Defective Interfering genomes: do they protect us against viral infections?

Oct ,24 2016

Viruses have learned to escape from cellular detection

Cells and viruses are in constant battle. Whereas the former have evolved to develop mechanisms to detect viral genomes, the latter have evolved to escape from such detection. Therefore, viruses need to be constantly optimally fit and only those that successfully escape from cellular detection are likely to survive to spread infection.

Cells possess distinct families of proteins whose function is to detect anything “foreign”. These are located mainly in the cellular membrane, endosomes and free in the cytoplasm. Two main cytoplasmic receptors that recognize viral nucleic acids are RIG-I and Mda5. They bind to nucleic acids that contain particular features that are not normally present in the host nucleic acids, thus distinguishing between host and virus genomes. Upon detection, RIG-I and/or Mda5 become activated, initiating a signalling pathway that culminates in the production of interferon (IFN). IFN is secreted and acts in both neighbouring uninfected cells and infected cells to initiate a signalling cascade in them that ends with the production of hundreds of proteins, many of them with antiviral activity. The result of this is the establishment of an antiviral state in cells that dramatically reduces viral replication and spread.

Viral replication consists of the generation of a faithful copy of the viral genome within the infected cell. Briefly, the viral polymerase reads along the viral genome from its 3’end to its 5’end in the cytoplasm, thus creating an antigenome that will be subsequently read by the viral polymerase to generate a genome identical to the previous one. One can think that more genomes imply more opportunities for cytoplasmic receptors to detect viral infection. Unfortunately, this is not always the case, as viruses have evolved mechanisms to avoid such detection. For example, many RNA viruses generate the nucleoprotein (NP), a viral protein used to encapsidate the viral genome simultaneously to its generation and thereby the viral genome is hidden and cannot be detected easily.

Viruses generate defective interfering (DI) genomes

But nothing is perfect. Viral polymerases of RNA viruses (viruses whose genome is formed by RNA) are prone to make errors in the replication process. One of these errors leads to the generation of what is known as defective interfering genomes (DIs). This occurs when the viral polymerase “jumps” from one template to another while reading, or from one part of the template to another, and then resumes elongation. This is not exclusive of RNA viruses, as potentially all viruses are capable of spontaneously generating DI genomes during their replication cycle.

“Jumping” from one part of the template to another nearer the 5’end generates a single internal deletion (multiple deletions are also possible), whereas “jumping” from one template to the nascent strand is called copyback or hairpin DI genome (Fig. 1, example of the replication of parainfluenza virus 5 and the generation of copyback DI genomes). No matter how it was generated, the DI genome lacks essential genes for replication. Therefore, the DI virus, containing the DI genome, needs a co-infection by a helper, non-defective (ND) virus that provides the essential proteins encoded by the missing genes, to be able to replicate within the cell. When this occurs, the DI genome has great advantage over the ND genome in the replication process (hence interfering with it) and DI viruses soon surpass in number those viruses containing ND genomes.






Fig. 1. Schematic of the replication process of an RNA virus. The genomic RNA of parainfluenza virus 5 (PIV5) is represented. The viral polymerase reads along the genomic RNA to generate the antigenomic RNA. The genomic RNA is read by the viral polymerase to generate more genomic RNA. It can occur that the viral polymerase jumps to the nascent strand, thus creating a stem-loop structure called Trailer copyback DI. When the jump occurs when reading the genomic RNA it is called Leader copyback DI.

DIs alert the immune system of the presence of viral infection

Coming back to the cytoplasmic receptors RIG-I and Mda5, they recognize determined features in the nucleic acids. RIG-I recognizes 5’ triphosphorylated double stranded RNA with blunt ends, and this happens to be the case of the copyback DI from PIV5 shown in Fig. 1, and also other DI genomes of certain viruses. Indeed, it has been shown that the cellular immune system is activated upon detection of DI genomes of PIV5 instead of normal, non-defective viral genomes.

Let’s suppose that cells are infected with PIV5. Once in the cytoplasm, the virus needs to counteract different mechanisms of cellular immune activation, as these mechanisms lead to the production of proteins that have a deleterious effect for the virus survival. Focusing on the replication process, we have already said that the new genome gets encapsidated as soon as it is generated, making it difficult for cytoplasmic receptors to recognize it. Further in time, some DI genomes will have been generated that will have formed DI viruses that subsequently go out of the cell to infect neighbouring cells and thus alert their immune system. This raises a problem for the virus, as the more DI viruses generated, the more likely the cells are to detect DI genomes and initiate an antiviral response that combats the overall viral infection.

Can DIs protect us from viral threat?

A lot of efforts have been made to take advantage of the ability of DIs to activate the IFN system. To date, no investigation is conclusive enough to ascertain that DIs can have therapeutic effects in humans, and numerous studies performed on animals have not yet yielded promising results.

The best studied DI RNA in terms of therapeutic purposes is an influenza DI RNA that could potentially protect against all influenza A subtypes.

In terms of developing DI viruses which could protect against other common virus infections, the best studied of them seems to be an influenza DI RNA that potentially could protect against all influenza A subtypes. Nevertheless, it is still not possible to modify a DI sequence to increase its protective ability, as DIs have not been extensively studied in these terms. Furthermore, it seems that the ability of the DI RNA to interfere with the helper virus is not the sole answer to establish a correlation between protection and interference, thus complicating the development of new therapeutics using DIs.

In summary, DI genomes play an important role in the virus life cycle, seriously affecting the spread of infection and the activation of the host immune system. They could represent a powerful advantage in developing new therapeutics against viral infection. Unfortunately, results in animal models have not performed as desired to conclude that DIs can be used as therapeutic agents. The good point is that we will soon see crucial advancements due to intense research in the field.



Barbalat, R., Ewald, S.E., Mouchess, M.L. & Barton, G.M. 2011. Nucleic acid recognition by the innate immune system. Annu Rev Immunol, 29:185-214.

Killip, M.J., Young, D.F., Gatherer, D., Ross, C.S., Short, J.A., Davison, A.J., Goodbourn, S. & Randall, R.E. 2013. Deep sequencing analysis of defective genomes of parainfluenza virus 5 and their role in interferon induction. J Virol, 87(9):4798-4807.

Marriott, A.C. & Dimmock, N.J. 2010. Defective interfering viruses and their potential as antiviral agents. Rev Med Virol, 20(1):51-62.