A novel mechanism for the acquisition of virulence by a human influenza A virus

Abstract

Cleavage of the hemagglutinin (HA) molecule by proteases is a prerequisite for the infectivity of influenza A viruses. Here, we describe a novel mechanism of HA cleavage for a descendant of the 1918 pandemic strain of human influenza virus. We demonstrate that neuraminidase, the second major protein on the virion surface, binds and sequesters plasminogen, leading to higher local concentrations of this ubiquitous protease precursor and thus to increased cleavage of the HA. The structural basis of this unusual function of the neuraminidase molecule appears to be the presence of a carboxyl-terminal lysine and the absence of an oligosaccharide side chain at position 146 (N2 numbering). These findings suggest a means by which influenza A viruses, and perhaps other viruses as well, could become highly pathogenic in humans.

Figure 1

HA cleavage mediated by the WSN NA. 293T cells were transfected with a plasmid expressing the WSN HA alone, the WSN HA and WSN NA, or the WSN HA and the NA from A/Hong Kong/1/68 (HK) (H3N2). Cells were labeled with [³⁵S]methionine at 24 h after transfection for 10 min and chased for 1 h. They were then incubated with trypsin (0.5 μg/ml), 1% FCS, or bovine (50 μg/ml) or human (5 μg/ml) plasminogen for 30 min. HAs were then immunoprecipitated from cell lysates by monoclonal antibodies to the WSN HA.

Figure 2

Polykaryon formation by cells expressing HA or both the HA and NA. 293T cells were transfected with plasmids expressing the WSN HA (a–c), WSN HA and WSN NA (d–f) or WSN HA and HK NA (g–i). At 24-h posttransfection, the cells were treated with 0.5 μg/ml trypsin (a, d, and g), 1% FCS (b, e, and h), or 5 μg/ml human plasminogen (c, f, and i) for 30 min. After exposure to PBS (pH 5.0), the cells were then incubated for 6 h, fixed with 3% formaldehyde, and stained with Giemsa solution.

Figure 3

Three-dimensional structure of the NA monomer illustrating the relative positions of the carboxyl-terminal amino acid residue (space filled) and oligosaccharide chain at position 146 (space filled with dots). The sialic acid is shown in red in the substrate binding pocket (stick). The last seven carboxyl-terminal residues (490–496) of the NA are shown in purple. (A) Top-to-bottom view. The NA is a tetramer, and the upper and right-hand sides of the depicted molecule face with other monomers. (B) Side view. In a tetramer, the right-hand side is internal and the left-hand side external. The fourfold axes are indicated by a square with an arrow. This figure is based on the NA structure of an N2 virus, A/Tokyo/3/67, as determined by Varghese et al. (23), rather than that of an N1 virus, which is not available. However, because the basic folding of the NA is essentially identical between type A and type B NAs (24), the basic folding of the N1 NA should not differ appreciably from the one shown.

Figure 4

Plasminogen binding to NA on the cell surface. (A) 293T cells were transfected with the WSN NA (WSN) gene, a mutant containing an alteration at the carboxyl-terminal residue, from lysine to arginine (K453R), or the HK NA gene and incubated for 24 h. Transfected cells were suspended in PBS containing 1% BSA and then incubated with 100 μg/ml human plasminogen. Bound plasminogen was detected by anti-human plasminogen antibody and fluorescein isothiocyanate-labeled anti-rabbit antibody. Cells were analyzed by flow cytometry. The thin line indicates control cells transfected with an expression plasmid with no insert, while the thick line refers to NA gene-transfected cells. (B) MDBK cells were infected with the WSN, WSN-HK, or WS virus. Twelve hours after infection, the cells were incubated with human plasminogen (10 μg/ml) and bound human plasminogen was detected as described above. MOCK, mock-infected cells.

Figure 5

Schematic representation of plasminogen-mediated HA cleavage. (A) Plasminogen binds to a lysine at the carboxyl terminus of the WSN NA. Bound plasminogen is activated to plasmin by a plasminogen activator (most likely of cellular origin). Enzymatically active plasmin then cleaves HA0 into HA1 and HA2 subunits. (B) If NA does not contain a lysine at the carboxyl terminus or an oligosaccharide chain is present in the vicinity, plasminogen does not bind to the NA and HA0 remains uncleaved.