Plasmids 101: Viral Vector Elements

Posted by Marcy Patrick on Jul 17, 2014 3:09:00 PM


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The use of viral vectors in research is beneficial for a number of reasons, including but not limited to: helping to get difficult-to-deliver DNA into mammalian cells, increasing the efficiency of gene transduction, allowing for control over which cells are infected through viral pseudotyping, and ease of vector cloning and modification. At the most basic level, viral vectors consist of a viral genome that has been adapted into a plasmid-based technology and modified for safety through the removal of many essential genes and the separation of the viral components. Read on for a brief description of the viruses used to make these vectors as well as a table defining the major elements found within the plasmids comprising the viral vector systems.

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Viral Vectors

Of the many viruses out there, only a subset are commonly used in the lab and we will discuss the three most prevalent: gamma-retrovirus, lentivirus, and adeno-associated virus. 

Gamma-retrovirus and Lentivirus

A retrovirus is an RNA virus that uses reverse transcriptase to make a DNA provirus which can be incorporated into the host's genome. There are two common genera of retrovirus used by scientists: Gamma-retrovirus (many times shortened to just "retrovirus") and lentivirus. From these two genera, the most studied types are Murine Leukemia virus (MLV) and human immunodeficiency virus-1 (HIV-1), respectively. Since these viruses are closely related, their life cycle and the required components are basically the same, although differences do exist at the sequence level. This means that plasmids containing virus-specific elements such as the LTRs or structural proteins are not interchangeable; however, other, more general, viral components such as a heterologous envelope or a post-transcriptional regulatory element can be used across either system. The most notable difference between gamma-retrovirus and lentivirus is the fact that lentivirus can infect both dividing and non-dividing cells, whereas gamma-retrovirus is restricted to dividing cells only. 

Adeno-associated Virus

Adeno-associated virus or AAV is a small parvovirus that infects humans and some other primate species, but is not currently known to cause disease. If AAV is exclusively used to infect human cells, it stably integrates at a specific chromosomal site as part of its lysogenic life cycle; however, the addition of a helper virus such as adenovirus or a helper plasmid containing the specific viral proteins E1A, E1B, E2A, E4, and VA will cause AAV to enter a lytic cycle and replicate. There are at least eleven different serotypes of AAV, with likely more to be discovered. The most studied and experimentally used serotype is AAV2; however, many factors including species and cell type may make other serotypes more desirable. 

Common Viral Vector Elements

When developing viral vectors, scientists strive for a number of features: low risk, high expression, large payload capacity, ability to infect target cells, no immune response from the host, and easy to develop/use in the lab. In virtually all viral expression systems employed by scientists, non-essential components are stripped away and the remaining native genes are spread over multiple plasmids to ensure safety. The choice for how the elements are divided up is dependent on whether the component needs to be provided in cis (on the same plasmid) or in trans (on a separate plasmid) as your insert. The tables below list some components most commonly found in viral vector systems.  

Gamma-retroviral and Lentiviral Elements:

 Element  Plasmid type Provided? Purpose
LTR Transfer in cis Long terminal repeats; U3-R-U5 regions found on either side of a retroviral provirus (see below). Cloning capacity between the LTRs is ~8.5kb, but inserts bigger than ~3kb are packaged less efficiently.
U3 Transfer in cis Unique 3’; region at the 3’ end of viral genomic RNA (but found at both the 5’ and 3’ ends of the provirus). Contains sequences necessary for activation of viral genomic RNA transcription.
R Transfer in cis Repeat region found within both the 5’and 3’ LTRs of retro/lentiviral vectors. Tat binds to this region.
U5 Transfer in cis Unique 5’; region at the 5’ end of the viral genomic RNA (but found at both the 5’ and 3’ ends of the provirus).
5' LTR Transfer in cis Acts as an RNA pol II promoter. The transcript begins, by definition, at the beginning of R, is capped, and proceeds through U5 and the rest of the provirus. Third generation vectors use a hybrid 5'LTR with a constitutive promoter such as CMV or RSV.
TAR Transfer (2nd generation only) in cis Trans-activating response element; located in the R region of the LTR and acts as a binding site for Tat. 
3' LTR Transfer in cis Terminates trascription started by 5’ LTR by the addition of a poly A tract just after the R sequence.
cPPT Transfer in cis

Central polypurine tract; recognition site for proviral DNA synthesis. Increases transduction efficiency and transgene expression.

Psi (Ψ) Transfer in cis RNA target site for packaging by Nucleocapsid.
RRE Transfer in cis Rev Response Element; sequence to which the Rev protein binds.
WPRE Transfer in cis Woodchuck hepatitis virus post-transcriptional regulatory element; sequence that stimulates the expression of transgenes via increased nuclear export.
Gag Packaging  in trans Precursor structural protein of the lentiviral particle containing Matrix, Capsid, and Nucleocapsid components.
Pol Packaging in trans Precursor protein containing Reverse Transcriptase and Integrase components.
Rev Packaging (on separate plasmid from Gag/Pol in third generation systems) in trans  Binds to the Rev Response Element (RRE) within unspliced and partially spliced transcripts to facilitate nuclear export.
Tat Packaging (second generation only) in trans Trans-activator; binds TAR to activate transcription from the LTR promoter.
VSVG Envelope in trans Vesicular stomatitis virus G glycoprotein; Broad tropism envelope protein used to psuedotype most lentiviral vectors.

Please see our previous post "Your Lentiviral Plasmid FAQs Answered" or our Lentiviral FAQ webpage for more detailed information on lentiviral vectors including Biosafety FAQs. 

AAV Elements:

 Element  Plasmid type Provided? Purpose
ITR Cloning in cis Inverted terminal repeat; 145 bases each. Symmetry of ITRs is required for efficient multiplication of the AAV genome. Forms a T-shaped hairpin that serves as the origin of viral DNA replication. Contains D region required for packaging. Cloning capacity between the ITRs is ~4kb.
Rep Packaging in trans Packaging proteins with four possible variants: Rep78, Rep68, Rep52, and Rep40; Required for genome replication and necessary for integration. Rep proteins from most serotypes can be interchangeably used with any ITR serotype* 
Cap Packaging in trans Structural capsid proteins with three variants: VP1, VP2, and VP3; VP1 possesses phospholipase A2 activity, which is likely necessary to release the AAV particles from late endosomes. VP2 and VP3 are crucial for correct virion assembly. Determines the serotype/ viral tropism.

*Exception is AAV5, which requires AAV5 Rep and AAV5 ITRs for packaging.

 


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Topics: Plasmid Elements, Plasmids 101, Viral Vectors

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