Article

Helper Virus and Binary Vector System in Mammalian Expression System

Helper Virus  (Defective Phage help another Defective Phage)

AUTHOR : NEHA SHARMA

As  mentioned before defective phage helps another defective phage means it helps the virus or virusoid (non self replicating ssRNAs) which are not able to replicate their genetic material on their own and ultimately help in development of a fully infectious agent.  Helper viruses are all from the family Sobemoviruses.

Basic functions of Helper virus

1. It helps the virus by restoring the activity of the viral gene 
2. By providing enzyme for replication
3. Enabling to form a protein coat

Helper Virus in Virusoids

Virusoids are called satellite viruses which encode for the capsid protein but depend on the helper virus for replication. 

The virus capsid of virusoids are packaged in the helper viral cells without affecting or interfering with the replication of the helper virus . 

 Example :  Hepatitis Delta Virus (HDV) is a virusoid that is circular in structure with 1700 nucleotides and it leads to the synthesis of HDV assosciated proteins. 

The Hepatitis B Virus (HBV)  is a helper virus of Hepatitis D Virus ( HDV) which together leads to more severe effects on the liver. 

Helper virus for expression of Adeno-associated Virus (AAV) 

Adeno-associated virus  ( AAV ) is a small , non enveloped and 25nm in size. It contains ssDNA as a genetic material. It is composed of 3 viral proteins VP1,VP2  and VP3. AAV is a type of satellite virus which replicates only in the cells that are coinfected with adenoviruses .

Adenovirus plays a different roles in the AAV replication in host cell are :

• AAV promoter activation, drives cells to S-phase
• promotes AAV second-strand synthesis
• AAV promoter regulation, AAV genome replication, Rep splicing, capsid protein production.
• Prevents  degradation of AAV capsids & Rep52

Adeno-associated Virus as shown in figure undergo two phases in respect of presence of helper virus or not 

1^st Phase ( absence of helper Virus ): Latent Phase

1. When AAV infects human cells in absence of these helper viruses its ssDNA genome is copied into dsDNA by host cell DNA Polymerase .
2. Then integrated into a single region Chromosome 19 where it doesn’t have any deleterious effects.

2^nd Phase (Presence of Helper Virus): Lytic Phase

In this phase 2 , presence of helper virus leads to the successful production of activated AAV viral particles. The helper virus activates P5 , P40 and P19 promoters which are important for Rep and Cap genes for its activation.

Role of P5 and P19 promoter:  P5 leads to Rep 68 and 78 transcription , P19 leads to Rep 40 and 52 transcription and P40 facilitates the activation of Cap gene .

These lytic phase events included : Rep mediated excision from host genome : Rep 78 leads to complete arrest of host cell in S Phage and Rep 68 acts as site specific endonucleases which leads to unwinding of DNA.

1. Replication of Viral genome : Replication is done by Rep78 by oligomeric behaviour which leads to initiation of replication . E2A , E1b55K and E4 are the helper adeno viral genes which promote the replication of Viral genome. 
2. Expression of viral Gene : VA RNA are the main helper viral gene which leads to the expression of viral genome along with the Rep genes .
3. Packaging and release of fully formed AAV : Rep 40 and Rep 52 are the type of DNA helicases essential for accumulation and packaging of the genome in capsid . P40 promoters facilitate the activation of Cap genes which encode for three viral proteins VP1,VP2 and VP3 which constitute capsid formation for viral genome packaging.

After the successful packaging of viral genome in capsid , AAV is released from the host cell by helper virus via inducing Cell Lysis. 

BINARY VECTOR IN MAMMALIAN CELL

Binary Vector System used in mammalian cell expression systems to help the virus which are not able to express on their own.

Binary Vector System consists of two plasmid that are involved in gene transformation , the first vector is a helper plasmid and second vector contains Gene of Interest 

Adeno-associated Virus (AAV)

Adeno-associated virus vector has advantages: low immunogenicity that is almost non pathogenic for in vivo conditions , easy propagation , infection of most cell types and long term gene expression. 

AAV genome size is 4.7kb and single stranded DNA and it consists of two open reading frame for Rep gene and Cap gene and containing inverted terminal repeats (145bp long ) on both the sides which leads to synthesis of Complementary DNA for the transfer plasmid.

In this figure , 3 cassette are :

1. Adeno-associated Virus genome (wild AAV)

It consists of ITR at both ends ,rep gene, cap gene , promoters and poly A tail 

2. Adeno-associated Virus Vector plasmid (AAV Vector )

It consists of ITR of 145 bp at both ends , promoter ,transgene that is gene of interest . poly A tail . In this ITR Sequences serve as a initiation of viral genome replication , ability to form hairpin like structure which allows independent synthesis of second DNA strands 

3. Helper plasmid 

It consists of promoter P5 for activation of rep and cap gene , adenovirus genes like E2, VA and E4 .It is formed from the Helper Virus Adenovirus for the generation of E4, E2a and VA gene which are responsible AAV DNA replication and packaging of genome and leads to the production of active viral components.

In this figure , AAV plasmid and Helper plasmid are cotransfected to HEK-293 Cells . AAV Plasmid leads to the replication of transgene with the help of a replicase gene (rep gene ) and ITR present on both ends. Helper plasmid containing Cap gene leads to the formation of capsid . After this packaging takes place and  produces active viral particles .

1. Indicates formation of AAV plasmid and Helper Plasmid
2. Indicates cotransfection into host cell
3. Cap gene function as a production of capsid structural protein
4. Indicates the replication of transgene in host cell and assembly of regulatory proteins and structural proteins
5. Indicates production of active viral particles and cell lysis with the help of Helper Virus.

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