Retroviruses – Friends or Foes?
The word ‘retrovirus’ evokes images of HIV, the AIDS pandemic and a desperate worldwide effort to defeat this mighty adversary. But on the flip side, scientific research has managed to tame the virus and use it as a tool for advancement.
Retroviruses are commonly used to introduce genes into mammalian cells to express or knockdown genes of interest. In addition, retroviruses are being developed for use in gene therapy. Though the use of retroviruses raises safety issues, new molecular and genetic techniques of vector design circumvent concerns and make them widely available for safe use in the lab and as potential gene therapy agents.
Pros and cons of retroviral vectors
Compared to other modes of gene delivery such as classical chemical based transfection, electroporation, and microinjection, retroviruses have the following characteristics:
- The DNA integrates into the host cell genome and replicates stably with genomic DNA instead of remaining in a plasmid form in the cytoplasm
- They have low immunogenicity
- Lentiviruses (as opposed to standard modified retroviruses like MMLV) can infect both dividing and quiescent cells
- Smaller insert carrying capacity
- Risk of insertional mutagenesis – accidental insertion of retroviral DNA into an unintended site causing disruption of host gene function
- Lower titers (than other viral vectors)
- Low replication efficiency in self-inactivating (SIN) vectors
Special concerns for using retroviral vectors
In 1990, Nobel laureate Howard Temin, who co-discovered the reverse transcriptase enzyme, detailed the risks and safety considerations associated with retroviral vectors, but stated that “safety considerations should not hold up further human trials of retrovirus vectors”.
However, immediately after in 1992, in a pre-clinical trial in rhesus monkeys, a retrovirally-transduced bone marrow transplant led to development of fatal lymphomas in 3 out of 10 recipients, the cause being formation of replication-competent viruses. Thus, the theoretical chance of formation of replication-competent virus particles that cause infections and of viral integration into the host genome causing oncogenesis and mutations cannot be ignored.
This has led to intense research and development of viral vector strategies and newer technologies that make retroviral work safer. Some of these are:
Retroviral vectors used in research are made replication-deficient. Genes required for viral infection but unnecessary for packaging and transduction are deleted. Viral structural genes that are incorporated into the plasmid do not contain a ? (psi) sequence (which essentiallyhelps to incorporate the RNA into viral particles) thus making replication-deficient viruses.
The viral packaging genes essential for viral particle production and transduction are split into multiple plasmids to decrease the frequency of recombination events between the genes that lead to formation of replication-competent viruses. Thus, three and even four different plasmids are utilized to transfect packaging cell lines and produce viral particles.
Higher biosafety is ensured in newer systems due to genetic modifications in the transfer plasmid making it easy to integrate into the host genome but prevent activation afterwards (called a SIN or self-inactivating vector).
Depending on the type of viral envelope proteins, three types of viruses can be made:
- Pantropic – can infect cells of all species
- Ecotropic – can infect cells of mouse and rat origin
- Amphotropic – can infect human, mouse and rat cells
Ecotropic viruses are the safest to use, as they cannot infect human cells. (Though consider the fact that ecotropic and amphotropic viruses are less stable than pantropic viruses).
All retroviral work is carried out in a BSL2 (or BSL3 for certain experiments like work with HIV) containment facility and is strictly governed by institutional guidelines.
Tips for keeping safe
If you are going to be working with retroviruses:
- Get trained to work with viruses, viral particles and biological material
- Wear the correct PPE (Personal protection equipment)
- Be informed as to emergency response and spill procedures
- Be extra careful with sharps and bio hazardous materials
- Know the right protocols for waste disposal and management
- Follow exact experimental protocols and safety procedures
- Report accidents, spills and unusual incidents
- Mosier D E (2004). Introduction for “Safety Considerations for Retroviral Vectors: A Short Review”. Applied Biosafety. 9(2):68-75.
- Temin (1990). Safety considerations in somatic gene therapy of human disease with retrovirus vectors. Human Gene Therapy. 1:111-123.
- Donahue et al (1992). Helper virus induced T cell lymphoma in nonhuman primates after retroviral mediated gene transfer. J. Exp. Med. 176:1125-1135.
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I don’t understand the paragraph about retroviruses being replication deficient. You say “Genes required for viral infection but unnecessary for packaging and transduction are deleted”. First, what is the difference between infection and transduction? Then you go on to say “Viral structural genes that are incorporated into the plasmid do not contain a ? (psi) sequence (which essentiallyhelps to incorporate the RNA into viral particles)”. The first sentence says that packaging is not affected, but the second sentence says that structural genes lack the Psi sequence which is required for packaging. How is replication prevented, and how does that affect the use of retrovirus as a tool for gene expression/knock-down?