top of page

What is a Viral Vector?

Viruses are one of the simplest forms of life in nature. They consist only of a minimal number of protein (and in some cases lipid) layers, which encapsulate the viral genome, coding for all instructions necessary for its assembly and spread within living cells.  Alteration of the viral genome can be exploited to introduce exogenous genes into target cells, in vitro and in vivo. Such engineered viruses are termed ‘viral vectors’ and they are commonly used as research tools, as well as potential vehicles for the treatment of certain genetic diseases in gene therapy trials. To render them safer, multiple mutations and modifications were introduced into these vectors, restricting their replication and pathogenicity. These vectors effectively infect many types of mammalian cells (and may be expressed in a cell-type specific manner) and can be used to over-express specific genes, including fluorescent markers, or to silence target genes via an RNAi mechanism. Virus-mediated gene transfer (called ‘transduction’) is relatively stable over long periods of time and has almost no toxic effects on target cells; properties which make it a popular and rapidly expanding technique.



How are Vectors Engineered?

Engineering a new virus begins with a DNA plasmid, which contains essential sequences taken from the original wild-type virus, which enable the packaging of the specific genomic sequence by the capsid proteins. These elements usually flank the genomic sequence to-be expressed and they differ between virus families, which means that each such plasmid can be packaged only by the viral proteins of the same family, e.g. a protein in an AAV plasmid cannot be packaged into a lentivirus. 

Examples of to viral plasmids; Lentivirus (left) and AAV (right). Both plasmids express EGFP under a CMV promoter as well as a U6 promoter, designed for expression of shRNA sequences. Each construct contains cloning sites which enable easy replacement of each of the elements (promoter, coding sequence or fluorescent marker).

How are Viruses Assembled?

Once the viral plasmid, containing the desired gene, has been engineered, it will be transfected into a culture of HEK293T cells, along with several other plasmids termed 'helpers'. These helper plasmids code for the viruses capsid, regulation and envelope proteins. Each of these genes is located on a different plasmid in order to reduce the chances for a recombination that could yield replication-compatent virions. By separating the viral genome into three, or more, plasmids, the chance for such a recombinatin is virtually non-existant.


Once all the plasmids have been successfully taken up by the cells, they will start producing all the proteins needed to assemble a mature virus. These proteins are capable of recognizing the special elements that are embedded in the vector plasmid and encapsulate only starnds of RNA, in the case of lentivirus, or DNA with AAV. The mature, infectious virions can then be collected from the culture and prepared for use.


While some viruses, such as the lentivirus bud out of the cell and can be collected directly from the culture medium, others, like the AAV, accumulate within the cells and can only be collected following lysis of the transfected cells.


Example of a HEK293 culture trasfected with a viral plasmid which expresses GFP under a CMV promoter. Transfection efficiency regularly exceeds 90% 

What is the difference between the different types of vectors?

The different vectors are based on differnt types of viruses and as such they retain both the advantages and the limitations of the viruses from which they derive:


Viruses from this family use the enzyme 'reverse transcriptase' in order to transform RNA to DNA, an attribute which enables them to incorporate their genetic material into the genome of its host. Once there, the virus's DNA can remain stable and hidden from the cell's defences, until such moment when it is activated and starts to replicate within the infected cell.

The main member of this group that is used as a vector is the murine leukemia virus (MLV) which is one of the simplest members of this family. This virus, however, is not efficiently expressed in non-dividing cells.



Lentiviruses are members of the retrovirus family. Lentiviral vectors are based on the structure and mechanism of HIV: In it's native form, this virus is an extremely difficult challange to control due to its ability to incorporate its genome into that of the host cell. In addition, the lentivirus is one of the few members of this family which can incorporate its genetic material into the genome of non-dividing cells, such as neurons. 

In order to increase the variety of cells these vectors can infect, the virus's envelope protein, which allows HIV to find and infect T-cells with high specificity, has been replaced with a protein from a different virus - the vesicular stomatitis virus G protein (or VSV-G for short), which is able to recognize many different classes of cells.



Adeno-Associated Virus

A schematic illustration of a virus from the familly Retroviridae along with a diagram elaborating on the virus's method for replication within the host cell. Click on the image to view a larger version. 

Adeno-associated virus, or AAV, is a small, non-pathogenic satelite virus of larger viruses such as adeno and herpes virus and can only replicate in cells already infected by them. Due to its small size, it is possible to reach very high concentrations of this virus, which allow for very efficient transduction in-vivo.

AAV also posseses the ability to incorporate its genetic material into the genome of its host but only in dividing cells. However, since this virus is non-pathogenic, host cells are not in a hurry to dispose of it and expression of the transgene remains high and stable, even in non-dividing cells.


bottom of page