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 DNAGene of Interest

DNA Loaded Syringe

Introduction

•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 DNAGene of Interest

No New Viral Particles are CreatedInfection dose not spread

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.

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.

ADENOVIRUS - Structure

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.

Entry of the nuclear material inside the host nucleus

Immediate Early Intermediate Late

Genome organization of Virus

Involved in the replication of the virus

E1A

Machinery for viral DNA replication and the ensuing transcription of late genes

Metabolism of virusmRNA and providefunctions that promoteVirus DNA replication andshut- off of host proteinSynthesis,

E3,E1B,E2A,E2B,E4

IX, IVa2 L1,L2,L3,L4,L5

Onset of DNA synthesis

Involved in modulatingThe immune response ofInfected cells.

Adenovirus replication is a two phaseEvent, early and late, occurring before andafter DNA replication respectively

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.

• 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.

Construction of recombinant Adenovirus

Construction of recombinant Adenovirus

Construction of recombinant Adenovirus

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

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.

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.