4.1. IAV Infection
Influenza viruses are single-stranded, negative-sense, enveloped RNA viruses of the orthomixoviridae
family with a segmented genome composed of eight independent RNA fragments, each one encoding for structural and non-structural proteins. According to the antigenic differences between the nucleoprotein (NP) and matrix (M) protein, influenza viruses can be classified into three types, namely, A, B and C. Although all three types of influenza viruses can naturally infect humans, only the type A virus has a wide range of animal host species, including birds, swine, horses and other mammals 
, whereas the identification of influenza B and C viruses in animal hosts is sporadic 
IAVs have been extensively studied due to their ability to cause highly contagious diseases in humans and animals (such as poultry, swine and horses), with potentially fatal outcomes 
. Their intrinsic nature is to continuously change the antigenicity by accumulating point mutations on the surface glycoproteins to escape the existing immunity established by previous infection or vaccination (so-called “antigenic drift”) 
. Furthermore, they cause pandemics by the so-called “antigenic shift”, during which new antigenic subtypes are introduced, by segment reassortment, into an immunologically naïve host population. Further adaptations occur to facilitate transmission in the new host species 
. Although many global pandemics and major epidemics have occurred at regular intervals during human history 
, during the last century, however, four pandemics have been documented in 1918, 1957, 1968 and 2009 
. Then, due to the replicating nature of influenza viruses and the pressure of the immune response, the pandemic viruses progressively evolve into seasonal viruses that acquire mutations to escape the immune response elicited in the previous year 
. These antigenic changes require an annual update of the seasonal vaccine composition 
. Interestingly, every 38–40 years, a replacement of the normally circulating seasonal virus with a completely new virus occurs that is not recognized by memory B and T lymphocytes and, thus, causes a pandemic, as most of the population is immunologically naïve 
Influenza virus infections induce both innate and adaptive host immune responses, which ultimately result in the abortion of virus replication 
. Innate immunity and adaptive immunity profoundly differ from each other in terms of responsiveness, specificity and functionality. Innate immunity is the first line of defense against IAV that is specialized in controlling primary infection and induces the adaptive response through the production of co-stimulatory molecules, such as type I IFN, that exhibit antiviral, anti-proliferative and immunomodulatory functions 
. Thus, antibody-mediated immunity and cellular-mediated immunity become activated and completely neutralize the virus.
4.3. TRIM22 and IAV Evolution
A wide range of proteomic and genome-wide RNAi-based screens have been used to identify host factors that are partners of NPs and RNPs in viral replication, as reviewed in 
. However, few factors have been extensively characterized. TRIM22 has the peculiarity of being able to restrict seasonal, but not pandemic, influenza virus replication in vitro 
. Despite the fact that the NP is a highly conserved protein, differently from the hemagglutinin protein that mediates entry into cells, and that it is the target of neutralizing antibodies 
, in comparison with seasonal pandemic virus sequences, four lysine (K) mutations were identified in seasonal viruses, whereas pandemic viruses were endowed with arginine (R) residues (Figure 2
Figure 2. Evolution from pandemic to seasonal IAV has shaped TRIM22 restriction. Pandemic viruses are resistant to TRIM22 inhibition as their NP is endowed with four arginine (R) residues that progressively mutate into lysine (K) residues, becoming the target of the U3 ubiquitin ligase activity of TRIM22. The transition of R into K is dependent on viral polymerase errors that generate viral quasispecies either characterized by one, two, three or four K residues. However, a bottleneck of transmission favors the emergence of an IAV NP susceptible to TRIM22 restriction. This phenomenon is likely related to the general rule of viral evolution which endows the virus with the ability to become more transmissible and less pathogenic.
These four R-to-K changes progressively accumulated in approximately 90 years of IAV circulation in humans when sequences from the original pandemic 1918 H1N1 virus were compared with those of the following seasonal strains until 2009, when a new pandemic H1N1 virus emerged. The modeling of the atomic NP 3D structure showed that the four lysine residues are exposed to the solvent and therefore are potential targets of TRIM22 ubiquitination 
. Concerning the other possible roles of the amino acid R-to-K changes, it has been previously reported that none of these residues are involved in the bipartite nuclear localization signal 
, binding to viral RNA 
and viral polymerases 
, but they are mainly correlated with the host specificity of the virus 
. In this regard, two sites, i.e., 98 and 422, are part of cytotoxic T lymphocyte (CTL) epitopes 
. As only two of the four R-to-K variations are likely the result of CTL escape, other selective forces must contribute to the NP variation.
Of relevance is the potential role of adaptive mutations in the IAV animal host that can render viruses resistant to human restriction factors and, thus, have the advantage of being transmitted to humans. In this regard, human myxovirus resistance A (MxA) has been described as a potent restriction factor of avian IAVs 
; however, the 1918 and 2009 pandemic H1N1 viruses have acquired a cluster of mutations in the NP that inactivates MxA restriction 
. Mutations conferring MxA resistance are absent in avian IAVs; however, these mutations have been acquired in avian-derived viruses circulating in swine 
. As pandemic strains are also resistant to TRIM22 restriction, NP adaptation in the swine host could also explain their lack of susceptibility to TRIM22 restriction. However, during IAV evolution in humans, TRIM22 acquires the ability to interact with the NP and adaptive mutations in the NP that render IAVs sensitive to TRIM22 restriction. Indeed, TRIM22 directly interacted with the NP of susceptible IAV strains both in a cotransfection system and during in vitro
infection, and this interaction was followed by TRIM22-mediated downregulation and ubiquitination of the viral protein 
. In contrast, the 2009 pandemic virus and the viral strains that are resistant to TRIM22 activity were unable to interact with TRIM22. Experiments based on the mini-replicon genome system demonstrated that the four NP R-to-K mutations are the main determinants of TRIM22 sensitivity 
In order to elucidate the mechanisms that IAV has adopted to escape restriction factors, Juan Ortin’s laboratory demonstrated that, in the absence of the selection pressure exerted by IFNs, serial passages of IAV promoted the introduction of mutations that allowed the virus to increase replication fitness 
. However, in the absence of any constraint such as that of IAV cultivation in eggs or cell cultures, many of the adaptative mutations acquired during viral passages were purged from the viral population during or shortly after infection, as demonstrated in a human challenge study 
. In the presence of selection pressure and bottleneck of transmission, IAV may acquire adaptative mutations that could lead to increased susceptibility to restriction factors, including TRIM22, thereby resulting in a less efficient viral replication.
In conclusion, TRIM22 is an IFN-dependent restriction factor of human-adapted IAV, whereas it does not function as a barrier for pandemic viral strains. During replication in animal hosts, the pandemic strains undergo a number of amino acid changes in the NP that render them resistant to TRIM22 restriction and favor their transmission and human-to-human spread. Overall, the genetic variations in the NP gene will be useful for monitoring the viruses and preparing effective prevention and control strategies for potential pandemic influenza outbreaks.