Compounds with anti-influenza activity: present and future of strategies for the optimal treatment and management of influenza. Part I: influenza life-cycle and currently available drugs

GASPARINI, R.; AMICIZIA, D.; LAI, PL.; BRAGAZZI, NL.; PANATTO, D.

In order for the virus to emerge from the endosome and for ribonucleoprotein (RNP) to be released into the cytosol, the envelope must fuse with the endosome membrane. This part of the life-cycle of the virus requires the acidity of the lumen of the endosome to decrease to a pH value of about 5 [103, 104] . In this process, a crucial role is played by M2, which not only acts on ion channels, thereby allowing acidification of the interior of the virus [105] , but also alters its conformation, resulting in changes of the curvature of the viral envelope [106, 107] . This leads to two important events, namely: the dissociation of the M1 protein from RNP and a dramatic change in the conformation of HA [108] , which can expose its fusion peptide. This peptide may allow fusion of the viral envelope and the endosome membrane and the release of RNP into the cytoplasm. The cleavage of HA occurs through a proteasic enzymatic action [109] . Proteases that can cause the cleavage of HA are widely distributed throughout the human body, and the precise role that each plays in cutting the HA is not exactly known. Nevertheless, proteases are known to belong to two main classes, namely trypsin-like enzymes or furinlike serine enzymes [109] . These enzymes are produced by the cells, particularly those of the respiratory system, in the presence of inflammation (proinflammatory cytokines/chemokines, neutrophils, etc.) [110] . The release of RNP into the cytosol is also enabled by the host's aggresome processing machinery (made up of dynein, dynactin and myosin II) [111] . Further molecules and pathways could take part in this process. M2 is more than just a simple ion channel. Indeed, it plays a multifaceted role in the entire viral life-cycle, as the mechanism of proton permeation, conductance and acidification is crucial to each different step and activity of the virus. After endocytosis, the low endosomal pH (in the range 5-6) activates the M2 channel prior to HA-mediated fusion, and favours dissociation of the M1 protein from the vRNPs, thereby facilitating the entry of RNPs into the nucleus. M2 is fundamental to genome unpacking and the release of the viral RNA during viral uncoating [112] . Moreover, M2 enables the viral RNA to package and facilitates virion scission and budding during viral maturation and assembly, replication and infection: during transport to the cell surface, in the trans-Golgi network (TGN) membrane, M2 acts as a proton-leak channel and prevents the activation of de novo synthesized HA by regulating the pH of the Golgi apparatus of the host cell and equilibrating its pH with the pH of the viral interior. Indeed, if it were not elevated in this way, the low pH would cause a conformational rearrangement of HA, whose intracellular cleavage would prematurely inactivate the new influenza viral progeny [113, 114] . M2, together with other proteins such as NS1 and HA, could play an additional role of fine-tuning the apoptosis of infected cells, thus favouring viral replication [115] . NS1 is involved in several biological tasks, such as mRNA splicing and translation, cell survival, and immune defence. In particular, as far as the type I interferon (IFN-I)-mediated response is concerned, it interacts with PACT/PRKRA (Protein activator of the interferon-induced protein kinase / Protein kinase, interferoninducible double stranded RNA dependent activator), which is an important cofactor for the IFN-I response elicited by the viral RNA-sensor