Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Summary

We describe the usage of a fluorescent reporter vaccinia virus that enables real-time measurement of viral infectivity and gene expression through the stage-specific expression of spectrally distinct reporter fluorophores. We detail a plate-based method for accurately identifying the stage at which virus replication is affected in response to small molecule inhibition.

Abstract

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. The family also includes the smallpox virus, Variola. Due to the complexity of poxvirus replication, many questions still remain regarding their gene expression strategy. In this article we describe the conceptualization and usage of recombinant vaccinia viruses that enable real-time measurement of single and multiple stages of viral gene expression in a high-throughput format. This is enabled through the use of spectrally distinct fluorescent proteins as reporters for each of three stages of viral replication. These viruses provide a high signal-to-noise ratio while retaining stage specific expression patterns, enabling plate-based assays and microscopic observations of virus propagation and replication. These tools have uses for antiviral discovery, studies of the virus-host interaction, and evolutionary biology.

Introduction

Traditionally, virus expression is studied using molecular biology techniques (e.g. northern blotting, western blotting, microarray hybridization, etc.) ¹. While these methods are capable of providing detailed information with respect to categorizing expression changes of individual mRNAs or proteins, they are typically not amenable to real-time and high-throughput processes. Alternative approaches using fluorescence-based reporters have been applied previously when working with poxviruses; however, their development and usage has been motivated by varied aims. Several such methods were designed for selection of recombinant viruses ^2,....

Protocol

1. Plate Cells

1. Dissociate HeLa cells from plate and dilute in growth media (DMEM, 2 mM L-glut, 10% FBS) to approximately 2.0 x 10⁵ cells/ml. Dispense 100 μl/well in a black-walled, clear flat-bottom 96-well plate (20,000 cells/well).
2. Incubate cells for 24 hr until confluent in a 37 °C incubator + 5% CO₂.

2. Infect Cells

1. Dilute virus and infect cells.
   1. Thaw TrpV (Triple virus; Early Venus, Intermediate mCherry, Late TagBFP) and PLV (Promoter-less Venus) fluorescent virus and disaggregate using sonication for 5 minutes. Alternatively, crude virus stocks can be....

Results

As an example of the typical usage of these viruses, the triple fluorescence-reporter virus was used to compare the point of inhibition of several well-defined poxvirus inhibitors.

HeLa cells were plated in tissue culture treated black-walled clear flat-bottom 96-well plates and incubated overnight. Confluent monolayers were infected at a multiplicity of infection (MOI) of 10 using either triple reporter virus (TrpV; Table 1) or promoter-less Venus (PLV) as detailed on the pla.......

Discussion

 Here, we have described the practical usage of a multi-stage vaccinia reporter virus (TrpV), which provides reproducible, real-time feedback measures of virus replication. By using well-defined poxvirus inhibitors, we were able to show that TrpV responds in a manner consistent with the understood mechanism of action for each inhibitor. While the TrpV virus provides the most comprehensive information about virus stage progression, two-stage (IREV, LREV) and single-stage (EV, IV, LV) viruses can also be used similarl.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco's Modified Eagle Medium (DMEM)	Gibco	11995-065
Fetal Bovine Serum - Optima, Heat Inactivated (FBS)	Atlanta Biologicals	S12450H
200 mM L-Glutamine, (L-glut)	Gibco	25030-081
96 Well Flat Clear Bottom Black Polystyrene TC-Treated Microplates	Corning	3603
384 Well Flat Clear Bottom Black Polystyrene TC-Treated Microplates	Corning	3712
Trypsin, 2X, Sterile, Irradiated	Worthington Biochemical Corp.	TRLVMF
Phosphate-Buffered Saline (PBS)	Gibco	10010-023
Dimethyl Sulfoxide, Cell Culture (DMSO)	American Bioanalytical	AB03091
1-β-D-Arabinofuranosylcytosine  (AraC), MW= 280 g/mol	SigmaAldrich Co	C6645
isatin β -thiosemicarbazone (IBT), MW= 234 g/mol	Fisher Scientific	NC9075202
Rifampicin, MW= 823 g/mol	SigmaAldrich Co	R3501
ST-246, MW= 376 g/mol	SIGA Labs, Corvallis, OR
8% Paraformaldehyde (formaldehyde) aqueous solution	Electron Microscopy Sciences	157-8
TempPlate RT optically clear film	USA Scientific	2978-2700
Opti-MEM Reduced Serum Medium	Gibco	31985-070