The 1918 viral influenza pandemic was one of the deadliest flu outbreaks in history with approximately 20-50 million deaths worldwide.1 Due to the emergence of the 2009 H1N1 influenza pandemic and the likelihood of future flu pandemics, the molecular characteristics accounting for the virulence of the 1918 virus are of great interest. Two viral envelope glycoproteins, Hemagglutinin (HA) and Neuraminidase (NA), have been the focus of numerous studies due to their abilities to affect the viral infectious cycle and host specificity. While the HA protein mediates viral attachment to the host cell membrane, the NA protein removes sialic acid on host cells and viral particles to promote viral release and prevent self-aggregation. The importance of the NA protein to the infectivity of the 1918 virus was demonstrated in a study in which the NA gene in the 1918 virus was replaced with that from a contemporary, less virulent H1N1 virus, Tx/91.1 Five days after inoculation, replacement of the NA gene reduced the infectivity titer at least 100-fold compared to the wild-type virus.
Scientists at R&D Systems have now identified structural features of the 1918 viral NA protein that may have contributed to the robust infectivity of the virus.2 The protein was purified from baculovirus-expressing Sf 21 insect cells and separated by gel filtration. It appeared as a monomer, dimer, and tetramer, but only the tetramer retained enzymatic activity. Significantly, the monomer and dimer could not be oligomerized into the tetramer in solution, suggesting that unique structural elements are required for NA oligomerization and activation. Differences in the molecular masses of the monomer and tetramer under reducing conditions led to an analysis of their N-glycosylation patterns. While treatment of the tetramer with a series of endoglycosidases revealed a different N-glycan profile than the monomer (Figure 1), N-deglycosylation reduced but did not abolish its enzymatic activity (Figure 2). The observed decrease in activity was largely due to destabilization of the tetramer, indicating that glycosylation is required for proper folding of the NA protein. In addition, the NA tetramer was found to be resistant to trypsin digestion in contrast to both the monomer and dimer (Figure 3). Further analysis revealed that the stalk region of the NA protein, a site which is typically vulnerable to host protease attack in other influenza viruses, contains five N-glycosylation sites and no trypsin cleavage sites in the 1918 viral NA. Both of these features likely protect the NA protein from cleavage by host proteases. As a point of comparison, NA from the less virulent Tx/91 virus contains two different N-glycosylation sites and three tryptic sites in its stalk region.2 These findings led to the speculation that a unique glycosylation pattern and trypsin resistance in the stalk of the NA protein may have allowed the 1918 virus to more robustly infect a wider range of tissues.
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Figure 1. The Active NA Tetramer has a Different N-glycan Profile than the Inactive Monomer. The NA tetramer was treated with different combinations of endoglycosidases under native conditions for 20 hours and then separated on a reducing SDS gel. While the monomer was only deglycosylated by PNGase F (P) and Endoglucosaminidase F3 (not shown), the tetramer was partially deglycosylated by all four endoglycosidases revealing a distinct N-glycan profile. P, PNGase F; F1, Endoglucosaminidase F1; F3, Endoglucosaminidase F3; and H, Endoglucosaminidase H. M = Molecular weight marker. |
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Figure 2. Deglycosylation of the NA Tetramer Reduces but does not Abolish Its Enzymatic Activity. The enzymatic activity of the NA tetramer was measured after 20 hours (aqua bars) and 44 hours (purple bars) of treatment with different combinations of endoglycosidases. P, PNGase F; F1, Endoglucosaminidase F1; F3, Endoglucosaminidase F3; and H, Endoglucosaminidase H |
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Figure 3. The NA Tetramer is Resistant to Trypsin Digestion. The NA monomer, dimer, and tetramer were either untreated (-) or treated (+) with trypsin and then separated by SDS-PAGE. Proteins were visualized by staining the gel. Trypsin is indicated by the arrow. M = Molecular weight marker. |
References
- Pappas, C. et al. (2008) Proc. Natl. Acad. Sci. USA 105:3064.
- Wu, Z.L. et al. (2009) Biochem. Biophys. Res. Commun. 379:749.
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This symbol denotes references that cite the use of R&D Systems products.