In 1918  the“Spanish flu” killed, within two months, at least 50 million people (3-5% of the world population): an apparently innocent virus caused the most devastating pandemic in history. Three more pandemics have occurred in the last and the new centuries: the “Asian” (1957), the “Hong Kong” (1968) and the more recent “Swine influenza” (2009).

The causal agent of these pandemics belongs to the family of influenza A viruses, the same that evolves into the classic seasonal flu (with generally mild symptoms that regress in about a week). How is it possible that two viruses belonging to the same family can cause such a different pathology severity?

Researchers of the “Viral Pathogens and Biosafety” Unit of the San Raffaele Hospital in Milan, Italy have uncovered a new piece of this puzzle. The results are fascinating and innovative and, 100 years since the first pandemic, contribute to the solution of the mystery that is still unsolved.  Isabel Pagani, first author of the research and PhD student of the course in Molecular Medicine of the San Raffaele University in Milan, and Dr. Elisa Vicenzi, Head of Unit.


Influenza A virus has a spheroidal shape and is equipped with a membrane (which separates it from the external environment) derived from the infected cell. Anchored in the membrane there are two key viral proteins, i.e. HA (hemagglutinin) and NA (neuraminidase). In nature, there are 16 HA and 9 NA that can be combined to generate influenza virus subtypes (eg H1N1, H2N2, H3N2 etc). Inside the viral particle it is contained its genetic information of the virus, composed of 8 RNA segments; each of them associated with other viral proteins. For the purpose of this research, it is important to underline the role of the nucleoprotein (NP).

Simplified scheme of influenza A virus. Adapted from Horimoto, T. et al., 2005
Simplified scheme of influenza A virus. Adapted from Horimoto, T. et al., 2005


Isabel Pagani recalls: “This research takes inspiration from a previous study of our research Unit carried on mostly by Dr. Andrea Di Pietro, at the time also a student of this PhD in our Unit. The published study showed how an intracellular protein induced by Interferon, the TRIM22 protein, was able to inhibit seasonal influenza virus replication. In fact, when a virus enters the cells, it stimulates the synthesis of Interferon, which promotes the activation of dozens of anti-viral response genes, including TRIM22. Our study showed that this protein directly interacts with the viral nucleoprotein and promotes its association with several ubiquitin molecules. Ubiquitin is a kind of “label” recognized by the proteasome (the intracellular degradation machinery): TRIM22, therefore, induces the degradation of a component of the virus indispensable for its replication”.

Photo Credit: ShutterStock
Photo Credit: ShutterStock

The influenza virus is a zoonotic virus, i.e. it can be transmitted from the animals-to-humans. “In the animal hosts, flu viruses of different subtypes can combine to give rise to a new viral particles: if the human immune system is naïve to the new virus it is unable to recognize and neutralize it thus leading to a pandemics, i.e. an epidemy on a global scale”.


Our question was whether the same antiviral mechanism based on the induction of TRIM22 could also contain the infection of the pandemic influenza virus”. To deepen this study, the San Raffaele researchers collaborated with Dr. Nadia Naffakh of the Pasteur Institute in Paris, who provided a sample of 2009 Swine influenza virus for testing. “We infected some cells with this virus, and evaluated the response to TRIM22: we found that TRIM22 was not able to contain pandemic virus infection in vitro”.

Why is this? “We know that protein ubiquitination – which then causes nucleoprotein degradation of the nucleoprotein in the proteasome – requires the presence of a specific  amino acid called lysine (indicated by the letter K). By making a comparison between the seasonal and the pandemic influenza viruses, we observed that in pandemic viruses, 4 lysines are replaced by 4 arginines, another amino acid (indicated by the letter R) that, however, cannot be used for protein ubiquitination. This means that:

  1. In the seasonal influenza virus, the nucleoprotein has 4 lysines, target of TRIM22 ubiquitination reaction. In this way, the proteasome degrades the viral nucleoprotein causing the inhibition of viral replication.
  2. In the pandemic virus, the nucleoprotein has 4 arginines: TRIM22 no longer finds the residues for the ubiquitination reaction. Thus, the viral nucleoprotein is not degraded, and virus replication can proceed undisturbed.

Thanks to the complex technique called “reverse genetics”, we have created several mutants of the nucleoprotein” specifies Isabel Pagani “and we observed that only the “quadruple mutant”, i.e. the virus with all 4 lysines replaced by arginines, was no longer susceptible to the inhibition induced by TRIM22”.



Dr. Vicenzi says: “During the last century, the virus has evolved from pandemic to seasonal, passing through various intermediate states. Inspired by a letter published in Nature in 1970 by the geneticist John Maynard Smith, the 4 arginine-to-lysine mutations of the nucleoprotein are best understood by analogy with a word game. The object of the game is to pass from one word (= a viral protein), to another of the same length by changing one letter at a time (= an amino acid change) with the request that the intermediate words are meaningful (= that all viruses are able to replicate)”.

In the example, in 4 steps we get from “WORD” to “GENE”.

Our research has shown a very similar mechanism: from the 4 arginines present in the pandemic virus, resistant to the action of TRIM22, the virus has evolved by changing one amino acid at a time, until reaching the 4 lysines of the seasonal influenza virus that made it now susceptible to TRIM22 inhibition”.

We are also interested in studying the reaction of our immune system to viral infection” concludes Dr. Vicenzi. “Our immune system responds to influenza virus by producing antibodies that can neutralize the virus before it infects cells, but also by inducing cytotoxic T cells, which recognize and kill infected cells. Of the two, the antibody response has always been the most studied because the flu vaccines are based on it. Our work suggests that the evolution of pandemic to seasonal virus has been strongly influenced by a collaboration among different components of the immune response to viruses including the cytotoxic response of T lymphocytes (forcing the conversion of arginine to lysines) and TRIM22, the molecule induced by Interferon. The general implications of our study are part of the global effort to achieve a “universal vaccine” that would protect against all Influenza viruses, both seasonal and pandemic”.

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