HCoV-MERS

216 Pages·2015·3.93 MB·English

Generation of a Human Middle East Respiratory Syndrome
Coronavirus (HCoV-MERS) Infectious Clone System by
Homologous Recombination of Bacterial Artificial Chromosomes

by

Aidan M. Nikiforuk B.A.Sc.

A Thesis submitted to the Faculty of Graduate Studies of

The University of Manitoba
in partial fulfillment of the requirements of the degree of

MASTER OF SCIENCE

Department of Microbiology

University of Manitoba

Winnipeg

Copyright © 2015 by Aidan Nikiforuk

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List of Abbreviations:

ACE-2: angiotensin-converting enzyme two
ARDS: acute respiratory distress syndrome
AUG: codon for methionine and site of translation initiation
BAC2: bacterial artificial chromosome number two, a plasmid vector
BAC: bacterial artificial chromosome
BCV: bovine coronavirus
BGT: rabbit beta-globin terminator sequence, stops the transcription of RNA-dependent
RNA polymerase
BHK-21: baby hamster kidney cells
BtCoV: bat coronavirus
CD26: cluster of differentiation twenty-six, also identified as dipeptidyl peptidase IV
cDNA: complementary DNA, synthesized from RNA by reverse transcriptase PCR
CL-3: containment level 3 laboratory
CMV: cytomegalovirus promoter
CPE: cytopathic effect or cytopathogenic effect
CSCHAH: Canadian Science Center for Human and Animal Health
CSM-Trp: complete supplement medium without tryptophan
DI: defective interfering; may refer to a viral particle or genome
DMEM: Dulbecco’s modified Eagle medium
DNA: deoxyribonucleic acid
dNTPs: deoxynucleotide triphosphates
DPP4: dipeptidyl peptidase IV also known as CD26
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E: coronavirus envelope protein
ECDC: European Center for Disease prevention and Control
ECMO: extracorporeal membrane oxygenation
EMC: Erasmus Medical Center
EMC/2012: an isolate of the HCoV-MERS virus
ER: endoplasmic reticulum
EVD: Ebola virus disease
GFP: green fluorescent protein
Gpt: guanine phosphoribosyltransferase
HA: hemagglutinin, influenza glycoprotein
HCoV: human coronavirus
HCW: health care worker
HDVr: hepatitis delta virus ribozyme
HE: hemagglutinin esterase
HKU4: bat coronavirus Hong-Kong university isolate four
HKU5: bat coronavirus Hong-Kong university isolate five
ICTV: International Committee on Taxonomy of Viruses
IFN: interferon
Kb: kilo-base pairs of DNA
KSA: Kingdom of Saudi Arabia
LB: Luria-Bertani medium for the growth of bacteria
M: coronavirus membrane protein
mBAC4: plasmid construct containing the same mutation in HCoV-MERS genome as
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pYES1L14
mBAC6: final plasmid construct of the HCoV-MERS genome
mBACL23: sub-cloning plasmid construct used to correct mutation in mBAC4
MEM: Minimal Essential Medium for mammalian cell culture
MERS: Middle East respiratory syndrome
MF1: MERS fragment one
MF2: MERS fragment two
MF3: MERS fragment three
MHV: murine hepatitis virus
MODS: multiple organ distress syndrome
mRNA: messenger ribonucleic acid
N: coronavirus nucleoprotein
NS6: HCoV-MERS non-structural protein number six
Nsp: viral non-structural protein
ORF: open reading frame
ORF1a/b: first open reading frame in a coronavirus genome
ORF1b: a RT-qPCR assay used to detect the HCoV-MERS genome
PCR: polymerase chain reaction
pYES1L: a bacterial artificial chromosome plasmid used for in vivo homologous
assembly of the HCoV-MERS fragments (MF1, MF2 and MF3).
pYES1L14: plasmid construct containing the same mutation as mBAC4
RBD: receptor binding domain
RDRP: RNA-dependent RNA polymerase
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RecA: essential protein for the repair and maintenance of DNA in bacteria
RFP: red fluorescent protein

RGS: reverse genetic system
RNA: ribonucleic acid

RT-PCR: reverse-transcriptase polymerase chain reaction

RT-qPCR: real-time quantitative polymerase chain reaction
RTC: coronavirus replication transcription complex

S: coronavirus glycoprotein

SARS: severe acute respiratory syndrome
sBAC: HCoV-SARS infectious cDNA clone system

SDS: sodium dodecyl sulfate

sgRNA: sub-genomic ribonucleic acid
SOC: super optimal catabolite repression broth

SOE-PCR: splice by overlap extension polymerase chain reaction

SSA: single-strand annealing
T7: bacteriophage promoter

TAQ: thermus aquaticus polymerase
TGEV: transmissible gastroenteritis coronavirus
TRS: coronavirus transcription regulatory sequence

upE: a RT-qPCR assay used to detect the HCoV-MERS genome
UTR: un-translated region
vvHCoV: vaccinia virus based coronavirus reverse genetics system

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Abstract

Coronaviruses have caused high pathogenic epidemics within the human population on
two occasions; in 2003 a coronavirus (HCoV-SARS) caused severe acute respiratory syndrome
and in 2012 a novel coronavirus emerged named Middle East respiratory syndrome (HCoV-
MERS). Four other species of coronavirus circulate endemically in the human population
(HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1), which cause more benign
respiratory disease than either HCoV-SARS or HCoV-MERS. The emergence of HCoV-MERS
provides an additional opportunity to study the characteristics of coronaviruses. Reverse genetics
can be used to study an organism’s phenotype by logical mutation of its genotype. Construction
of an infectious clone construct provides a means to investigate the nature of HCoV-MERS by
reverse genetics. An HCoV-MERS infectious cDNA clone system was constructed to use for
reverse genetics by homologous recombination of bacterial artificial chromosomes (BACs). This
system should aid in answering remaining questions of coronavirus genetics and evolution as
well as expedite the development of vaccines and prophylactic treatments for HCoV-MERS.

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Acknowledgements

This work would not have been possible without the support of my family, mentors,
colleagues and friends. I would like to particularly acknowledge Dr. Steven Theriault, Dr.
Deborah Court, Dr. Darwyn Kobasa and Dr. Karen Brassinga for acting as mentors, providing
me with guidance and the opportunity to learn. To Bradley Cook I would like to extend gratitude
for teaching me many laboratory fundamentals and being willing to discuss ideas and results on
any Sunday. Todd Cutts worked to keep the lab running smoothly and I would like to thank him
for training me to work in containment level 3. Anders Leung introduced me to the project,
providing much technical knowledge and “encouragement”. Finally, I would like to mention Dr.
Charlene Ranadeherra and Mable “wing-sum” Hagan because they played music in the lab, were

always keen to share pens and kept Anders in check.

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Dedication
I would like to dedicate this work to all of the people who contributed to my education in
school and more importantly in life. Foremost, my parents Doreen Docherty and Andrew
Nikiforuk deserve recognition for their sacrifice and love. I would also like to acknowledge
Allison Black, Dimitar Kashchiev and the lovely Veronica Izydorcyzk ()..(). I hope that I can

contribute to your lives, as you have to mine.

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Table of Contents
List of Abbreviations: …………………………………………………………………………II
Abstract: ……………………………………………………………………………………….III
Acknowledgments: …………………………………………………………………………….IV
Dedication: ……………………………………………………………………………………..V
List of Figure and Tables: ……………………………………………………………………XII
Copyright Permissions:………………………………………………………………………XIII
Chapter One: Literature Review ....................................................................................................... 14
1.0.0.0: The Nature of Zoonotic Disease ..................................................................................................... 14
1.1.1.0 Introduction ...................................................................................................................................................... 15
1.1.2.0 Order: Nidovirales .......................................................................................................................................... 16
1.1.3.0 Families of the Nidovirales: Arteriviridae, Coronaviridae, Mesoniviridae and Roniviridae . 17
1.1.4.0 Subfamily: Coronavirinae ........................................................................................................................... 17
1.1.5.0 Genus and Lineage: Betacoronavirus, Lineage C ................................................................................ 18
1.2.0.0: Epidemiology of the Middle East Respiratory Syndrome (MERS) ...................................... 19
1.2.1.0: Advent of the HCoV-MERS Epidemic: ................................................................................................ 19
1.2.2.0 Zoonotic Epidemiology: ............................................................................................................................... 20
1.2.3.0 Human-to-Human Transmission of HCoV-MERS .............................................................................. 26
1.4.0.0: Biological Structure of Coronaviruses ......................................................................................... 34
1.4.1.0 Genome Organization ................................................................................................................................... 36
1.4.2.0 Translated Regions ......................................................................................................................................... 39
1.4.3.0 ORF 1a/b Proteins .......................................................................................................................................... 39
1.4.4.0 Glycoprotein (Spike Protein) ...................................................................................................................... 40
1.4.5.0 Envelope and Membrane Proteins ............................................................................................................ 43
1.4.6.0 Nucleocapsid Protein..................................................................................................................................... 43
1.4.7.0 Accessory Proteins ......................................................................................................................................... 44
1.4.8.0 Un-Translated Regions ................................................................................................................................. 45
1.4.9.0 The Coronavirus Leader Sequence, 5’ Un-translated Region (UTR) ............................................ 45
1.4.10.0 Coronavirus 3’ Un-translated Region (UTR) ...................................................................................... 48
1.4.11.0 Coronavirus Transcription Regulatory Sequence (TRS) ................................................................. 48
1.5.0.0: Coronavirus Life Cycle .................................................................................................................... 51
1.5.1.0 Viral Entry ........................................................................................................................................................ 51
1.5.2.0 Transcription and Genome Replication ................................................................................................... 53
1.5.3.0 Discontinuous Negative-Strand Transcription ...................................................................................... 56
1.5.4.0 Coronavirus Genome Replication ............................................................................................................. 59
1.5.5.0 Virus Assembly and Release ...................................................................................................................... 60
1.5.6.0 Overview of Coronavirus Replication ..................................................................................................... 61
1.6.0.0: Reverse Genetics Systems ................................................................................................................ 64
1.6.1.0 Coronavirus Reverse Genetic Systems .................................................................................................... 66
1.6.2.0 Homologous Recombination of DNA for Assembly of Plasmid Vectors .................................... 80
1.6.3.0 In vivo Homologous Recombination ........................................................................................................ 80
1.6.4.0 In vitro Homologous Recombination ....................................................................................................... 82
1.6.5.0 Conclusion ........................................................................................................................................................ 84
1.7.0.0 Declaration of Research Intent and Hypothesis .......................................................................... 84
1.7.0.1 Research Intent ................................................................................................................................................ 84
1.7.0.2 Hypothesis ........................................................................................................................................................ 85
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1.7.0.3 Significance of Research .............................................................................................................................. 85
Chapter Two: Materials and Methods ............................................................................................. 86
2.1.0.0 Materials and Methods ........................................................................................................... 86
2.1.1.0 General Techniques ............................................................................................................................ 86
2.1.1.1 Cell Culture ...................................................................................................................................................... 86
2.1.1.2 Gel-Electrophoresis and Extraction of PCR Products ........................................................................ 87
2.1.1.3 Enzymatic Restriction Digestion of DNA .............................................................................................. 88
2.1.1.4 Amplification of DNA with TAQ Polymerase for TOPO-TA Cloning .................................... 88
2.1.1.5 Screening of Escherichia coli (E.coli) Colonies by Polymerase Chain Reaction ...................... 89
2.1.1.6 Screening of Saccharomyces cerevisiae (S.cerevisiae) Colonies by Polymerase Chain
Reaction ........................................................................................................................................................................... 89
2.1.1.7 Electroporation of E.coli cells with Plasmid DNA .............................................................................. 90
2.1.1.8 Glycerol Stock Preparation of E.coli cells .............................................................................................. 91
2.1.1.9 Isolation of Plasmid Vectors from E.coli cells ...................................................................................... 91
2.1.1.10 Sequencing of Polymerase Chain Reaction Amplicons and Plasmid Constructs .................... 92
2.1.2.0 Strategy for Molecular Cloning of the HCoV-MERS cDNA Infectious Clone System ..... 92
2.1.2.1 Overview ........................................................................................................................................................... 92
2.1.2.2 Synthesis of HCoV-MERS Synthetic Gene Fragments ..................................................................... 93
2.1.2.3 Selection of Bacterial Artificial Chromosome Vector ........................................................................ 93
2.1.2.4 Site Directed Mutagenesis of the TrueBlue-BAC2 Vector ............................................................... 94
2.1.2.5 Primer Design .................................................................................................................................................. 97
2.1.2.6 Splice-by-Overlap Extension Polymerase Chain Reaction ............................................................... 97
2.1.2.7 High-Order Genetic Assembly of the HCoV-MERS Genome ..................................................... 101
2.1.2.8 Sub-cloning the HCoV-MERS Genome .............................................................................................. 103
2.1.2.9 Correction of the HCoV-MERS NS6 Mutation in the mBAC4 Vector ..................................... 106
2.1.2.10 Summary of HCoV-MERS cDNA Infectious Clone System Construction ........................... 109
2.1.3.0 Rescue of the HCoV-MERS virus from the cDNA Infectious Clone Construct ................ 111
2.1.3.1 Rescue of Coronavirus Infectious cDNA Clone Systems in Mammalian Cell Culture ........ 111
2.1.3.2 Viral RNA Isolation.................................................................................................................................... 112
2.1.3.3 Production of HCoV-MERS cDNA From Viral Genomes Extracted by RNA Isolation ..... 112
2.1.3.4 Confirmation of HCoV-MERS Virus Rescue by PCR Amplification of
the Viral RDRP Gene ............................................................................................................................................... 113
2.1.3.5 Phylogenetic Analysis of the HCoV-MERS RDRP Gene Fragment .......................................... 113
2.2.0.0 Results ...................................................................................................................................... 116
2.2.1.0 Objective One .................................................................................................................................... 116
2.2.1.1 Construction of the HCoV-MERS cDNA Genome From Synthesized Gene Fragments ..... 116
2.2.2.0 Objective Two .................................................................................................................................... 119
2.2.2.1 Assembly of the HCov-MERS cDNA Genome Fragments Using S.cerevisiae ...................... 119
2.2.2.2 Electroporation of HCoV-MERS pYES1L Constructs into E. coli Cells .................................. 123
2.2.2.2 Restriction Digest of HCoV-MERS pYES1L Maxi-Preparations ............................................... 125
2.2.2.3 Site Directed Mutagenesis of the True Blue-BAC2 Vector to Remove PvuI and BstEII
Restriction Enzyme Digest Sites ........................................................................................................................... 127
2.2.2.4 Sub-Cloning of the HCoV-MERS Genome Between pYES1L20 and TrueBlue-BAC2
ΔPvuI/ΔBstEII Vectors ............................................................................................................................................ 129
2.2.2.5 In-vitro Homologous Recombination of the HCoV-MERS mBAC4 Plasmid to Correct The
NS6 G11194A Mutation .......................................................................................................................................... 132
2.2.3.0 Objective 3 .......................................................................................................................................... 134
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