Viral Transduction - Part 1

Viral Transduction 

A method to introduce genetic material into cells using viruses as vectors 

What are the two ways DNA/RNA can be expressed?

DNA/RNA can be either permanently integrated into the genome of the target cell or transiently expressed 

Why would viral delivery be preferred over other methods?

• transduction is easier 

• viral integration is more reliable than traditional transfection

• less toxic to cells than transfection, preserving cell viability 

What are the two types of non-enveloped viruses?

• adeno-associated virus

• adenovirus 

What type of virus is a lentivirus?

Enveloped

What is the protein that all viral genomes are located in?

capsid

What is the lipid bilayer that surrounds the capsid?

Envelope

Which viruses have an envelope?

Retrovirus and Lentivirus

What is the viral genome for retroviruses (RV) and lentiviruses (LV)?

RNA

What is the viral genome for adeno-associated viruses (AAV)? 

ssDNA 

What is the viral genome for Adenoviruses (Ad)?

dsDNA

What is the capacity for RV/LV?

~ 8 kb

What is the capacity for AAV?

~ 4.7 kb

What is the capacity for Ad?

~ 7.5 kb

Which virus type can be integrated into the host genome?

RV/LV

Can AAV and Ad be integrated into the host genome?

No

What is the gene expression like in RV/LV?

Stable, good for long term dividing cells

What is the gene expression like in AAV?

Transient, it can be stable or long term in non-dividing cells

What is the gene expression like in Adenoviruses?

Transient

Which virus types trigger LOW immune responses?

RV/LV, AAV

Do adenoviruses trigger a high or low immune response?

High

The pathology of Adenoviruses ____

Respiratory diseases

How retrovirus (RV) and Lentivirus (LV) enter the cell?

1. The virus enters the cell via the glycoprotein envelope binding cell surface receptors

2. Membrane fusion occurs releasing the RNA genome into the cytoplasm

3. RNA is turned into dsDNA via viral reverse transcriptase

4. dsDNA enters the nucleus via nuclear pore (LV) or when the cell divides (RV)

5. The virus integrates into the host cell genome

What are the 5 parts of a LV? 

1. Long Terminal Repeat (LTR) 

2. Gag

3. Pol

4. Env

5. Accessory genes (Rev) 

What are the 4 parts of a RV?

1. LTR

2. Gag

3. Pol

4. Env

What does the LTR do?

Facilitate integration of the sequence into the host genome, function as enhancers and promoters

Gag

Structural protein

Pol

Reverse transcriptase and integrase

Env

Envelope protein

Accessory genes (Rev) 

Facilitate nuclear transport 

What are the 4 lentivirus plasmids?

1. Packaging plasmid (gag and pol)

2. Regulatory plasmid (rev)

3. Envelope plasmid (VSVG)

4. Transgene plasmid (contains gene wanted to express)

The production of Lentivirus

1. Use of four plasmids

2. Transfection of cells (293T)

3. Collection of the supernatant 48 hrs later

4. Filtration and ultracentrifugation to collect the viral particles

The 9 steps of Adeno-Associated Virus (AAV) production

1. Virus bind to receptors on target cells

2. The virus is taken into the endosome through endocytosis

3. After release from the endosome, virions are ubiquitylated, proteosome degraded or taken to the nucleus

4. The virus travels to the nucleus

5. Viral genome is uncoated and the genome is released

6. AAV single stranded DNA is converted into double stranded DNA

7. Transcription

8. mRNA is exported out of the nucleus

9. Translation and expression of the transgene

The AAV genome

1. ITR

2. Rep

3. Cap

ITR

Inverted Terminal Repeats - function as the viral origin of replication and packaging signal 

Rep Gene

gene for replication and packaging of AAV genome

Cap gene 

gene for encoding proteins (VP1, VP2, VP3) of the viral capsid

Characteristics of AAV

• does not integrate

• can infect human and other mammals but not currently known to cause disease

• low immunogenicity

• transduce dividing and non dividing cells

• need helper virus or plasmid to replicate

AAV Serotypes 

Multiple serotypes that allow virus to target specific cells 

• can be done via organ specific injections or capsid modification 

Construction of recombinant AAV

1. ITR

2. Replace the Rep and Cap gene by a promoter and a transgene