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Reverse Genetics of
RNA Viruses
Chang Won Lee, DVM, [email protected]
Phone: 330-263-3750
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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 modernvirology
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RG of RNA viruses
Generation or recovery (rescue) of
infectious virus from cloned cDNA
RNA cDNA infectious virus
Infectious Clone
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In vitro-transcribed RNA
cDNA in vector
OR
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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
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)
Rhabdoviridae: 11-15 kb(Rabies)Paramyxoviridae: 15-16 kb
(Newcastle Disease)
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)
Non-segmented Segmented
+
vesense
-vesens
e
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Polarity
Plus-stranded RNA viruses
- deproteinated genomes of these viruses are ableto utilize the host cell machinery to initiate their lifecycle
Negative-stranded RNA viruses
- requires encapsidation with the viral nucleoproteinbefore it can serve as a functional template toinitiate transcription/replication
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Schematic Diagram of
RG SystemsIn vitro transcribed RNA Purified NP and P proteins
vRNA
RNP complex
mRNA
Infectious Virus
Ampr
P T
pHH21Pol I
Ampr
P T
pCR3.1CMV pA
Transcription plasmid Expression plasmids for NP and Ps
+OR OR
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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 reversetranscriptase and DNA polymerase- require systematic assembly of large RNA genome
- difficult to produce in vitro transcripts devoid ofvector derived sequences
Cloning- instability of full-length cDNA clones in bacteria
Sequence verification VPM 700May06
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Plus-stranded RNA
viruses Poliovirus infectious clone (1981)
- Racaneillo and Baltimore, Science 214:916
- cloned in bacterial plasmid pBR322
Coronavirus
- Almazan et al., 2000 (PNAS, 97:5516)- Yount et al., 2000 (J Virol, 74:10600)
- Thiel et al., 2001 (J Gen Virol, 82:1273)
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Engineering the largest RNA virus genomeas an infectious bacterial artificial
chromosome (Almazan et al. 2000)
Cloning of thecDNAs into a BAC
Nuclear expression
of RNA
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Strategy of systematicassembly of large RNAand DNA genomes
(Yount et al. 2000)
Simple and rapidapproach
- Mutagenesis and
systematic cloning
- Adjoining cDNA
subclones
- In vitro transcription
- RNA transfection
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A cDNA copy of the humancoronavirus genome cloned
in vaccinia virus (Thiel etal. 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 replicationand packaging of the genomic RNA
- the viral RNA polymerase is essential fortranscribing both mRNA and complementary,positive-sense antigenomic template RNA- both genomic and antigenomic RNAs exist as viralribonucleoprotein (RNP) complexes
In 1994 (Schnell et al., EMBO, 13:4195-4203)- the rescue of the first NS RNA virus, rhabdovirusrabies virus, starting entirely from cDNA
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Rescue of non-segmented
negative-stranded virusesT7 PolymeraseExpression System- Vaccinia virus
- or Cell lines
Transctiption plasmidfor genomic RNA
Expression plasmidsFor NP, P, etc.
Helper virus
OR
+ +
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 ofinfected cells
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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)
Virionconstituents
Infected cells
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Structure of influenza
virus
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Binding
Endocytosis
Conformationchange of HA
Fusion
Uncoating
Replication Glycosylation
Budding
Life cycle
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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 andtermination of transcription (Neumann et al., 1994)
Ampr
P T
pHH21
-235 -130 -40 +12 +30
+1
UCE Core T1 T2
Influenza viral cDNA
Promoter Terminator VPM 700May06
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Plasmid-Based
Reverse Genetics293T
Neumann et al. PNAS
96:9345-9350, 1999
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Hoffmann, 2000
Bidirectional pol I - pol II transcription system
Hoffmann et al. PNAS, 97:6108-6113, 2000
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P T
P T
P T
P T
P T
P T
P T
P T
P A
P A
P A
P A
PB2PB1PAHANPNAMNS
PB2PB1PA
NP
Transcription Plasmid Expression Plasmid
Transfection
Amplification
Infectious virus
T P
T P
T P
T P
T P
T P
T P
T P
PB2PB1PAHANPNAMNS
Bi-directional Pol I-Pol II Plasmid
CMV
CMV
CMV
CMV
CMV
CMV
CMV
CMV
293T or Vero
MDCK or MDBK
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Ampr
P T
pHH21
Ampr
T P
Bi-pCR3.1CMV pA
Generation of RNA polymerase I constructs
AGTAGAA....TTTTGCT
TCATCTT....AAAACGA
CGTCTCNTATTAGTAGAA....TTTTGCTCCCNGAGACG
GCAGAGNATAATCATCTT....AAAACGAGGGNCTCTGC
GGGTTATTGGAGACGGTACCGTCTCCTCCCCCC
CCCAATAACCTCTGCCATGGCAGAGGAGGGGGG
GGGT TCCCCCC
CCCAATAA GGG
TATTAGTAGAA....TTTTGC
TCATCTT....AAAACGAGGG
GGGTTATTAGTAGAA.TTTTGCTCCCCCC
CCCAATAATCATCTT.AAAACGAGGGGGG
ORInfluenza viral cDNA
Viral RNA
PCR
BsmBIBsmBI
P TInfluenza viral cDNA
BsmBI
RT-PCR
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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
P T
pHH21
Ampr
Bi-pCR3.1CMV pA
OR
ECE
293T
Rescued Virus
Transfection
Amplification
2-3 days
2-3 days
MDCK
4 or 9Expression
Plasmid+
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References
Boyer and Haenni. 1994, Virology198:415-426
Walpita and Flick. 2005, FEMSMicrobiol Letters 244:9-18
Neumann and Kawaoka. 2001,
Virology 287:243-250
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