In this study, we present evidence that variation in the P2 position of the HA cleavage site from an H1N1 influenza virus is directly linked to viral pathogenicity. In particular, we show that the high neurovirulence of the A/WSN/33 virus in mice is partially due to a tyrosine substitution at the P2 position, compared to the conserved serine residue shared by almost all other H1 HAs. In vitro, H1 HA with tyrosine or phenylalanine at the P2 position, which represents an optimized substrate, can be cleaved more efficiently by the serine protease plasmin than an HA with a serine residue at this position. Having an optimized substrate sequence for plasmin cleavage is crucial for in vitro viral multicycle replication in serum-free MDBK cells and also for in vivo replication in mouse brain. In both cases, the available plasmin or plasminogen might be very limited compared to the plasminogen normally present in cell culture serum, explaining why HA cleavage and virus propagation under serum-free conditions is greatly affected by the sequence at the cleavage site.
We suggest that A/WSN/33 and A/NWS/33 viruses, selected by extensive passaging of parental A/WS/33 virus in mouse brain, have not only accumulated mutations in the NA gene to recruit plasminogen and locally enhance plasminogen availability but have also selected mutations in HA to facilitate plasmin-mediated cleavage by the cell-associated plasminogen. Our results indicate that a bulky aromatic residue at the P2 position of WSN HA contributes greatly to viral neurovirulence, most likely because it allows efficient HA cleavage by plasminogen residing in brain tissue (4
). In contrast, the proteases responsible for WSN HA cleavage in respiratory organs might have no preference for specific residues at the P2 position, as lung tissue expresses a variety of trypsin-like proteases (2
) that are less likely to have a preference for specific residues at P2 position. While trypsin-like enzymes have been shown to act as candidate proteases for HA cleavage in the brain (19
), our data indicate that plasmin/plasminogen is a major discriminating factor that allows A/WSN/33 and A/NWS/33 viruses to spread efficiently in the brain.
As host proteases are required for influenza virus replication in vivo
, the exogenous addition of trypsin in the cell culture medium is often necessary for propagating influenza viruses in vitro
. However, several cell lines, for example, human HepG2 cells and certain MDCK cells, have been shown to support influenza virus replication in the absence of trypsin because these cell lines can either express HA-activating proteases or recruit proteases from tissue culture serum (31
). In contrast to HepG2 and MDCK cells, the trypsin-independent replication of influenza virus in MDBK cells is unusual because support for viral replication is restricted to A/WSN/33 and is also WSN NA dependent. This suggests not only that WSN NA can facilitate the recruitment of host cell proteases but also that WSN HA may consist of special structural elements to facilitate its cleavage.
In our study, we demonstrate that WSN HA cleavage in MDBK cells is mediated by the serine protease plasmin and that a tyrosine or phenylalanine residue at the P2 position of the HA cleavage site is critical for plasmin-mediated cleavage. Because plasminogen/plasmin are critical proteases involved in many biological functions, including fibrinolysis and wound healing as well as tissue homeostasis, the production and activation of plasminogen/plasmin are tightly regulated (44
). In the host, zymogen (plasminogen) is synthesized mainly by liver cells, and activation from plasminogen to the active form of plasmin depends mainly on its cleavage by plasminogen activators, which include both tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) (34
). Although it has been shown previously that WSN NA can recruit plasminogen to infected cells, it is not yet clear what factor activates the recruited plasminogen to plasmin, either in vitro
or in vivo
. To date, we cannot define the source for the plasmin associated with the MDBK cells in our study. However, we speculate either that MDBK cells can secrete plasminogen or that MDBK cells express higher levels of plasminogen activators at the cell surface, which can facilitate the production of plasmin from residual plasminogen even after extensive washing of cells with PBS. It has been reported that some nonhepatic cells express higher levels of mRNAs corresponding to plasminogen (48
); however, we have failed to detect plasminogen at the MDBK cell surface or in conditioned culture medium by Western blotting, indicating that the level of the expressed plasminogen from MDBK cells is very low. Regardless of the source of plasminogen, the presence of WSN NA can further enhance the concentration of plasminogen locally around WSN HA by recruiting plasminogen by means of the viral NA, consistent with our hypothesis that WSN HA, which represents an optimized plasmin substrate, can be efficiently cleaved and activated.
A trypsin-independent phenotype has also been described for the 1918 pandemic H1N1 influenza virus (8
), and similarities in HA cleavage between 1918 influenza virus and WSN have been suggested. However, the 1918 virus NA has been shown to be unable to recruit plasminogen (8
), and also the 1918 virus HA has no bulky aromatic residue at position P2 to allow it to be cleaved more efficiently by plasminogen (Fig. ). The mechanisms underlying HA cleavage therefore appear to differ between A/WSN/33 and 1918 influenza viruses. It may be possible that the 1918 virus NA recruits a different protease and that the recruited protease specifically recognizes the HA of the 1918 virus or that the enzymatic activity associated with 1918 virus NA allows HA to have different glycan modifications—which may subsequently enhance the access of the proteases to HA. In the future, a similar approach described in our study might be applied to the investigation of the molecular mechanism of 1918 influenza virus trypsin-independent viral replication and may help us understand the high virulence of 1918 influenza virus.
While A/WSN/33 and A/NWS/33 are laboratory-adapted viruses, a functional role for activation of influenza virus by plasmin/plasminogen is also likely in clinical situations. In addition to the activity of endogenous tissue plasminogen, coinfecting bacteria present in the respiratory tract, such as Staphylococcus
spp., produce plasminogen activators that can be used to cleave HA (5
). It has also been reported that plasmin-mediated activation may not be a unique feature of the HA of A/WSN/33, as other influenza viruses such as A/Udorn/72 (H3), may recruit plasminogen to the surface of the virus particle via annexin II on the viral envelope (24
). In the case of influenza A/WSN/33 virus, a maximal effect of the plasmin/plasminogen system has likely resulted from multiple genomic mutations in the selection of the virus from A/WS/33 (with mutations in the NA as well as in HA distal to the cleavage site and potentially in other gene segments). The data presented here suggest that the acquisition of a bulky hydrophobic residue (such a tyrosine or phenylalanine) at the P2 position of the HA cleavage site may have profound effects on the pathogenic potential of any influenza virus.
Although influenza virus is mainly pneumotropic in humans, it can occasionally cause encephalopathy or encephalitis (11
). Influenza viral antigen and genome have been detected in cerebrospinal fluid and brain tissue in human lethal cases, and influenza virus has also been speculated to contribute to the higher occurrence of central nervous system disorders in humans during and following pandemics (13
). To date, the proteases that might be responsible for influenza virus HA cleavage in the human brain are poorly understood. Although it has been shown that trypsin I isolated from rat brain and murine mini-plasmin accumulated in the cerebral capillaries were capable of cleaving HA (23
), their role in influenza virus neuropathogenesis has not been established in vivo
in humans. In the future, it will be of great interest to further define the role of plasmin and the activation of plasminogen in influenza virus neurovirulence in clinical situations. It will also be of critical importance to monitor surveillance data of influenza virus isolates for changes in the P2 position of the HA cleavage site (and in other positions) that may allow enhanced virus activation by proteases such as plasminogen/plasmin and increased virulence and spread in the host.