Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

Abstract

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be "rescued" when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Introduction

The ability of a virus to productively replicate in a particular host is in part governed by the availability of host cell factors that support viral entry and replication1. The virus-host range can also be dictated by the capacity of a virus to counter cellular antiviral defenses that would otherwise impede viral replication2,3. It is the outcome of these complex virus-host interactions that ultimately decide whether a virus will be able to complete its life cycle in a particular host. Given the potentially pathogenic consequences for the host if viral replication ensues, it is critica....

Protocol

1. General Lymantria dispar (LD652) Cell and Virus Culture

  1. LD652 cell culturing and plating
    1. To culture L. dispar-derived LD652 cells, maintain a monolayer of cells in a growth medium (Table of Materials) incubated at 27 °C under normal atmosphere. Maintain the cells in 10 cm tissue-culture-treated dishes and passage the cells upon reaching 80% confluency.
    2. To plate, dislodge adherent LD652 cells from the plate by pipetting.......

Representative Results

As an example of live-cell imaging applications to monitor VSV rescue upon VACV coinfection, LD652 cells were plated in an 8-well chambered dish and then mock-infected or infected with VSV-DsRed (MOI = 1) in the presence or absence of VACV-FL-GFP (MOI = 25). Because VSV-DsRed expresses DsRed as a free protein and is not fused to structural VSV proteins (Figure 1A), it is only detected after VSV entry and gene expression initiates. All cells were then labeled .......

Discussion

Here we have described simple fluorescence- and luminescence-based assays to screen for conditions that rescue VSV replication in restrictive lepidopteran cell cultures. The abortive infection of VSV in lepidopteran cells creates an excellent signal-to-noise ratio when assaying for VSV gene expression. For example, the LU signals detected in lysates from single VSV-LUC infections were ~1,000-fold higher than in mock-infected lysates, yet these signals only changed approximately twofold over a 72-h time course. In contras.......

Acknowledgements

D.G. was supported by funding from the University of Texas Southwestern Medical Center's Endowed Scholars Program. The authors thank Michael Whitt (The University of Tennessee Health Science Center) and Sean Whelan (Harvard Medical School) for the provision of VSV-DsRed and VSV-LUC. The authors also thank Gary Luker (University of Michigan Medical School) for the kind gift of the VACV-FL-GFP strain.

....

Materials

NameCompanyCatalog NumberComments
6-well tissue culture platesCELLTREAT229106
24-well tissue culture platesCELLTREAT229124
10 cm tissue culture dishesCorningC430167
Grace’s Insect MediumSigmaG8142
EX-CELL 420Sigma14420C
Fetal Bovine Serum - OptimaAtlanta BiologicalsS12450
Growth medium1:1 mixture of Grace's Insect Medium and EX-Cell 420 Serum-Free Medium also containing 1 % antibiotic-antimycotic solution and 10 % Fetal bovine serum
Antibiotic-Antimycotic Solution (100×)SigmaA5955
Dulbecco’s Phosphate Buffered Saline (DPBS)SigmaD8662
Serum Free Media (SFM)Thermo Fisher10902096
Cytosine arabinosideSigmaC1768
Transfection reagentThermo Fisher10362100
Corning cellgro DMSO (Dimethyl Sulfoxide)Corning25950CQC
Reporter lysis buffer 5XPromegaE3971
Luciferase Assay ReagentPromegaE1483
96-Well MicroplatesCorning3915
Mouse anti-FLAG antibodyWako014-22383
Rabbit anti-firefly luciferase antibodyAbcamab21176
Mouse anti-actin antibodySigmaA2066
Mouse anti-VSV MN/AN/ADr. John Connor (Boston University)
Mouse anti-VACV I3LN/AN/ADr. David Evans (University of Alberta)
8-well Chambered dishLab-Tek II155409
Cell viability dyeThermo FisherC12881
FLUOstar microplate readerBMG LabtechFLUOstar
Confocal microscopeOlympusFV10i-LIV
Image analysis softwareOlympusv1.18cellSens software
Eppendorf 5702 ventilated centrifugeEppendorf22628102
Odyssey Fc Infrared Imaging SystemLi-COR BiosciencesOdyssey Fc
LD652 cellsN/AN/ADr. Basil Arif (Natural Resources Canada)
BSC-40 cellsATCCCRL-2761
BHK cellsATCCCCL-10
HeLa cellsATCCCCL-2
BSC-1 cellsATCCCCL-26
in vitro transcription and purification kitThermo FisherAM1626
PCR purification kitQiagen28104

References

  1. Nomaguchi, M., Fujita, M., Miyazaki, Y., Adachi, A. Viral tropism. Frontiers in Microbiology. 3, 281 (2012).
  2. Werden, S. J., McFadden, G. The role of cell signaling in poxvirus tropism: the case of the M-T5 host range protein of myxoma ....

Explore More Articles

ArbovirusViral ImmunologyHost FactorsViral Immune EvasionLuminescence AssayFluorescent AssayLepidopteran Insect CellsVSV LuciferaseVaccinia VirusReporter Lysis Buffer

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2025 MyJoVE Corporation. All rights reserved