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Mutations in the receptor-binding site of the hemagglutinin of pandemic influenza A(H1N1) 2009 viruses have been detected sporadically. An Asp222Gly (D222G) substitution has been associated with severe or fatal disease. Here we show that 222G variants infected a higher proportion of ciliated cells in cultures of human airway epithelium than did viruses with 222D or 222E, which targeted mainly nonciliated cells. Carbohydrate microarray analyses showed that 222G variants bind a broader range of α2-3-linked sialyl receptor sequences of a type expressed on ciliated bronchial epithelial cells and on epithelia within the lung. These features of 222G mutants may contribute to exacerbation of disease.
Although the majority of disease cases have been mild, the pandemic influenza A(H1N1) 2009 (H1N1pdm) virus has caused a substantial number of severe and fatal infections (2). Mutants with a D222G or D222E substitution (D225G or D225E in the H3 numbering system) in the receptor-binding site of the virus hemagglutinin (HA) have been detected sporadically (1), and the D222G substitution has been observed to correlate with cases of severe or fatal disease (1, 3, 9, 14). Cell surface receptors for influenza viruses are sialyl glycans (α2-3 Sia or α2-6 Sia) with terminal sialic acid linked α2-3 or α2-6, respectively, to a penultimate galactose. These differ in distribution in the tissues and cells of different species. The sialyl glycans are differentially recognized by the HAs of human and animal influenza viruses and are critical determinants of host range and tissue tropism (16). Using an experimental system of differentiated cultures of human tracheobronchial epithelial cells (HTBE) for studying influenza virus cell tropism, we and others have established that in the initial stages of infection, seasonal human influenza viruses which recognize α2-6 Sia receptors infect mainly nonciliated cells, whereas avian viruses which recognize α2-3 Sia receptors predominantly infect ciliated cells (8, 17, 22).
Previous analyses of human and swine influenza H1N1 viruses (5, 15, 21) and preliminary studies of H1N1pdm viruses (24) have indicated that amino acid substitutions in the HA at position 222 may affect the specificity of receptor binding. This, in turn, would be predicted to determine the range of cell types in human respiratory tissues infected by the viruses (17, 20, 22, 23). We have therefore examined the influence of the D222G and D222E substitutions on the cell tropism of H1N1pdm viruses in HTBE cultures (Table (Table1).1). Five viruses were isolated from clinical material in MDCK cells and passaged solely in these cells. Two of these, A/Hamburg/5/2009 (Ham) (4) isolated from a case of mild infection and A/Moldova/G186/2009 (Mol) from a serious but nonfatal infection, had 222D. A/Dakar/37/2009 (Dak) isolated from a mild case of the disease had 222E. Two isolates from fatal cases, A/Lviv/N6/2009 (Lvi) and A/Norway/3206-3/2009 (Nor), had 222G. A sixth virus tested, A/Hamburg/5/2009-e (Ham-e), was derived from Ham by egg passage and plaque purification in MDCK cells and differed by a single substitution, D222G.
The preparation of differentiated HTBE cultures, viral infection of the cultures for cell tropism analysis, and double immunostaining for viral antigen and cilia of ciliated cells were done as described previously (17). Infected cells were counted in the epithelial segment that included 15 to 30 consecutive microscopic fields containing between 5 and 20% ciliated cells relative to the total number of superficial cells. Percentages of infected ciliated cells relative to the total number of infected cells were calculated for each segment. Ten segments per culture were analyzed, and the results were averaged.
Two distinctive patterns of cell tropism were observed (Fig. (Fig.11 and Table Table1).1). The viruses with 222D (Mol and Ham) and 222E (Dak) showed a pattern of cell tropism typical of seasonal influenza A and B viruses (17, 22) infecting predominantly nonciliated cells known to be rich in α2-6 Sia sequences (17): less than 5% of infected cells were ciliated. In contrast, the three viruses with 222G, Lvi, Nor, and Ham-e, infected both ciliated and nonciliated cells, and 20% or more of the infected cells were ciliated and known to express α2-3 Sia sequences (11, 17). This change in cell tropism, with a 5- to 10-fold increase in the infection of ciliated cells, thus correlated with the presence of the D222G substitution in the HA, and other amino acid differences, in particular D222E, had little or no effect. Furthermore, there were no differences between the amino acid sequences of the neuraminidases (NA) of the 222D, 222G, and 222E viruses which might have an impact on cell tropism: the NA sequences of Mol, Nor, Lvi, and Dak were identical.
To investigate whether changes in receptor binding specificity could account for the distinct cell tropism of the 222G variants, we performed carbohydrate microarray analyses (Fig. (Fig.22 and Table Table2;2; see Fig. S1 and S2 and Table S1 in the supplemental material). The virus preparations were analyzed in the absence of or following inactivation by treatment with beta-propiolactone; the conditions used (4) had no perceptible effect on the receptor-binding profiles. Virus suspensions were concentrated by pelleting, adjusted to contain equivalent concentrations of viruses as assessed by HA titration with human red blood cells and gel electrophoresis with immunoblotting, and stored at 4°C in phosphate-buffered saline (pH 7.4) containing 0.05% sodium azide. The microarray analyses were performed as described previously (4) using the same array series of lipid-linked probes (see Table S1 in the supplemental material). Unless stated otherwise, the viruses were analyzed at an HA titer of 2,000.
For all of the viruses, the intensities of binding to α2-6 Sia sequences were greater overall than the intensities of binding to the α2-3 Sia sequences. There were, however, marked differences between the two 222D viruses, Mol and Ham, and the three 222G variants, Lvi, Nor, and Ham-e, in binding to the α2-3 Sia sequences (highlights are in Table Table2).2). With the 222D viruses, relatively low intensities of binding to α2-3 Sia sequences were detected and they bound mostly to α2-3 Sia sequences that were modified with fucose (Fuc) on the outer N-acetylglucosamine (GlcNAc), as in the blood group-related antigens sialyl Lewisa (SLea) and SLex (probes 28, 29, and 31) and/or with sulfate (SU) on GlcNAc (probes 27 and 35, Table Table2;2; see Fig. S2b in the supplemental material). This is in accord with our previous study of Ham (see Fig. S3 in the supplemental material for reference 4). In contrast, the 222G mutants not only bound more strongly to these α2-3 Sia sequences but bound to additional sequences, such as the VIM-2 antigen sequence (probe 39) with Fuc on internal GlcNAc and to sequences lacking Fuc or SU (probes 23 and 24, Table Table2;2; see Fig. S2a in the supplemental material). All of the pdm viruses investigated here showed greater binding to the 6SU-SLex sequence (probe 35) than to the analogue lacking SU (probe 31) and 6′SU SLex (probe 33, Table Table2).2). This is a property shared with highly pathogenic poultry viruses (6, 7). The pattern of binding to the α2-6 Sia sequences was largely unchanged (Fig. (Fig.2;2; see Fig. S2c in the supplemental material).
As passage in MDCK cells tends to select “complementary” amino acid changes such as K154E or G155E in addition to the single D222G mutation present in the virus of the clinical specimen, two more viruses were investigated as controls for the effects of this substitution in Lvi, the double mutant (G155E D222G). These were A/Athens/16606/2009 (Ath) and A/Lisbon/120/2009 (Lis), which possess the G155E substitution in the absence of D222G. The binding profiles observed for Ath and Lis (see Fig. S3 in the supplemental material) indicated that the 155 substitution did not contribute to the increased α2-3 Sia binding of Lvi, which was therefore due exclusively to the D222G substitution.
The D222E mutant Dak exhibited a carbohydrate-binding profile that was intermediate between those of the 222D and 222G viruses. Compared to the 222D viruses (Mol and Ham) that targeted preferentially nonciliated cells, Dak displayed slightly increased binding to some α2-3 Sia sequences. It was clearly distinguishable from the 222G variants by weaker or negligible binding to a number of other α2-3 Sia sequences, for example, probes 24 and 33 and the VIM-2 antigen sequence, probe 39 (Fig. (Fig.22 and Table Table2;2; see Fig. S1 and S2 in the supplemental material). These are properties that Dak shared with 222D viruses. The similarities in receptor binding and cell tropism of the 222E and 222D viruses are consistent with their circulation in the population, in contrast to the 222G variants that have emerged sporadically and do not appear to be transmitted readily to other individuals (18).
There is thus a clear correlation between enhanced binding to α2-3 Sia sequences by the 222G variants and increased infection of ciliated epithelial cells. The increased capacity of 222G mutant viruses to infect ciliated epithelial cells prominent along the entire airway epithelium would be predicted to interfere with the important mucociliary clearance function of these cells and increase the severity of disease. Another human pathogen, Mycoplasma pneumoniae, which can also cause severe respiratory disease targets the microvilli of ciliated cells in the human bronchus (10) that express the VIM-2 antigen (12, 13). The enhanced capacity of the 222G variants to target α2-3 Sia receptors present in relatively larger amounts on ciliated epithelial cells of the tracheobronchial epithelium (11, 17) and on cells in bronchioles and alveoli (20) may also contribute to more severe pulmonary infection, as suggested by the more frequent identification of 222G variants in specimens from the lower respiratory tract (3), and may explain why they are infrequently transmitted. It is also pertinent to note that the D222G substitution was identified in the HAs from two of five victims of the 1918 pandemic (19). Glycan array analyses of recombinant HAs from one of the 1918 222G mutant viruses (A/New York/1/18) showed (21) a narrow profile of binding to certain α2-3 Sia sequences which had an additional negative charge such as SU or sialic acid. The pattern was more restricted than the repertoire of α2-3 Sia sequences bound by the 222G 2009 pdm viruses that we have investigated here. The New York variant showed little binding to α2-6 Sia sequences, in contrast to the strong and broad α2-6 Sia binding profiles of the 2009 pdm viruses observed here and in an earlier study (24). These differences between the 1918 and 2009 pdm viruses are most likely a reflection of differences in other residues in the receptor-binding pocket.
Whether the selection of the D222G mutation is a cause or a consequence of more severe lower respiratory tract infection is still to be resolved. It is evident, however, that its emergence is likely to exacerbate the severity of disease. The altered receptor specificity and distinctive cell tropism of the D222G mutants of H1N1pdm are hallmarks of a more dangerous pathogen, emphasizing the importance of close monitoring of the evolution of these viruses.
We thank Y. Zhang for mass spectrometric analyses of oligosaccharides and carbohydrate probes; M.S. Stoll for the software for microarray data analysis, storage, and presentation; C. Herbert for assistance in preparation and purification of the oligosaccharides and lipid-linked probes; Z. Xiang and T. Hou for DNA sequence analyses; and M. Eickmann for antiserum against H1N1pdm.
This work was supported by grants from the Wellcome Trust (WT085572MF) to T.F., A.H., and M.M.; the UK Medical Research Council (G0600512), the Biotechnology and Biological Sciences Research Council (BB/G000735/1), the UK Research Council Basic Technology Initiative Glycoarrays (GRS/79268) and Translational Grant (EP/G037604/1), and the NCI Alliance of Glycobiologists for Detection of Cancer and Cancer Risk (U01 CA128416) to T.F.; the Deutsche Forschungsgemeinschaft (SFB 593) to H.-D.K.; and the Von Behring-Röntgen-Stiftung and the LOEWE Program of the State of Hesse Universities of Giessen and Marburg Lung Center to M.M. S.W., R.D., and V.G. are supported by the UK Medical Research Council. A.S.P. is a fellow of the Fundação para a Ciência e Tecnologia (SFRH/BPD/26515/2006, Portugal).
Published ahead of print on 8 September 2010.
†Supplemental material for this article may be found at http://jvi.asm.org/.
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