Reverse Genetics - Part I

8/2/2019 Reverse Genetics - Part I

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Reverse Genetics of

RNA Viruses

Chang Won Lee, DVM, [email protected]

Phone: 330-263-3750

VPM 700May06
mailto:[email protected]:[email protected]


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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
http://vir.sgmjournals.org/content/vol82/issue6/images/large/0821273002.jpeg


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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

Chang-Won Lee, 1996 VPM 700May06


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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
http://www.pnas.org/content/vol96/issue16/images/large/pq1692169003.jpeg


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Plasmid-Based

Reverse Genetics293T

Neumann et al. PNAS

96:9345-9350, 1999
http://www.pnas.org/content/vol96/issue16/images/large/pq1692169003.jpeghttp://www.pnas.org/content/vol96/issue16/images/large/pq1692169003.jpeg


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Hoffmann, 2000

Bidirectional pol I - pol II transcription system

Hoffmann et al. PNAS, 97:6108-6113, 2000
http://www.pnas.org/content/vol97/issue11/images/large/pq1101336001.jpeghttp://www.pnas.org/content/vol97/issue11/images/large/pq1101336001.jpeg


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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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Reverse Genetics - Part I

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