Reverse Genetics of Reverse Genetics of RNA VirusesRNA Viruses
Chang Won Lee, DVM, Ph.D.Chang Won Lee, DVM, Ph.D.
[email protected]@osu.edu
Phone: 330-263-3750Phone: 330-263-3750
VPM 700May’06
Reverse Genetics (RG)Reverse Genetics (RG)
The creation of a virus with a full-The creation of a virus with a full-length copy of the viral genomelength copy of the viral genome
The most powerful tool in modern The most powerful tool in modern virology virology
VPM 700May’06
RG of RNA virusesRG of RNA viruses
Generation or recovery (Generation or recovery (rescuerescue) ) of infectious virus from cloned of infectious virus from cloned cDNAcDNA
RNA cDNA infectious virus
“Infectious Clone”
VPM 700May’06
In vitro-transcribed RNA
cDNA in vectorOR
Nature of RNA virusesNature of RNA viruses
Polarity (+ sense or – sense)Polarity (+ sense or – sense) Size of the genomeSize of the genome Segmented or notSegmented or not Site of replication (nucleus or Site of replication (nucleus or
cytoplasm)cytoplasm)
VPM 700May’06
Families of RNA Families of RNA VirusesViruses
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
+ve s
en
se
-ve s
en
se
PolarityPolarity
Plus-stranded RNA virusesPlus-stranded RNA viruses- deproteinated genomes of these viruses are - deproteinated genomes of these viruses are able to utilize the host cell machinery to able to utilize the host cell machinery to initiate their life cycleinitiate their life cycle
Negative-stranded RNA virusesNegative-stranded RNA viruses- requires encapsidation with the viral - requires encapsidation with the viral nucleoprotein before it can serve as a nucleoprotein before it can serve as a functional template to initiate functional template to initiate transcription/replication transcription/replication
VPM 700May’06
Schematic Diagram of Schematic Diagram of RG SystemsRG Systems
In 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
VPM 700May’06
Construction of a full-Construction of a full-length cDNA clonelength cDNA clone Long and tedious!Long and tedious! Require the presence of the entire viral sequenceRequire the presence of the entire viral sequence
- published sequence- published sequence- or sequencing new isolate- or sequencing new isolate
cDNA synthesis cDNA synthesis - require thermostable and high fidelity reverse - require thermostable and high fidelity reverse transcriptase and DNA polymerasetranscriptase and DNA polymerase- require systematic assembly of large RNA genome- require systematic assembly of large RNA genome- difficult to produce in vitro transcripts devoid of - difficult to produce in vitro transcripts devoid of vector derived sequencesvector derived sequences
CloningCloning- instability of full-length cDNA clones in bacteria- instability of full-length cDNA clones in bacteria
Sequence verificationSequence verification
VPM 700May’06
Plus-stranded RNA Plus-stranded RNA virusesviruses Poliovirus infectious clone (1981)Poliovirus infectious clone (1981)
- Racaneillo and Baltimore, Science - Racaneillo and Baltimore, Science 214:916214:916
- cloned in bacterial plasmid pBR322- cloned in bacterial plasmid pBR322
CoronavirusCoronavirus
- Almazan et al., 2000 (PNAS, 97:5516)- Almazan et al., 2000 (PNAS, 97:5516)
- Yount et al., 2000 (J Virol, 74:10600)- Yount et al., 2000 (J Virol, 74:10600)
- Thiel et al., 2001 (J Gen Virol, 82:1273) - Thiel et al., 2001 (J Gen Virol, 82:1273) VPM 700May’06
Engineering the largest RNA virus Engineering the largest RNA virus genome as an infectious bacterial genome as an infectious bacterial artificial chromosome (Almazan et al. artificial chromosome (Almazan et al. 2000)2000)
Cloning of the Cloning of the cDNAs into a BACcDNAs into a BAC
Nuclear Nuclear expression of RNAexpression of RNA
Strategy of systematic Strategy of systematic assembly of large RNA assembly of large RNA and DNA genomesand DNA genomes((Yount et al. 2000)Yount et al. 2000)
Simple and rapid Simple and rapid approachapproach
- Mutagenesis and - Mutagenesis and
systematic cloningsystematic cloning
- Adjoining cDNA- Adjoining cDNA
subclonessubclones
- In vitro - In vitro transcriptiontranscription
- RNA transfection- RNA transfection
A cDNA copy of the A cDNA copy of the human coronavirus human coronavirus genome cloned in genome cloned in vaccinia virus (Thiel et vaccinia virus (Thiel et al. 2001)al. 2001)
In vitro DNA ligationIn vitro DNA ligation Clone into vaccinia Clone into vaccinia
virusvirus In vitro transcriptionIn vitro transcription RNA transfectionRNA transfection Cytoplasmic expressionCytoplasmic expression
Negative-stranded Negative-stranded RNA virusesRNA viruses DifficultiesDifficulties
- precise 5’ and 3’ ends are required for - precise 5’ and 3’ ends are required for replication and packaging of the genomic RNAreplication and packaging of the genomic RNA- the viral RNA polymerase is essential for - the viral RNA polymerase is essential for transcribing both mRNA and complementary, transcribing both mRNA and complementary, positive-sense antigenomic template RNApositive-sense antigenomic template RNA
- both genomic and antigenomic RNAs exist as - both genomic and antigenomic RNAs exist as viral ribonucleoprotein (RNP) complexesviral ribonucleoprotein (RNP) complexes
In 1994 (Schnell et al., EMBO, 13:4195-4203)In 1994 (Schnell et al., EMBO, 13:4195-4203)
- the rescue of the first NS RNA virus, - the rescue of the first NS RNA virus, rhabdovirus rabies virus, starting entirely from rhabdovirus rabies virus, starting entirely from cDNAcDNA
VPM 700May’06
Rescue of non-Rescue of non-segmented negative-segmented negative-stranded virusesstranded viruses
T7 PolymeraseExpression System- Vaccinia virus - or Cell lines
Transctiption plasmid for genomic RNA
Expression plasmids For NP, P, etc.
Helper virus
OR
+ +
Infectious virusVPM 700May’06
Rescue of Influenza Rescue of Influenza VirusVirus
Family : Family : OrthomyxoviridaeOrthomyxoviridae Genera Genera influenza A virusinfluenza A virus
influenza B virusinfluenza B virus influenza C virusinfluenza C virus thogotovirusthogotovirus
Segmented RNA genomeSegmented RNA genome Negative polarityNegative polarity Replicates in the nucleus of Replicates in the nucleus of
infected cellsinfected cells Chang-Won Lee, 1996 VPM 700
May’06
GenomesGenomes
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
Structure of influenza Structure of influenza virusvirus
VPM 700May’06
Binding Endocytosis Conformati
on change of HA
Fusion Uncoating Replication Glycosylati
on Budding
Life cycle
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Key to generation of Key to generation of influenza virusinfluenza virus vRNA encapsidated by NP must be transcribed
into mRNA by the viral polymerase complex The vRNP complex is the minimal functional
unit
VPM 700May’06
Helper virus-based Helper virus-based methodmethod
VPM 700May’06
RNA polymerase IRNA polymerase I
A nucleolar enzyme, which transcribes ribosomal RNAA nucleolar enzyme, which transcribes ribosomal RNA- In growing cells, rRNA accounts for 80% of the total RNA- In growing cells, rRNA accounts for 80% of the total RNA
A Replacement of the rDNA template with a cDNA encoding A Replacement of the rDNA template with a cDNA encoding an influenza viral gene did not impair the precise initiation an influenza viral gene did not impair the precise initiation and termination of transcription (Neumann and termination of transcription (Neumann et alet al., 1994)., 1994)
Ampr
P T
pHH21
-235 -130 -40 +12 +30 +1
UCE Core T1 T2
Influenza viral cDNA
Promoter Terminator VPM 700May’06
Plasmid-BasedReverse Genetics
293T
Neumann et al. PNAS 96:9345-9350, 1999
Hoffmann, 2000
Bidirectional pol I - pol II transcription system
Hoffmann et al. PNAS, 97:6108-6113, 2000
P TP TP TP TP TP TP TP T
P AP AP AP A
PB2PB1PAHANPNAMNS
PB2PB1PANP
Transcription PlasmidExpression Plasmid
Transfection
Amplification
Infectious virus
T PT PT PT PT PT PT PT P
PB2PB1PAHANPNAMNS
Bi-directional Pol I-Pol II Plasmid
CMV
CMV
CMV
CMV
CMV
CMV
CMV
CMV
293T or Vero
MDCK or MDBK
VPM 700May’06
Ampr
P T
pHH21
Ampr
T P
Bi-pCR3.1CMV pA
Generation of RNA polymerase I constructs
AGTAGAA....TTTTGCTTCATCTT....AAAACGA
CGTCTCNTATTAGTAGAA....TTTTGCTCCCNGAGACGGCAGAGNATAATCATCTT....AAAACGAGGGNCTCTGC
GGGTTATTGGAGACGGTACCGTCTCCTCCCCCCCCCAATAACCTCTGCCATGGCAGAGGAGGGGGG
GGGT TCCCCCCCCCAATAA GGG
TATTAGTAGAA....TTTTGC TCATCTT....AAAACGAGGG
GGGTTATTAGTAGAA…….TTTTGCTCCCCCCCCCAATAATCATCTT…….AAAACGAGGGGGG
ORInfluenza viral cDNA
Viral RNA
PCR
BsmBIBsmBI
P TInfluenza viral cDNA
BsmBI
RT-PCR
VPM 700May’06
Cell lines for Cell lines for transfectiontransfection 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 efficiencyVPM 700May’06
Tranfection EfficiencyTranfection Efficiency
MDCK Vero 293T
VPM 700May’06
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+
VPM 700May’06
ReferencesReferences
Boyer and Haenni. 1994, Virology Boyer and Haenni. 1994, Virology 198:415-426198:415-426
Walpita and Flick. 2005, FEMS Walpita and Flick. 2005, FEMS Microbiol Letters 244:9-18Microbiol Letters 244:9-18
Neumann and Kawaoka. 2001, Neumann and Kawaoka. 2001, Virology 287:243-250Virology 287:243-250
VPM 700May’06
