Novel insights into proteolytic cleavage of influenza virus hemagglutinin

Abstract

The influenza virus hemagglutinin (HA) mediates the first essential step in the viral life cycle, virus entry into target cells. Influenza virus HA is synthesised as a precursor protein in infected cells and requires cleavage by host cell proteases to transit into an active form. Cleavage is essential for influenza virus infectivity and the HA-processing proteases are attractive targets for therapeutic intervention. It is well established that cleavage by ubiquitously expressed subtilisin-like proteases is a hallmark of highly pathogenic avian influenza viruses (HPAIV). In contrast, the nature of the proteases responsible for cleavage of HA of human influenza viruses and low pathogenic avian influenza viruses (LPAIV) is not well understood. Recent studies suggest that cleavage of HA of human influenza viruses might be a cell-associated event and might be facilitated by the type II transmembrane serine proteases (TTSPs) TMPRSS2, TMPRSS4 and human airway trypsin-like protease (HAT). Here, we will introduce the different concepts established for proteolytic activation of influenza virus HA, with a particular focus on the role of TTSPs, and we will discuss their implications for viral tropism, pathogenicity and antiviral intervention.

Figure 1

Structural rearrangements associated with influenza virus hemagglutinin cleavage by host cell proteases. The HA precursor HA0 is cleaved into two subunits HA1 (red) and HA2 (blue) by cellular proteases which recognise either a multibasic or monobasic cleavage site located in a loop between HA1 and HA2. Multibasic cleavage sites harbour multiple arginines and/or lysines and are found in the HA‐proteins of HPAIV. Monobasic cleavage sites consist of a single arginine (Arg 344 (purple) in case of the 1918 influenza virus) or lysine and are found in the HA‐proteins of human influenza viruses and LPAIV. Upon proteolytic cleavage, the fusion peptide (green) in HA2 is liberated from HA1 and the HA is primed for activation by low pH, which involves insertion of the fusion peptide into a negatively charged pocket located adjacent to the cleavage site‐bearing loop in HA0. The subunits HA1 and HA2 remain covalently linked by a single disulfide bond (yellow), the newly formed C‐terminus of HA1 and N‐terminus of HA2 are labelled in purple. (A) Schematic representation of HA cleavage. (B) Structural changes associated with cleavage of the 1918 influenza HA (A/South Carolina/1/18 (H1N1), Accession‐No: ADD17229, HA0 (1RD8.pdb), HA1 + HA2 (1RUZ.pdb)

Figure 2

Activation of the influenza virus hemagglutinin in different cellular compartments. The influenza virus HA binds to alpha 2–3 linked (avian viruses) or alpha 2–6 linked (human viruses) sialic acids presented by proteins or lipids on the host cell surface. Upon endocytic uptake of virions, the acidic environment of the endosome triggers HA‐driven fusion of the viral and the endosomal membrane. After fusion the viral ribonucleoproteins are released into the host cell cytoplasm and transported to the nucleus, where viral genomic and messenger RNAs are synthesised. The viral membrane proteins HA, NA and M2 are synthesised in the secretory pathway. The HA proteins of HPAIV are cleaved in the Golgi apparatus/TGN by pro‐protein convertases of the subtilisin family, like furin or PC6, and cleaved HA is incorporated into progeny particles. The HA proteins of human influenza viruses and LPAIV can be cleaved by different proteases and in different cellular locations. HA can be transported to the cell surface, incorporated into virions in an uncleaved form and cleavage can subsequently be mediated by soluble proteases like plasmin in the extracellular space or by unknown serine protease(s) in endosomal vesicles of target cells. Alternatively, HA can be cleaved by the TTSPs TMPRSS2, TMPRSS4 and HAT, and cleavage could either occur en route to the cell membrane (TMPRSS2) or upon insertion into the target cell membrane (HAT). The cellular location where HA is cleaved by TMPRSS4 is at present unclear.

Figure 3

Structural organisation of the hemagglutinin‐activating enzymes TMPRSS2, TMPRSS4 and HAT.