Reverse Genetics of RNA Viruses

Chang Won Lee, DVM, Ph.D. Lee.2854@osu.edu Phone: 330-263-3750

VPM 700 May06

Reverse Genetics (RG)

The creation of a virus with a fulllength 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

cDNA In vitro-transcribed RNA OR cDNA in vector Infectious Clone
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RNA

infectious virus

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

Non-segmented 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) 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)

+ve sense -ve sense

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 OR Transcription plasmid
Ampr

Purified NP and P proteins OR Expression plasmids for NP and Ps

Ampr

pHH21
Pol I P

pCR3.1
CMV P
T pA

RNP complex vRNA mRNA

Infectious Virus

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Construction of a fulllength 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 VPM 700
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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 genome as an infectious bacterial artificial chromosome (Almazan et al. 2000)

Cloning of the cDNAs into a BAC Nuclear expression of RNA

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

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

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, 13:4195-4203) - 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

Transctiption plasmid for genomic RNA

T7 Polymerase Expression System - Vaccinia virus - or Cell lines

Expression plasmids For NP, P, etc. OR

Helper virus

Infectious virus

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Rescue of Influenza Virus

Chang-Won Lee, 1996

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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Genomes
RNA segments (bp)
1. Polymerase (basic) 2 (2341) 2. Polymerase (basic) 1 (2341) 3. Polymerase (acidic) (2233)

Protein (aa)
PB2 (759) PB1 (757) PA (716)

4. Hemagglutinin (1775)
5. Nucleoprotein (1565) 6. Neuraminidase (1413) 7. Matrix (1027)

HA (565)
NP (498) NA (454) M1 (252) M2 (97)

Virion constituents

8. Nonstructural (890)

NS2(NEP)(121) NS1 (230)

Infected cells

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
P T

+1
-235 -130 -40 +12 +30

Influenza viral cDNA

UCE

Core

T1

T2 VPM 700 May06

Promoter

Terminator

Plasmid-Based Reverse Genetics

293T

Neumann et al. PNAS 96:9345-9350, 1999

Bidirectional pol I - pol II transcription system

Hoffmann, 2000

Hoffmann et al. PNAS, 97:6108-6113, 2000

Transcription Plasmid Expression Plasmid

Bi-directional Pol I-Pol II Plasmid

PB2 PB1 PA HA NP NA M NS

P P P P P P P P

T T T T T T

PB2 PB1 PA NP

P P P P

A A A A

T T

PB2 CMV T PB1 CMV T PA CMV T HA CMV T NP CMV T NA CMV T CMV T M NS CMV T

P P P P P P P P

Transfection 293T or Vero

Amplification MDCK or MDBK
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Infectious virus

Generation of RNA polymerase I constructs

Ampr
pHH21
P T

Ampr
OR
CMV

Viral RNA RT-PCR

P pA Influenza viral cDNA AGTAGAA....TTTTGCT TCATCTT....AAAACGA

Bi-pCR3.1
T

BsmBI
GGGTTATTGGAGACGGTACCGTCTCCTCCCCCC CCCAATAACCTCTGCCATGGCAGAGGAGGGGGG

PCR
CGTCTCNTATTAGTAGAA....TTTTGCTCCCNGAGACG GCAGAGNATAATCATCTT....AAAACGAGGGNCTCTGC

BsmBI
GGGT CCCAATAA TCCCCCC GGG

BsmBI
TATTAGTAGAA....TTTTGC TCATCTT....AAAACGAGGG

GGGTTATTAGTAGAA.TTTTGCTCCCCCC CCCAATAATCATCTT.AAAACGAGGGGGG

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 pHH21

4 or 9 Expression Plasmid

OR

CMV

Bi-pCR3.1
pA

Transfection
293T MDCK

2-3 days

Amplification

ECE

2-3 days

Rescued Virus

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References

Boyer and Haenni. 1994, Virology 198:415-426 Walpita and Flick. 2005, FEMS Microbiol Letters 244:9-18 Neumann and Kawaoka. 2001, Virology 287:243-250

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