Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins

Summary

Generation of recombinant rotaviruses from plasmid DNA provides an essential tool for the study of rotavirus replication and pathogenesis, and the development of rotavirus expression vectors and vaccines. Herein, we describe a simplified reverse genetics approach for generating recombinant rotaviruses, including strains expressing fluorescent reporter proteins.

Abstract

Rotaviruses are a large and evolving population of segmented double-stranded RNA viruses that cause severe gastroenteritis in the young of many mammalian and avian host species, including humans. With the recent advent of rotavirus reverse genetics systems, it has become possible to use directed mutagenesis to explore rotavirus biology, modify and optimize existing rotavirus vaccines, and develop rotavirus multitarget vaccine vectors. In this report, we describe a simplified reverse genetics system that allows the efficient and reliable recovery of recombinant rotaviruses. The system is based on co-transfection of T7 transcription vectors expressing full-length rotavirus (+)RNAs and a CMV vector encoding an RNA capping enzyme into BHK cells constitutively producing T7 RNA polymerase (BHK-T7). Recombinant rotaviruses are amplified by overseeding the transfected BHK-T7 cells with MA104 cells, a monkey kidney cell line that is highly permissive for virus growth. In this report, we also describe an approach for generating recombinant rotaviruses that express a separate fluorescent reporter protein through the introduction of a 2A translational stop-restart element into genome segment 7 (NSP3). This approach avoids deleting or modifying any of the viral open reading frames, thus allowing the production of recombinant rotaviruses that retain fully functional viral proteins while expressing a fluorescent protein.

Introduction

Rotaviruses are major causes of severe gastroenteritis in infants and young children, as well as the young of many other mammalian and avian species¹. As members of the Reoviridae family, rotaviruses have a segmented double-stranded RNA (dsRNA) genome. The genome segments are contained within a nonenveloped icosahedral virion formed from three concentric layers of protein². Based on sequencing and phylogenetic analysis of the genome segments, nine species of rotavirus (A−D, F−J) have been defined³. Those strains comprising rotavirus species A are responsible for the vast major....

Protocol

1. Media preparation and cell culture maintenance

1. Obtain baby hamster kidney cells constitutively expressing T7 RNA polymerase (BHK-T7) and African green monkey kidney MA104 cells.
   NOTE: BHK-T7 (or BSR-T7) cells are not commercially available, but are a common cell line of laboratories using reverse genetics to study RNA virus biology. The BHK-T7 cell line used in this protocol was obtained from Dr. Ursula J. Buchholz (National Institutes of Health, Bethesda, MD, USA), a co-developer of the original BHK cell line expressing T7 RNA polymerase²⁹. MA104 cells are available from European Collection of Authenticated Cell Cultures ....

Results

The reverse genetics protocol described in this article proceeds through multiple distinct steps: (1) co-transfection of BHK-T7 cells with rotavirus pT7 transcription vectors and a pCMV/NP868R expression plasmid, (2) overseeding of transfected BHK-T7 cells with MA104 cells, (3) amplification of recombinant viruses present in BHK-T7/MA104 cells lysates using MA104 cells, and (4) plaque isolation of recombinant virus using MA104 cells (Figure 2). In our hands, the protocol is efficient, yieldi.......

Discussion

In our laboratory, we routinely rely on the reverse genetics protocol described herein to produce recombinant SA11 rotaviruses. With this approach, individuals with little experience in molecular biology techniques or working with rotaviruses recover recombinant viruses even on their first attempt. We have generated close to 100 recombinant viruses following this protocol, including those with genomes that have been re-engineered to express foreign proteins (e.g., FPs) and that contain sequence additions, deletions, and .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Baby Hamster Kidney - T7 RdRP (BHK-T7) Cells			Contact: ubuchholz@niaid.nih.gov
Bio-Rad 8-16% Tris-Glycine Polyacrylamide Mini-Gel	Bio-Rad	45608105
Cellometer AutoT4 viable cell counter	Nexcelom
ChemiDoc MP Gel Imaging System	Bio-Rad
Chloroform	MP	194002
Clarity Western Enhanced Chemiluminescence (ECL) Substrate	Bio-Rad	170-5060
Competent E.coli DH5alpha Bacteria	Lucigen	60602-2
Complete Protease Inhibitor	Pierce	A32965
Disposable Transfer Pipettes, Ultrafine Extended Tips	MTC Bio	P4113-11
Dulbecco's Modified Eagle Medium (DMEM)	Lonza	12-604F
Eagle's Minimal Essential Medium, 2x (2xEMEM)	Quality Biological	115-073-101
Ethanol, Absolute (200 proof)	Fisher Bioreagents	BP2818-500
Ethidium Bromide Solution (10 mg/ml)	Invitrogen	15585-011
Fetal Bovine Serum (FBS)	Corning	35-010-CV
Fetal Bovine Serum (FBS), Heat Inactivated	Corning	35-011-CV
Flag M2 Antibody, Mouse Monoclonal	Sigma-Aldrich	F1804
GenEluate HP Plasmid Midiprep Kit	Sigma	NA0200-1KT
Geneticin (G-418)	Invitrogen	10131-027
Gibco FluroBrite DMEM	ThermoFisher	A1896701	DMEM with low background fluorescence
Glasgow Minimal Essential Medium (GMEM)	Gibco	11710-035
Goat Anti-Rabbit IgG, Horseradish Peroxidase (HRP) Conjugated	Cell-Signaling Technology	7074S
Guinea Pig Anti-NSP3 Antiserum	Patton lab	lot 55068
Guinea Pig Anti-VP6 Antierum	Patton lab	lot 53963
Horse Anti-Guinea Pig IgG, Horseradish Peroxidase (HRP) Conjugated	KPL	5220-0366
Horse Anti-Mouse IgG, Horseradish eroxidase (HRP) Conjugated	Cell-Signaling Technology	7076S
iNtRON Biotechnology e-Myco Mycoplasma PCR Detection Kit	JH Science	25235
Isopropyl alcohol	Macron	3032-02
L-glutamine Solution (100x)	Gibco	25030-081
Luria Agar Powder (Miller's LB Agar)	RPI research products	L24020-2000.0
Medium 199 (M199) Culture Medium	Hyclone	Sh30253.01
Minimal Essential Medium -Eagle Joklik's Forumation (SMEM)	Lonza	04-719Q
Monkey Kidney (MA104) Cells	ATCC	ATCC CRL-2378.1
NanoDrop One Spectrophotometer	ThermoScientific
Neutral Red Solution (0.33%)	Sigma-Aldrich	N2889-100ml
Non-Essential Amino Acid Solution (100x)	Gibco	11140-050
Novex 10% Tris-Glycine Polyacrylamide Mini-Gel	Invitrogen	XP00102BOX
Nuclease-Free Molecular Biology Grade Water	Invitrogen	10977-015
NucleoSpin Gel and PCR Clean-Up Kit	Takara	740609.25
Opti-MEM Reduced Serum Medium	Gibco	31985-070
Pellet pestle (RNase-free, disposable)	Fisher	12-141-368
Penicillin-Streptomycin Solution, (100x penn-strep)	Corning	30-002-Cl
Phosphate Buffered Saline (PBS), 10x	Fisher Bioreagents	BP399-20
Porcine Trypsin, Type IX-S	Sigma-Aldrich	T0303
PureYield Plasmid Miniprep System	Promega	A1223
Qiagen Plasmid Maxi Kit	Qiagen	12162
Qiagen Plasmid Midi Kit	Qiagen	12143
QIAprep Spin Miniprep Kit	Qiagen	27104
SA11 pT7 Transcription Vectors	Addgene	89162-89172
SA11 pT7/NSP3 Transcription Vectors Expressing Fluorescent Proteins			Contact: jtpatton@iu.edu
SeaKem LE Agarose	Lonza	50000	For gel electrophoresis
SeaPlaque agarose	Lonza	50100	For plaque assay
Superscript III One-Step RT-PCR kit	Invitrogen	12574-035
Trans-Blot Turbo Nitrocellulose Transfer Kit	Bio-Rad	170-4270
Trans-Llot Turbo Transfer System	Bio-Rad
TransIT-LTI Transfection Reagent	Mirus	MIR2306
Tris-Glycine-SDS Gel Running Buffer (10x)	Bio-Rad	161-0772
Triton X 100	Fisher Bioreagents	BP151-500
Trizol RNA Extraction Reagent	Ambion	15596026
Trypan blue	Corning	25-900-CI
Trypsin (0.05%)-EDTA (0.1%) Cell Dissociation Solution	Quality Biological	118-087-721
Tryptose Phosphate Broth	Gibco	18050-039
Tween-20	VWR	0777-1L
Vertrel VF solvent	Zoro	G0707178
Zoe Fluorescent Live Cell Imager	Bio-Rad