Adenovirus structure,Recombinant Adenovirus construction etc.

2. Infectious Viruses: A Genetic “Syringe”
• Viruses are composed of genetic material
encapsulated in a protein coat.
• 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 (rDNA) to infect
that gene into the target cell.
Virus
Target Cell
Target Cell Infected With Viral
DNA Containing The Gene of
Interest
Cell’s DNA
Viral DNA
Gene of Interest
DNA
Loaded
Syringe
Introduction

3. •The viral DNA does not contain the viral genes needed to make more viruses.
Virus
Target Cell
Target Cell Infected With Viral
DNA Containing The Gene of
Interest
Cell’s DNA
Viral DNA
Gene of Interest
No New Viral Particles are Created
Infection dose not spread

4. ADENOVIRUS- Classification
• DNA viruses first isolated from adenoidal tissue in 1953
• Approximately 100 serotypes have been recognized, at least 47 of
which infect humans.
• Subdivided into 6 subgroups based on heamagglutination (A-F).
• Human pathogens belong to 49 serotypes.
• Common serotypes:- 1-8, 11, 21, 35, 37, 40.

5. ADENOVIRUS - Structure
o Non-enveloped DNA virus
o Icosadeltahedrons.
o 70-90 nm in size.
o Linear ds DNA genome (36kb) with a terminal protein (molecular mass=55 kDa)
o capsid comprises 240 capsomeres, which consist of hexons and pentons.
o 12 pentons : have a penton base and a fiber.
o fiber : viral attachment proteins can act as a hemagglutinin.
o penton base and fiber are : toxic to cells, carry type-specific antigens.

6. Adsorption and entry into host
• Entry of adenovirus into host cell involves two sets of interactions
between the virus and the host cell.
1. Coxsackivirus adenovirus receptor(CAR).
2. Specialized motifs in the penton base protein interacts with an
integrin molecule.

7. Entry of the nuclear material inside the host nucleus

8. Genome organization of Virus
Onset of DNA
synthesis
Immediate Early Intermediate Late
E1A
Involved in the
replication of
the virus
Machinery for viral DNA
replication and the ensuing
transcription of late genes
Metabolism of virus
mRNA and provide
functions that promote
Virus DNA replication and
shut- off of host protein
Synthesis,
E3,E1B,E2A,
E2B,E4
IX, IVa2 L1,L2,L3,L4,L5
Involved in modulating
The immune response of
Infected cells.
Adenovirus replication is a two phase
Event, early and late, occurring before and
after DNA replication respectively

9. Importance of sequential gene expression
• These proteins have three major roles
1. To alter the expression of host proteins that are necessary for DNA
synthesis(E4)
2. To activate other virus genes(such as virus encoded DNA polymerase).
3. To avoid premature death of the infected cell by the host immune
response.(E3)
• Ψ site - packaging signal
• Inverted repeats present at the two ends of the linear dsDNA.

10. Construction of recombinant Adenovirus
• First generation contains a deletion in the E1 region encoding products.
• Second generation also has a deletion in the E2 or E4 locus in addition to that of E1.
• Third generation called ‘gutless’ vectors contains only cis-acting terminal repeats and
a packaging signal.

11. Construction of recombinant Adenovirus

12. Construction of recombinant Adenovirus

13. Cre/loxP system
• The Cre recombinase, a naturally occurring site-specific recombinase of
bacteriophage P1
• Recognizes a 32-bp sequence named loxP
• Cre can efficiently mediate site-specific recombination using two loxP sites
separated by sequences of variable lengths
• The recombination events include deletion, insertion, and inversion of the
sequences between the loxP sites

14. ADVANTAGES OF ADENOVIRUSES VECTORS
• Adenovirus can infect dividing as well as quiescent cells with equally high
efficiency
• The Adenovirus is ubiquitous- it has been isolated from a large number of
different species with over 100 known serotypes.
• Can rapidly infect a large range of human cells
• Low pathogenicity in humans.
• Can hold large segments of DNA.
• Genome does not undergo rearrangement at high rates.
• DNA is easy to manipulate with current recombinant DNA techniques.

15. Disadvantages
• Host immune system plays a pivotel role in adenovirus mediated gene transfer.
• Short term expression particularly in dividing cells.
• Limited transduction of the cells with reduced or no expression of attachment
and internalization receptor.
• Transient expression foreign genes.