Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

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 replication^2,3. 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.......

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.......

Materials

Name	Company	Catalog Number	Comments
6-well tissue culture plates	CELLTREAT	229106
24-well tissue culture plates	CELLTREAT	229124
10 cm tissue culture dishes	Corning	C430167
Grace’s Insect Medium	Sigma	G8142
EX-CELL 420	Sigma	14420C
Fetal Bovine Serum - Optima	Atlanta Biologicals	S12450
Growth medium			1:1 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×)	Sigma	A5955
Dulbecco’s Phosphate Buffered Saline (DPBS)	Sigma	D8662
Serum Free Media (SFM)	Thermo Fisher	10902096
Cytosine arabinoside	Sigma	C1768
Transfection reagent	Thermo Fisher	10362100
Corning cellgro DMSO (Dimethyl Sulfoxide)	Corning	25950CQC
Reporter lysis buffer 5X	Promega	E3971
Luciferase Assay Reagent	Promega	E1483
96-Well Microplates	Corning	3915
Mouse anti-FLAG antibody	Wako	014-22383
Rabbit anti-firefly luciferase antibody	Abcam	ab21176
Mouse anti-actin antibody	Sigma	A2066
Mouse anti-VSV M	N/A	N/A	Dr. John Connor (Boston University)
Mouse anti-VACV I3L	N/A	N/A	Dr. David Evans (University of Alberta)
8-well Chambered dish	Lab-Tek II	155409
Cell viability dye	Thermo Fisher	C12881
FLUOstar microplate reader	BMG Labtech	FLUOstar
Confocal microscope	Olympus	FV10i-LIV
Image analysis software	Olympus	v1.18	cellSens software
Eppendorf 5702 ventilated centrifuge	Eppendorf	22628102
Odyssey Fc Infrared Imaging System	Li-COR Biosciences	Odyssey Fc
LD652 cells	N/A	N/A	Dr. Basil Arif (Natural Resources Canada)
BSC-40 cells	ATCC	CRL-2761
BHK cells	ATCC	CCL-10
HeLa cells	ATCC	CCL-2
BSC-1 cells	ATCC	CCL-26
in vitro transcription and purification kit	Thermo Fisher	AM1626
PCR purification kit	Qiagen	28104