sv40 and retroviral vector

1. Animal viral vectors
Dr. Manikandan Kathirvel M.Sc., Ph.D., (NET)
Assistant Professor,
Department of Life Sciences,
Kristu Jayanti College (Autonomous),
(Reaccredited with "A" Grade by NAAC)
Affiliated to Bengaluru North University,
K. Narayanapura, Kothanur (PO)
Bengaluru 560077

2. Animal viral vectors
Animal viruses can be divided into DNA and RNA viruses, depending on the
nature of their genomes.
Animal viruses have to recognize a specific host cellular receptor for entry during
infection. Host receptor binding is the initial step of virus life cycle and could be an
effective target for preventing virus infection.

3. SV40 vector
1. A good example is the ds DNA Simian Virus 40. This monkey pathogen has a
small genome (5243 nt), encodes six-seven protein products, contains overlapping
genes, some of which are the result of alternate splicing.
2. Belongs to polyoma viruses group.
3. The virus was first isolated as a contaminant from a monkey kidney culture used
for the production of poliovirus vaccine.

5. 1. SV40 is a spherical virus, It is roughly 40-45 nm in diameter.
2. The genome of SV40 is like a mini chromosome with a circular, double stranded 5,243
bp chromosomes,
3. Its genome is very small and can be easily modified for gene therapy purpose.
4. Its genome has ds circular DNA wrapped up as nucleosome with the help of histone
proteins
5. The genome of SV40 consists of early proteins, late proteins and regulatory proteins.
6. Early proteins are non structural while late proteins are structural proteins; which
encodes 5 proteins, viz.,
-small-t,
-large-t (both early protein),
-VP1, VP2 and VP3 (VP= virion protein)- (late gene protein)
-has an origin of replication(about 80 bp) and is complexed with histones to
form chromatin.
Large-T is essential for viral replication,
while VP1, VP2 and VP3 form the viral capsid.
7. In laboratory, it is multiplied in cultured kidney cells of African green monkey; infected
cells lyse after 4 days releasing upto 10 raise to the power 5 virions/cell.
8. SV40 plasmids (vectors) can be packaged only if their DNA is within the range of 3900 to 5300 bp.

6. SV40- DNA virus:
Simian vacuolating virus 40 or simian virus 40 were shortly known as SV40 which was the
first eukaryotic DNA virus was SV40, for which a complete nucleotide sequence and a
detailed understanding of transcription were available. SV40 is a DNA virus that has the
potential to cause tumors in animals.
1. SV40 is a good example of how a small genome can be extensively utilized. The
genome is 5.2kb in size encodes six-seven protein products (gp1-gp7), contains
overlapping genes, some of which are the result of alternate splicing and contains two
sets of genes: Early and Late genes;
2. the early genes, expressed early in the infection cycle and coding for proteins involved
in viral DNA replication, and
3. the late genes, coding for viral capsid proteins.
4. Both strands of the 5243 nt dsDNA contains genes.
Early genes: (SV40gp6 and SV40gp7 genes )
The two early genes are encoded from the negative strand. These genes are actually
represented in a single mRNA that is alternatively spliced to encode the small and large T
antigens, proteins critical for launching the replication of the genome.
Late genes: (SV40gp3, SV40gp4 and SV40gp5 genes produce the three proteins
found in the viral capsid)
Alternatively splicing is also key for the late genes that encode the structural components
of the virus. In this case, VP2 and VP3 are products of the alternative splicing of the same
mRNA. Finally, the VP1 gene overlaps the VP2/VP3 gene region.

8. Simian Virus 40
• Good example of a small genome
• Shows how a small genome can be extensively utilized
• Features:
o 5243 nt dsDNA genome
ƒ Both strands contains genes
• Five of the six genes overlap
ƒ “Life-cycle specific” regions
• Early genes
o Negative strand genes important for early development of new virions.
o Genes transcribed in a single mRNA
o mRNA is alternatively spliced
o Encode the small and large T-antigens
o Proteins critical replication of the genome
• Late genes
o Encoded on the positive strand
o Two genes (VP2 and VP3) overlap
o A single mRNA for these genes
o Alternative splicing produces unique mRNAs
o Proteins critical for the structure of the virion

9. Life cycle
1. The cell infection starts by the binding of the SV40 virus to a receptor on the cell
membrane. This receptor has been identified as the major histocompatibility complex
2. After binding to the cell surface, polyomavirus capsid undergoes endocytosis and is
transported to the nucleus where the viral DNA is uncoated and transcription of the
early region begins.
3. The primary transcript from the early region is alternatively spliced to give two mRNAs
that encode large Tag and small tag.
4. Tag is a nuclear phosphoprotein of 94 kD and it is an essential factor for viral DNA
replication. It binds to the viral origin of replication (ori) where it promotes unwinding
of the double helix and recruitment of cellular proteins that are required for DNA
synthesis, including DNA polymerase-α and replication protein A.
5. Large Tag is thought to stimulate the cell cycle through its ability to bind to several
cellular proteins that are involved in crucial signal transduction pathways that control
cell cycle progression and apoptosis.
6. The role of the small tag in the polyomavirus life cycle is less clear.
7. Analysis of SV40 deletion mutants revealed that tag is not essential for lytic infection
in culture.

10. SV40 as vector:
1. SV40 is a well-known virus, vectors are easy-to-make, and can be produced in titers of
10(12) IU/ml.
2. The genome of SV40 contains very little non-essential DNA so it is necessary to insert
the foreign gene in place of essential viral genes and to propagate the recombinant
genome in the presence of a helper virus.
3. The early protein coding genes can be replaced by the gene of interest. Capacity of
the transgene is limited to 5 Kb. Long lasting transgene expression. It has been
reported that the transgene expression once established will remain lifelong.
4. This virus is capable of infecting several mammalian species, following a lytic cycle in
some host and a lysogenic cycle in others.
5. They also efficiently transduce both resting and dividing cells, deliver persistent
transgene expression to a wide range of cell types, and are nonimmunogenic.

11. Disadvantages:
1. SV40 recombinant DNA molecules is severely constrained by the facts that the viral
genome is small, 5.24 kb, and that the packaging limits are strict. Such systems
cannot, therefore, be used for the analysis of most eukaryotic genes.
2. SV40 plasmids (vectors) can be packaged only if their DNA is within the range of
3900 to 5300 bp.
3. Present disadvantages of rSV40 vectors for gene therapy are a small cloning
capacity and the possible risks related to random integration of the viral genome
into the host genome.

12. Retroviral vector

14. Retroviral vector
• Single stranded RNA genome
• Has two copies of the genome, which resemble eukaryotic mRNAs.
• The viral genome is reverse transcribed by reverse transcriptase into a DNA double-strand
copy inside the host cells. This process is called as Reverse transcription.
Features of RV vector
o Contains gene for replication, expression and packaging (ψ sequences).
o Gene of interest may inserted in the nonessential coding region or it may replace some
essential gene (gag).
o genomes are used as vectors, generally as shuttle vectors.
-Retrovirus genomes commonly contain these three open reading frames that encode for
proteins that can be found in the mature virus.
•Group-specific antigen (gag) codes for core and structural proteins of the virus,
•polymerase (pol) codes for reverse transcriptase, protease and integrase and
•envelope (env) codes for the retroviral coat proteins.

15. During the process of reverse transcription, sequences from the termini of viral RNA are
duplicated to generate long terminal repeats(LTRs).
These long terminal repeats contain both the promoter and the polyadenylation signal
for the transcription of viral mRNAs.
The specificity of proviral DNA integration is also determined by the long terminal
repeats.
Although retroviruses can integrate at many sites within the cellular genome, integrative
recombination always occurs at particular sites at the ends of the LTRs.
The sequences appropriately inserted between the two LTRs will be integrated intact.
After integration, the DNA genome behaves
like any cellular gene being transcribed and
replicating only if the cell replicates. Thus
the cell expresses the therapeutic gene and
no further viral replication is possible.

17. 1. Retroviral vectors are
created by removal
of the retroviral gag,
pol, and env genes.
2. Retroviral vectors
resemble their
parent virus except
that the genome
encodes as a
therapeutic gene or
gene of interest
instead of the viral
structural proteins.
Cloning and Packaging
Cloning and Packaging

18. Production of packaged retrovirus vector RNA.
1. The packaging cell line has two separate retroviral gene regions on its chromosomes;
one contains the gag gene, and the other contains the pol and env genes.
2. In each of these inserts, transcription is driven by sequences within the 5' long terminal
repeat (5' -LTR) region.
3. Both virus DNA segments lack the encapsidation sequence (ψ) that is required for
packaging a retroviral genome into a viral capsid.
4. The packaging cell line synthesizes viral proteins, but because there is no encapsidation
(Δψ) sequence within either of the retroviral mRNAs, empty viral capsids are produced.
5. The viral proteins continue to be synthesized after the transfection of a packaging cell
line with a full-length retroviral vector carrying a remedial (therapeutic) gene (Gene X)
and a selectable marker gene (Neor).
6. The full-length RNAs from the retrovirus vector sequence are replicated, and because
they have an encapsidation region (ψ), they are packaged into viral capsids.
7. The released viral particles are replication defective because they do not have a pol
gene.

19. 1. The packaging cell line has two separate
retroviral gene regions on its chromosomes;
one contains the gag gene, and the other
contains the pol and env genes.
2. In each of these inserts, transcription is
driven by sequences within the 5' long
terminal repeat (5' -LTR) region.
3. Both virus DNA segments lack the
encapsidation sequence (ψ) that is required
for packaging a retroviral genome into a viral
capsid.
4. The packaging cell line synthesizes viral
proteins, but because there is no
encapsidation (Δψ) sequence within either
of the retroviral mRNAs, empty viral capsids
are produced.
5. The viral proteins continue to be
synthesized after the transfection of a
packaging cell line with a full-length retroviral
vector carrying a remedial (therapeutic) gene
(Gene X) and a selectable marker gene
(Neor).
6. The full-length RNAs from the retrovirus
vector sequence are replicated, and because
they have an encapsidation region (ψ), they
are packaged into viral capsids.
7. The released viral particles are
replication defective because they do
not have a pol gene.

21. Bovine Papillomavirus DNA
Vectors

22. Bovine Papillomavirus DNA Vectors
BPVs are small non-enveloped viruses with an icosahedral capsid around 50–60
nm in diameter.
BPV is capable of transforming certain mammalian cells including rat, bovine,
hamster, and mouse cell lines in culture.
They can cause skin tumour equine sarcoid in horses and donkeys.
All BPVs have a circular double-stranded DNA genome.
The multicopy mode of plasmid replication provides for natural amplification of
cloned genes and, consequently, the possibility of higher levelsof expression.
Papillomavirus transformed cells don't contain integrated viral DNA rather they
contain between 50 and 300 copies of unintegrated, circular viral DNA.

23. Genome of BPV:
1. All BPVs have a circular double-stranded DNA genome of 7.3–8.0 kb.
2. The open reading frames (ORFs) are all located on one strand, and are divided into early
and late regions. The early region encodes nonstructural proteins E1 to E8. There are
three viral oncoproteins, E5, E6 and E7.
3. The late region encodes structural proteins L1 and L2. There is also a non-coding long
control region (LCR).

24. The BPV1 genome is a double-stranded DNA molecule of 7945 nucleotides whose sequence was first
determined by Chen et al. and by Stenlund et al.
The viral open reading frames
Ten viral ORFs have been identified, eight early ORFs which are required for early viral functions,
replication and transformation.
Lowy et al. showed that a 69 % BamHI-HindIII fragment is capable of inducing cellular transformation.
Coding information required for transformation must therefore be contained in this region and ORFs
from this region are referred to as early (E) ORFs
and two late ORFs LI and L2 code for the major and minor viral capsid proteins which span the 31 %
non-transforming region (coordinates 4171-7095) with a termination codon at coordinate 5593.

25. Shuttle vectors consisting of BPV and pBR322 sequences, and capable of replication in
both mouse and bacterial cells, are therefore of great value in animal cell
biotechnology.

26. The characteristics of the three basic BPV
vectors described in the text are shown.
Number 1 is the basic 69 % transforming
fragment plus the gene of interest.
Number 2 consists of the 69 % fragment, a
stimulating gene sequence, bacterial
sequences and the gene of interest.
Number 3 contains the complete BPV genome,
bacterial sequences and the gene of interest.
All three types of vector have in fact been used
successfully for the expression of foreign genes
with little or no difference in the levels of
expression.
Illustration of the three basic BPV vectors

27. 1. Interestingly, transformation efficiency of the 69 % BamHI-HindIII is slightly less than
that obtained when the full genome is used. If bacterial sequences are linked to the 69
% fragment then transformation efficiency falls substantially.
2. Transformation efficiency is however restored by the addition of certain eukaryotic
genes
3. which have 'stimulatory properties' (e.g. β-globin, rat pre-proinsulin, and rat growth
hormone).
4. These initially puzzling observations are now best explained by the work of Lusky &
Botchan who mapped a BPV enhancer in the 31 % non-transforming region.
5. This enhancer is implicated in the replication of the virus. It is presumed to activate
transcription at the origin of replication. Removal of the enhancer leads to reduced
replication of the genome and a consequent decrease in transformation efficiency.
6. Genes like β-globin and pre-proinsulin must therefore possess some sort of enhancer
function which can replace the enhancer function in the 31 % non-transforming region.