Viral vector

by
Imran Amin
 Virus
The word “virus” is of Latin origin with the meaning of slimy
liquid, poison, stench, and in addition even offensive taste, clearly
something unfavourable1

Viruses are obligate intracellular parasites and they are dependent

on the molecular machinery of their host for their replication

Living or nonliving??

viruses, especially bacteriophages, are the "most abundant

biological entities” on the planet
1
Webster’s Third New International Dictionary III pp 2556, 1981. Oxford English
 Infectious Viruses: A Genetic “Syringe”
Viruses are composed of genetic DNA
material encapsulated in a Loaded
protein coat. Syringe

Viruses inject their genetic

material into target cells.
Viruses infect target cells with their genetic material.

The viral DNA can be altered to contain a gene of interest to infect that
gene into the target cell.

Viral DNA Virus Target Cell Infected With Viral

Gene of Interest DNA Containing The Gene of
Interest

Cell’s DNA
Target Cell
 Viruses as Vector

•Any virus can potentially be used to express foreign genes

•Different viruses are better suited for different kinds of uses

•Integration may be important, such as in many gene therapy uses

•Larger viruses can express more and larger foreign genes but are
more difficult to manipulate

•The cis-acting promoters for genome replication and packaging

must be understood
 The ideal vector for gene transfer
• High concentration of virus allowing many cells to be infected

• Convenience and reproducibility of production

• Ability to infect and non-dividing cells

• Ability to integrate transduce into a site-specific location in the host

chromosome, or to be successfully maintained as stable episome

• Ability to target the desired type of cell

• No components that elicit an immune response

 Potential uses of viral vectors in animals
•Gene therapy to replace a missing or inadequate gene

•Cure for illness by expressing a reagent to, for example, kill

cancer cell

•Immunization by expressing an antigen from a pathogen

•Expression of gene in cultured cells for scientific study

 Viral vector in gene therapy
Gene therapy
The introduction of nucleic acids into cells for the purpose of
altering the course of medical condition or disease.

Administration
• ex vivo- cells removed, genetically modified,
transplanted back into a patient

• in vivo- direct transfer of genetic material into

patient
 Retroviruses
(including Lentivirus, HIV and MuLV based vectors)

Retrovirus

ssRNA Genome
•Single stranded RNA genome
•Lipid membrane enveloped Reverse Transcription
•Host range determined by envelope into dsDNA

proteins Random integration

into host genome

Host DNA

Host Cell
 The Retroviral Genome
LTR  LTR
gag pol env

Long Terminal Repeat (LTR): Necessary for integration into

host genome
 (Psi): packaging signal
gag: Packages viral genome into viral particles

pol: viral polymerase necessary for viral replication

env: viral envelope proteins, necessary for entry into host

cells, dictate host range
 Design of replication incompetent vectors

The viral vector is “gutted” as much as possible to

create room for the insert gene and to divide the viral
genome into cis- and trans- acting regions
Promoter and Insert Gene
LTR∆ LTR ∆
gag pol env Modified LTR
To impede the
Virus from
Performing more
than one round
of reverse
transcription
Transfer Packaging Envelope
Vector Vector Vector
“self inactivating”

Regulatory Structural &

Packaging Genes Viral envelope
Signals (LTR and
(may already be protein alters host
), promoter and
present in range of the viral
Insert Gene
packaging line) vector
 Viral Psuedotyping
Tropism: The ability of a virus to infect a particular type of host cell
Psuedotyping: Altering the viral envelope protein to alter tropism, thus
allowing the virus to infect cells it originally could not
Tropism Host Range Viral Envelope Receptor for Viral
Protein Envelope
Ecotropic Mouse / Rat Gap70 mCAT-1

Amphotropic Mammals 4070A Ram-1

/ Dualtropic / 10A1 / GALV

Pantropic All Animals VSV-G Phosphotidyl

serine
Phosphotidyl
inositol
GM3 ganglioside

Special care should be used when working with pantropic or amphotropic

viruses which can infect humans!
 Retroviral vectors
• Transcription could be under the control of LTRs or enhancer
promoter elements might be engineered in with the transgene.

• The chimeric genome is then introduced into a packaging cell,

which produces all of the viral proteins, such as the products of the
gag, pol and env genes, but these have been separated from the LTRs
and the packaging sequence.
• Only the chimeric genomes are assembled to generate a retroviral
vector.
• The culture medium in which these packaging cells have been
grown is then applied to the target cells, resulting in transfer of the
transgene.
 Retroviral vectors- limitations
• A critical limitation of retroviral vectors is their inability to infect
nondividing cells, such as those that make up muscle, brain, lung and
liver tissue.
• The cells from the target tissue are removed, grown in vitro and
infected with the recombinant vector, the target cells are producing
the foreign protein are then transplanted back into the animal (ex vivo
gene therapy).
• Problems with expression being shut off, prolonged expression is
difficult to attain.
• Expression is reduced by inflammatory interferons acting on viral
LTRs, as the retroviral DNA integrates, viral LTR promoters are
inactivated.
• Possibility of random integration of vector DNA into the host
chromosome.
 Introduction of Genes Into Animals
Viral vector Major limitations

Papova viruses Size; Host range

Papilloma viruses Size; Integration, Transformation

Adeno associated viruses Size; production

Adeno viruses Size; antigenicity, episomal DNA

Herpes/Vaccinia viruses Pathogenic, cytotoxic, lytic

Retroviruses Inability to infect post-mitotic cells

Lentiviruses Safety, integration

Virus-mediated gene transfer in plants
 Virus-mediated gene transfer
•Viral genome doesn’t integrate into plant genome.

•Viral vectors are episomal vectors, therefore they have high copy
number per cell and they are not subjected to the “position effect”.
The gene product is very rapidly accumulated.

•Viral genome sequences are excellent source of promoters,

enhancers and other components useful for designing gene vectors.

•Virus is systemically spread in the plant body.

•Generally have wide host range.

Viral vector as transient expression system
 Important plant viral vector

1. CaMV based vectors.

2. Geminivirus based vectors.
3. PVX based vectors.
Basic features
1. Broad host range, virulence, easy mechanical
transmission and seed transmission.

2. Carry additional genetic information within the

packaging limit. Viruses that are rod shaped or have
multipartite genome or contain a helper or satellite
component offer potential for carrying extra genetic
material.
3. Best for transient gene expression or hyper production of
certain products with out transformation
 Cauliflower mosaic virus (CaMV)

Member of Caulimoviridae

Peraretrovirus i.e viruses that contain a reverse

transcription stage in their replication cycle. This family
contains all plant viruses that consist of a double-stranded
DNA genome.
 CaMV

1. dsDNA virus. Packaged as nucleosome.

2. Mechanical and aphid mediated transmission
3. Virion DNA alone or cloned CaMV DNA is infectious
when simply rubbed on leaves
4. Up to 106 copies per cell. 3-4 weeks for systemic
infection through plant.

Gene I: plasmadesmata
movement
Gene VI: translation
transactivation
Gene V: reverse
transcriptase
Gene III/IV: assembly
Gene II/VI: inclusion bodies
 Expression of a bacterial gene in plants by using a viral vector

Several properties of the cauliflower mosaic virus (CaMV) indicate that it

could provide a useful vector for gene transfer in higher plants: (1) it has a
relatively small double-stranded genome that can be easily manipulated in
vitro; (2) cloned viral DNA is infectious when rubbed onto healthy leaves; (3)
virus spreads throughout the plant and can be found in most cells at high copy
number. Two regions of the CaMV genome open reading frames (ORFs) II
and VII do not seem to be essential for infection, as both can be either
deleted or expanded by small inserts of foreign DNA. No functional genes
have yet been introduced into these ORFs. Here we report the replacement of
CaMV ORF II by the R67 plasmid-encoded dihydrofolate reductase (DHFR)
gene; this gene (dhfr) confers resistance to methotrexate in Escherichia coli.
The chimaeric viral DNA can be stably propagated in turnip plants and the dhfr
gene is expressed, producing a functional enzyme.

Nature 310, 511 - 514 (09 August 1984)

 Limitations of CaMV vector
•Small insertions (10-30 bp) in various sites abolished infectivity.

•Only gene II could tolerate insertion of significant size and

could be entirely removed

•But the largest insert tolerated so far is 256-531 bp.

•Complicated polycistronic design (ATG of cloned DNA must

not interfere with the termination of gene I).

•Due to these limitations, CaMV vectors have not be widely

used.
 Geminiviruses

Single stranded circular DNA viruses with a size approx 2.8Kb

 Geminiviridae

• Mastrevirus

• Curtovirus

• Topocuvirus
Begomovirus
 Geminivirus vector
•Usually CP is replaced with gene of interest while beta
C1 and rep genes are replaced in case of satellite viruses

•Cabbage leaf curl virus (CabLCuV)

•Tomato golden mosaic virus (TGMV)
•African cassava mosaic virus (ACMV)
•Cotton leaf curl alphasatellite (CLCuA)
•Cotton leaf curl betasatellite (CLCuB)
•Tomato yellow leaf curl beta satellite (ToYLCCNB)
 Limitation
•Size limitation

•Multiple component inoculation in case of satellite virus vector

•Severe symptom

•Host range

•Difficult to inoculate
RNA virus for foreign gene expression

The vast majority of plants viruses consist of plus strand

RNA. Engineering gene expression vectors based on RNA
viruses has awaited the successful cloning of the viral
genome into in vitro transcription vectors from which
infectious transcripts can be obtained.
 Tobacco mosaic virus (TMV)
ssRNA virus of 6395 bases with 4 ORFs

5’ cap 126 / 183 kDa 30 kDa CP tRNA like

126 / 183 kDa: replicase complex translated from a

single frame (183 kDa is generated by readthrough of a
leaky amber termination codon of 130 kDa gene

30 kDa: movement protein

17.5 kDa: capsid protein

 Potato Virus X
•Potexvirus

•Positive sense stranded RNA

•Mechanical Transmissible

•Mild symptoms

•Can accommodate large genes

 PVX Vector

Gene of interest is transcribed by the double CP promoter

of PVX
PVX mediated expression of V2 gene in
Nicotiana benthamiana
PVX mediated expression of C5, beta C1
and GFP genes in Nicotiana benthamiana
 Limitation
Host range

Cross contamination (common for all mechanically

transmissible viruses)