transfection of siRNA pools targeting RNAs of the identified interaction partners, cells were infected with SARS-CoV-2 for 24 hours prior to fixation and imaging.…”

A Zika virus-responsive sensor-effector system inAedes aegypti

“…We focused on the NS2B/NS3 protease, which is required to process the virus polyprotein at well-defined sites not cleaved by cellular proteases. Previous studies have shown that recombinant proteins engineered to contain suitable cleavage sites can be selectively cleaved by virus-encoded protease, using proteases and corresponding cleavage sites from several viruses including DENV, leading to modest de-repression or translocation of a fluorescent reporter in transfected cells [4][5][6][7][8] .…”

“…Various protease-based sensors have been demonstrated in mammalian tissue culture cells [4][5][6][7][8] . These imply that our design could readily be adapted to other important mosquito-borne flaviviruses including dengue virus and yellow fever virus, simply by changing the protease-sensitive linker sequence.…”