Plasminogen-Binding Activity of Neuraminidase Determines the Pathogenicity of Influenza A Virus

Abstract

When expressed in vitro, the neuraminidase (NA) of A/WSN/33 (WSN) virus binds and sequesters plasminogen on the cell surface, leading to enhanced cleavage of the viral hemagglutinin. To obtain direct evidence that the plasminogen-binding activity of the NA enhances the pathogenicity of WSN virus, we generated mutant viruses whose NAs lacked plasminogen-binding activity because of a mutation at the C terminus, from Lys to Arg or Leu. In the presence of trypsin, these mutant viruses replicated similarly to wild-type virus in cell culture. By contrast, in the presence of plasminogen, the mutant viruses failed to undergo multiple cycles of replication while the wild-type virus grew normally. The mutant viruses showed attenuated growth in mice and failed to grow at all in the brain. Furthermore, another mutant WSN virus, possessing an NA with a glycosylation site at position 130 (146 in N2 numbering), leading to the loss of neurovirulence, failed to grow in cell culture in the presence of plasminogen. We conclude that the plasminogen-binding activity of the WSN NA determines its pathogenicity in mice.Influenza A viruses possess two virion surface glycoproteins, a hemagglutinin (HA) and a neuraminidase (NA). The HA binds to cell surface receptors and mediates fusion between the endosomal membrane and the viral envelope. The latter event requires cleavage of the HA into HA1 and HA2 subunits, thereby exposing the N-terminal hydrophobic region, which is thought to interact with the host membrane and trigger membrane fusion (32). Thus, influenza A viruses cannot infect host cells unless the HA is proteolytically cleaved (14,15).Although the virulence of influenza A viruses is controlled polygenically, the HA plays a pivotal role in determining the severity of infection in avian strains (8,11,26). The HA cleavage site sequences in virulent and avirulent avian influenza viruses differ; the former possess a series of basic amino acids at this site, while the latter do not (10, 13). The ubiquitous host proteases furin and PC6, which specifically recognize these multiple basic residues, cleave the HAs of virulent viruses, leading to systemic infection (12,27). By contrast, the HAs of avirulent viruses are not cleaved by these proteases because they lack the requisite series of basic residues at their cleavage sites. Instead, they are susceptible to proteases that are presumably localized in the respiratory and/or intestinal tract, thus leading to localized viral infection.All mammalian influenza viruses, excluding equine H7N7 viruses, have a single Arg residue at the HA cleavage site. Thus, the HAs cannot be cleaved by ubiquitous furin or PC6 protease, resulting in a localized infection. However, a mouseadapted human isolate, A/WSN/33 (WSN; H1N1), which is recognized as a neurovirulent strain, causes systemic infection when inoculated intranasally into mice (3). Studies with WSN-A/Hong Kong/68 (H3N2) reassortant viruses indicated that the NA gene determines WSN neurovirulence in mice by facilitating HA cleavage (24). Only a si...

PAR1 contributes to influenza A virus pathogenicity in mice

“…Interestingly, however, PAR1-AP did promote PLG-dependent HA cleavage in lung epithelial cultures, suggestive of a possible interaction of PAR1 signaling with the ability of IAV to become infectious and hence replicate. These findings are consistent with the prior observation that PLG contributes to the pathogenesis of IAV infection (27,28). Additionally, PAR1 signaling may promote PLG activation to plasmin (29,30), thereby providing a possible link to increased HA cleavage and IAV production.…”

“…PLG is an important mediator of lung inflammation (25,26) and is known to influence IAV virulence (27,28). Importantly, PLG binding to cells and activation may be controlled by PAR1 signaling (29,30).…”

Molecular cloning and characterization of two Helicobacter pylori genes coding for plasminogen-binding proteins

“…It is believed that tissue penetration and dissemination are facilitated by the acquisition of Pg on the bacterial surface and subsequent activation of this surface-associated Pg by a host-derived Pg activator such as tissue Pg activator (tPA) or urokinase Pg activator. Pg binding is used by the influenza A virus to enter host cells (17). The prion protein PrP(Sc) that is associated with transmissible spongiform encephalopathy has also been reported to bind Pg (18), although the physiological significance has been questioned (19).…”

Global Host Immune Response: Pathogenesis and Transcriptional Profiling of Type A Influenza Viruses Expressing the Hemagglutinin and Neuraminidase Genes from the 1918 Pandemic Virus

“…Recent work from our laboratories using such a surrogate system demonstrated that the NS1 protein of influenza A/Brevig Mission/1/18 virus expressed in the context of a recombinant influenza A/WSN/33 virus (WSN) was a more potent alpha/beta interferon (IFN-␣/␤) antagonist than the parental WSN NS1 protein in infected human respiratory epithelial (A549) cells (29). In addition to the IFN-antagonistic properties of the NS1 protein, the surface glycoproteins of influenza virus (HA and NA) have been shown to be important virulence factors in mice and birds (30,31,33,41,52,61,65,71,73). As the HA and NA sequences of the 1918 virus have been elucidated (57, 58), we next determined the contribution of the 1918 HA and NA genes to viral pathology in the mouse model by examining infection outcome, lung histology, and gene expression changes by expression microarray analysis.…”