Influenza virus hemagglutinin cleavage into HA1, HA2: no laughing matter.

What do an odd lab-derived strain of influenza, fowl plague virus, and the 1997 Hong Kong chicken flu have in common? The answer lies in how, and more specifically by what protease, the hemagglutinin protein of these various influenza strains is cleaved for viral activation. What does this have to do with the most devastating influenza virus of them all, the 1918 ‘‘Spanish’’ influenza? The connection may be a new and interesting mechanism for hemagglutinin cleavage proposed by Goto and Kawaoka in an article in this issue of Proceedings (1). In a series of experiments confirming and synthesizing 25 years of experimental data (2–4), Goto and Kawaoka (1) demonstrate specifically how the neuraminidase (NA) protein of influenza AyWSNy33 (H1N1), a curious variant of the first human influenza virus ever isolated, mediates hemagglutinin (HA) cleavage. Goto and Kawaoka (1) provide evidence for a model whereby the NA of WSNy33 directly binds plasminogen, sequestering it for cleavage activation. Subsequently, active plasmin cleaves and activates influenza hemagglutinin. Being able to sequester plasminogen for hemagglutinin cleavage allows the influenza virus to infect cells other than its usual targets. With the exception of WSNy33 and the few influenza strains named above, most influenza viruses replicate in a strictly limited subset of cells. Influenza A viruses are negativestranded RNA viruses. Like many other enveloped viruses, they code for a surface glycoprotein that must be cleaved by cellular proteases for activation. HA, a major influenza surface glycoprotein, is translated as a single protein, HA0. For viral activation, HA0 (assembled as trimers) must be cleaved by a trypsin-like serine endoprotease at a specific site, normally coded for by a single basic amino acid (usually arginine) between the HA1 and HA2 domains of the protein. After cleavage, the two disulfide-bonded protein domains produce the mature form of the protein subunits as a prerequisite for the conformational change necessary for fusion and hence viral infectivity (5). Influenza is a zoonotic disease, infecting a wide variety of warm-blooded animals, including birds and mammals. In aquatic birds, normal influenza replication takes place in the intestinal tract and tends not to cause symptoms. In mammals like humans and swine, influenza replication is limited to epithelial cells of the upper and lower respiratory tract. This tissue tropism is controlled to some extent by the limited expression of the appropriate protease for viral activation (6). In mammals, the suspected protease in the respiratory tract is tryptase Clara, a serine protease produced by nonciliated Clara cells of the bronchial and bronchiolar epithelia (6). Occasional avian influenza strains have been described with an insertion mutation at the cleavage site of HA, allowing HA to be cleaved by ubiquitously expressed proteases (furin and other subtilisin family proteases) (6). As a consequence, the virus can replicate throughout the bird’s body, producing necrotic foci in spleen, liver, lung, and kidney and encephalitic lesions in brain (7). These highly virulent strains have been observed in only two of the 14 described HA subtypes in birds (6) and include those influenza strains previously described as fowl plague viruses. They emerge only occasionally but can cause devastating mortality in poultry flocks (8). The insertion responsible for the ubiquitous cleavage adds additional basic amino acids at the cleavage site (9), with a minimal motif of RyL-X-RyL-R. Until recently, this mutation had been found only in avian viruses of the H5 and H7 subtypes, subtypes that were not thought to infect humans. This barrier was broken dramatically in 1997 in Hong Kong when 16 people were infected with an avian H5N1 influenza virus (10). Five people died of complications of infection, including the index case, a 3-year-old child who died with Reye’s syndrome (11). The AyHong Kongy156y97 (H5N1) virus isolated from the 3-year-old child possessed the cleavage site mutation typical of virulent avian influenza viruses (12, 13). Of the 12 patients clinically described (11), seven had pneumonia andyor acute respiratory distress syndrome. Gastrointestinal symptoms and impaired hepatic and renal function also were described. Whether the cleavage site mutation in these cases gave the virus the tissue pantropicity it showed experimentally in chickens (12) is not known, and definitive viral replication outside the respiratory tree in these patients was not observed (11). Nevertheless, it may be that the cleavage site mutation in these cases contributed to the lethality of the virus. Although the HA cleavage site mutation had not been found previously in humans, an influenza strain with the ability to replicate outside its normal host cells was described .50 years ago. WSNy33 was produced in 1940 by forcing the parent strain, WSy33, to replicate in mouse brain (14) to develop an animal model for the observed neurologic complications associated with the 1918 influenza. The strain was passaged extensively in ferrets, in chicken eggs, in mouse lung, and finally in mouse brain. Although this strain was believed initially to be specifically pneumotropic and neurotropic, producing a lethal encephalitis in mice, it recently has been shown to be pantropic or capable of systemic infection in mice (15). This leads us back to the paper by Goto and Kawaoka in this issue of Proceedings (1). They describe a functional model of how the HA protein of WSNy33 is cleaved more readily, synthesizing data from experiments going back over 25 years. One of the unusual properties of WSNy33 is its ability to undergo HA cleavage activation in tissue culture without the addition of exogenous trypsin. As early as 1973, Lazarowitz, et al. (2) were able to explain this observation with the finding that WSNy33 had its HA cleaved by serum plasmin. They