Reverse Genetics of RNA Viruses

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Reverse Genetics of RNA Viruses

• Chang Won Lee, DVM, Ph.D.
• Lee.2854_at_osu.edu
• Phone [phone]

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Reverse Genetics (RG)

• The creation of a virus with a full-length copy
  of the viral genome
• The most powerful tool in modern virology

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RG of RNA viruses

• Generation or recovery (rescue) of infectious
  virus from cloned cDNA

In vitro-transcribed RNA
cDNA
infectious virus
RNA
OR
cDNA in vector
Infectious Clone
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Nature of RNA viruses

• Polarity ( sense or sense)
• Size of the genome
• Segmented or not
• Site of replication (nucleus or cytoplasm)

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Families of RNA Viruses
Segmented
Non-segmented
Birnaviridae DS RNA 6 kb (IBD) Reoviridae DS
RNA 16-27 kb (Blue Tongue) Arenaviridae
ambisense 10.6 kb (LCV) Bunyaviridae 11-20
kb (Hanta) Orthomyxoviridae 10-13.6
kb (Influenza)
Arteriviridae 13-15 kb (PRRS) Caliciviridae
7.4-7.7 kb (Hepatitis E) Coronaviridae 27-32
kb (SARS) Flaviviridae 9.5-12.5 kb (West
Nile) Picornaviridae 7.2-8.4 kb (FMD) Rhabdoviri
dae 11-15 kb (Rabies) Paramyxoviridae 15-16
kb (Newcastle Disease)
ve sense
-ve sense
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Polarity

• Plus-stranded RNA viruses
• - deproteinated genomes of these viruses are
  able to utilize the host cell machinery to
  initiate their life cycle
• Negative-stranded RNA viruses
• - requires encapsidation with the viral
  nucleoprotein before it can serve as a functional
  template to initiate transcription/replication

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Schematic Diagram of RG Systems
In vitro transcribed RNA
Purified NP and P proteins

OR
OR
Transcription plasmid
Expression plasmids for NP and Ps
Ampr
Ampr
pHH21
pCR3.1
Pol I
CMV
pA
P
T
P
T
RNP complex
vRNA
mRNA
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Infectious Virus
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Construction of a full-length cDNA clone

• Long and tedious!
• Require the presence of the entire viral sequence
• - published sequence
• - or sequencing new isolate
• cDNA synthesis
• - require thermostable and high fidelity reverse
  transcriptase and DNA polymerase
• - require systematic assembly of large RNA
  genome
• - difficult to produce in vitro transcripts
  devoid of vector derived sequences
• Cloning
• - instability of full-length cDNA clones in
  bacteria
• Sequence verification

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Plus-stranded RNA viruses

• Poliovirus infectious clone (1981)
• - Racaneillo and Baltimore, Science 214916
• - cloned in bacterial plasmid pBR322
• Coronavirus
• - Almazan et al., 2000 (PNAS, 975516)
• - Yount et al., 2000 (J Virol, 7410600)
• - Thiel et al., 2001 (J Gen Virol, 821273)

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Engineering the largest RNA virus genome as an
infectious bacterial artificial chromosome
(Almazan et al. 2000)

• Cloning of the cDNAs into a BAC
• Nuclear expression of RNA

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Strategy of systematic assembly of large RNA and
DNA genomes(Yount et al. 2000)

• Simple and rapid approach
• - Mutagenesis and
• systematic cloning
• - Adjoining cDNA
• subclones
• - In vitro transcription
• - RNA transfection

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A cDNA copy of the human coronavirus genome
cloned in vaccinia virus (Thiel et al. 2001)

• In vitro DNA ligation
• Clone into vaccinia virus
• In vitro transcription
• RNA transfection
• Cytoplasmic expression

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Negative-stranded RNA viruses

• Difficulties
• - precise 5 and 3 ends are required for
  replication and packaging of the genomic RNA
• - the viral RNA polymerase is essential for
  transcribing both mRNA and complementary,
  positive-sense antigenomic template RNA
• - both genomic and antigenomic RNAs exist as
  viral ribonucleoprotein (RNP) complexes
• In 1994 (Schnell et al., EMBO, 134195-4203)
• - the rescue of the first NS RNA virus,
  rhabdovirus rabies virus, starting entirely from
  cDNA

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Rescue of non-segmented negative-stranded viruses
T7 Polymerase Expression System - Vaccinia virus
- or Cell lines
Expression plasmids For NP, P, etc.
Transctiption plasmid for genomic RNA


OR
Helper virus
Infectious virus
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Rescue of Influenza Virus

• Family Orthomyxoviridae
• Genera influenza A virus
• influenza B virus
• influenza C virus
• thogotovirus
• Segmented RNA genome
• Negative polarity
• Replicates in the nucleus of infected cells

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Chang-Won Lee, 1996
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Genomes
RNA segments (bp) Protein (aa)
1. Polymerase (basic) 2 (2341) PB2 (759)
2. Polymerase (basic) 1 (2341) PB1
(757) 3. Polymerase (acidic) (2233) PA
(716) 4. Hemagglutinin (1775)
HA (565) 5. Nucleoprotein (1565)
NP (498) 6. Neuraminidase (1413)
NA (454) 7. Matrix (1027)
M1 (252)
M2 (97) 8.
Nonstructural (890)
NS2(NEP)(121)
NS1 (230)
Virion constituents
Infected cells
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Structure of influenza virus
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Life cycle

• Binding
• Endocytosis
• Conformation change of HA
• Fusion
• Uncoating
• Replication
• Glycosylation
• Budding

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Key to generation of influenza virus

• vRNA encapsidated by NP must be transcribed into
  mRNA by the viral polymerase complex
• The vRNP complex is the minimal functional unit

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Helper virus-based method
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RNA polymerase I

• A nucleolar enzyme, which transcribes ribosomal
  RNA
• - In growing cells, rRNA accounts for 80 of the
  total RNA
• A Replacement of the rDNA template with a cDNA
  encoding an influenza viral gene did not impair
  the precise initiation and termination of
  transcription (Neumann et al., 1994)

Ampr
pHH21
T
P
1
-235 -130 -40

12 30
Influenza viral cDNA
UCE Core
T1 T2
Promoter
Terminator
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Plasmid-Based Reverse Genetics
293T
Neumann et al. PNAS 969345-9350, 1999
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Bidirectional pol I - pol II transcription system
Hoffmann, 2000
Hoffmann et al. PNAS, 976108-6113, 2000
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Expression Plasmid
Bi-directional Pol I-Pol II Plasmid
Transcription Plasmid
PB2 PB1 PA HA NP NA M NS
PB2 PB1 PA HA NP NA M NS
PB2 PB1 PA NP
T
P
P
T
CMV
P
A
T
P
P
T
CMV
P
A
T
P
P
T
CMV
P
A
T
P
P
T
CMV
P
A
T
P
P
T
CMV
T
P
P
T
CMV
T
P
P
T
CMV
T
P
P
T
CMV
Transfection
293T or Vero
Amplification
MDCK or MDBK
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Infectious virus
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Generation of RNA polymerase I constructs
Viral RNA
Ampr
Ampr
RT-PCR
OR
pHH21
Bi-pCR3.1
Influenza viral cDNA
CMV
pA
AGTAGAA....TTTTGCT TCATCTT....AAAACGA
P
T
T
P
PCR
BsmBI
GGGTTATTGGAGACGGTACCGTCTCCTCCCCCC CCCAATAACCTCTGCC
ATGGCAGAGGAGGGGGG
CGTCTCNTATTAGTAGAA....TTTTGCTCCCNGAGACG GCAGAGNATA
ATCATCTT....AAAACGAGGGNCTCTGC
BsmBI
BsmBI
TATTAGTAGAA....TTTTGC
TCATCTT....AAAACGAGGG
GGGT TCCCCCC CCCAATAA
GGG
GGGTTATTAGTAGAA.TTTTGCTCCCCCC CCCAATAATCATCTT.
AAAACGAGGGGGG
P
T
Influenza viral cDNA
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Cell lines for transfection

• Species-specificity of the RNA polymerase I
• - Human RNA Pol I 293T, Vero, Cos-1
• - Murine RNA Pol I BHK21
• - Chicken RNA Pol I CEFs, QT-6
• Replication of avian influenza virus
• - MDCK SJPL..Vero or 293T
• Transfection efficiency

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Tranfection Efficiency
MDCK
Vero
293T
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Ampr
Ampr
4 or 9 Expression Plasmid

Bi-pCR3.1
pHH21
OR
CMV
pA
P
T
Transfection
2-3 days
293T
MDCK
Amplification
ECE
2-3 days
Rescued Virus
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References

• Boyer and Haenni. 1994, Virology 198415-426
• Walpita and Flick. 2005, FEMS Microbiol Letters
  2449-18
• Neumann and Kawaoka. 2001, Virology 287243-250

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