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.

As the infection progresses, the apical membrane becomes rich in viral proteins, which together initiate the budding of the virus around the complex of RNPs, at the regions of the membrane where the extruded HA, the NA, and M1 and M2 proteins are concentrated [195] . Viral proteins are then delivered to the plasma membrane and assembled. Here, Rab11a [196] and HRB (HIV Rev-binding protein) [197] play an important role. HA is able to initiate the process of budding, but not to complete it. This requires the mediation of NA, M1 and M2 proteins [195] . It is also important to consider that, during the formation of the positive curvature of the cell membrane, the suitably assembled units of RNPs move toward the distal part of the viral bud, so that they can be properly wrapped by the viral envelope. As the budding process progresses, a stalk is generated which holds the virion to the cell. The viral envelope must then detach itself from the cell membrane. The M2 protein appears to be crucial to this process. Indeed, it is capable of generating a positive curvature of the membrane), which is necessary in order to enable the spherical virions to split off [195] . However, the virus is not yet free; it is bound to the cell by the binding of HA molecules with the units of sialic acid of the membrane surface. NA molecules must therefore detach the sialic acid from the cell surface in order to accomplish viral budding. The enzymatic mechanism of influenza virus sialidase has been studied by Taylor, who showed that the enzyme catalysis process is particularly complex and consists of four steps. During the first step the α-sialoside is distorted from a chair conformation to a pseudoboat conformation when the sialoside binds to the sialidase. The second step leads to sialosyl cation, an oxocarbocation intermediate. The third step is the formation of Neu5Ac, as α-anomer. The fourth step involves its mutarotation and the subsequent release of the thermodynamicallystable β-Neu5Ac [198] . Finally, these steps lead to sialic acid hydrolysis. Currently, 11 isoforms of NA are known; NA10 and NA11 have recently been isolated from bats and are not able to cleave sialic acid. Their precise role and mechanism are still unknown [54] . NA is further classified into two groups: group 1 (N1, N4, N5 and N8) and group 2 (N2, N3, N6, N7 and N9), based upon primary sequence [199] . Group 1 NAs contain a 150-cavity (formed by amino acids 147-151 of the 150-loop), an exposed pocket near the active catalytic site, whereas group 2 NAs lack this cavity [200] . Budding occurs via a VPS4 and VPS28 independent pathway [201, 202] .