Glycosylation at 11Asn on hemagglutinin of H5N1 influenza virus contributes to its biological characteristics
© The Author(s) 2017
Received: 24 June 2017
Accepted: 9 October 2017
Published: 21 November 2017
A stem glycosylation site of hemagglutinin (HA) is important to the stability of the HA trimmer. A previous study shows that the stem 10/11 overlap glycosylation site of the H5 subtype avian influenza virus may influence the cleavage of HA, whereas the exact site and its effect on virulence remain unclear. In this study, site-directed mutagenesis was used to generate single or double mutant rSY-Δ10(10NNAT), rSY-Δ11(10NNSA), and rSY-Δ10/11(10NNAA) of the overlapping glycosylation site (10NNST) on the HA of A/Mallard/Huadong/S/2005(SY). By using Western blot analysis, we show that both rSY-Δ11 and rSY-Δ10/11 mutant viruses had significant delay on HA cleavage and a reduced HA molecular mass compared to the wild-type virus rSY, while the rSY-Δ10 mutant virus exhibited a similar HA molecular mass to that of the wild-type virus rSY. Interestingly, both rSY-Δ11 and rSY-Δ10/11 mutant viruses reverted their glycosylation sites at 11N after passage, indicating that 11N is a true and critical glycosylation site. Compared to the wild-type virus rSY, rSY-Δ11 and rSY-Δ10/11 mutant viruses had decreased growth rates, reduced thermo- and pH-stability, decreased pathogenicity, and limited systemic spread. Therefore, our study suggests that the 11N glycosylation site plays a key role in HA cleavage, structural stability and pathogenicity in H5 subtype avian influenza virus.
H5 subtype avian influenza virus (AIV) infects not only poultry but also mammals worldwide [1–3], thus posing a threat to the poultry industry and to public health [4, 5]. Hemagglutinin (HA), a surface glycoprotein, plays an important role in the influenza life cycle [4, 6]. As the avian influenza virus evolves, glycosylation distribution of HA is becoming increasingly complicated [7, 8]. Glycosylation sites function differently depending on their location: the glycan near the antigen epitope may cause immune escape by disturbing antibody recognition [9–11]; the glycan near the cleavage sites may result in virulence reduction due to HA cleavage deficiency [12, 13]; the glycan near the receptor binding site may change its receptor affinity [14, 15]. Stem glycosylation of HA appears conserved, mainly attributed to the stability of the HA trimer [14, 16]. A previous study shows that there is a potential 10/11 glycosylation site overlap on the HA stem of the SY virus, which plays an important role in cleavage . However, the exact glycosylation site remains unclear. In this study, site-direct mutagenesis was used to delete the overlapping glycosylation site, so biological characteristics of the mutants could be determined.
Materials and methods
All animal studies were approved by the Jiangsu Administrative Committee for Laboratory Animals (Permission Number: SYXKSU-2007-0005) and complied with the Guidelines of Laboratory Animal Welfare and Ethics of Jiangsu Administrative Committee for Laboratory Animals.
Viruses and cells
Madin–Darby canine kidney (MDCK) cells, human embryonic (293T) cells and chicken embryo fibroblast (CEF) cells were maintained in Dubecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS, Foundation, Gemini) at 37 °C with 5% CO2. AIV A/Mallard/Huadong/S/2005 (SY, H5N1)  was propagated in 10-day-old specific-pathogen-free (SPF) embryonic chicken eggs.
Site-directed mutagenesis, virus rescue and identification
Mutagenesis primers for the hemagglutinin gene
Primer sequence (5′-3′)
Western blot analysis
To analyze the molecular mass of HA protein in the viruses , CEF cells were inoculated with the recombinant viruses at a multiplicity of infection (MOI) of 1 and incubated for 1 h at 37 °C with 5% CO2. The infected cells were washed three times with PBS and then fresh DMEM containing 2% FBS was added. At 12 h incubation, the cells were washed with pre-cooled PBS, scraped and lysed with 200 μL of lysis buffer (Thermofisher Scientific) individually on ice for 15 min. Total proteins were collected by centrifugation at 13 000 rpm at 4 °C for 10 min, subjected to 12% SDS-PAGE, and transferred to PVDF membrane. The membrane was blocked in 5% skimmed milk, incubated with mAb SYA9 and polyAb anti-M1 mouse serum, and then incubated with horseradish peroxidase-conjugated goat anti-mouse antibodies. The protein bands were developed using a chemiluminescence imaging analysis system. For time-point analysis, the CEF monolayer cells infected with each recombinant virus was taken at 12 h intervals from 12 to 72 h post-infection (hpi) and frozen in −80 °C. All collected cells were subjected to Western blot analysis.
Monolayer CEF cells were infected with each recombinant virus at an MOI of 1 in DMEM for 1 h. Then cells were washed to remove unbound viruses and fresh DMEM was added. The cells were incubated at 37 °C with 5% CO2 and supernatants were sampled every 12 h. After 72 hpi, TCID50 on CEF cells were determined for all samples .
Recombinant viruses were divided into nine 60 μL aliquots. All aliquots were exposed to 56 °C and each recombinant group was quickly cooled to 4 °C after 0, 5, 10, 15, 30, 60, 90, 120 and 150 min incubation . The titers of all aliquots were then tested by standard hemagglutination assay with 1% chicken red blood cells. All recombinant viruses were also diluted to the same TCID50 and incubated at 37 or 42 °C for 1, 3 and 5 days. The titers of all aliquots were tested by TCID50. In addition, methanol-inactivated recombinant viruses were incubated at 37 or 42 °C at a 2-h interval for 18 h. The titers of all samples were determined by hemagglutination assay.
Recombinant viruses were mixed with an equal volume of 100 mM acetate buffer (pH = 4.0 and pH = 5.0), 100 mM phosphate buffer (pH = 6.0), or neutral phosphate buffer (pH = 7.0) . After a 10-min incubation at 37 °C, the titers of all samples were determined by hemagglutination assay.
IVPI determination in chickens
Six-week-old SPF white leghorn chickens (10 per group) were injected intravenously with 0.1 mL of 1:10 diluted recombinant virus. Chickens were monitored daily for clinical signs of disease for 10 days, and the intravenous pathogenicity indices (IVPI) were calculated according to the OIE recommendation.
Virulence determination in mice
Eight-week-old BALB/c mice (5 per group) were infected intranasally with 104 or 106 EID50 of each virus in 50 μL PBS. The mice were weighed individually and monitored for signs of illness and mortality for 2 weeks. In addition, 8-week-old mice (6 per group) were infected intranasally with 104 EID50 of each virus in 50 μL PBS. Three mice from each group were euthanized on days 3 and 6 post-infection, and the lungs, brains, kidneys, spleens, hearts and livers were collected for virus titration [25–27].
The viral titers and antibody titers are expressed as the mean ± standard deviation. Statistical analyses were performed using a Mann–Whitney test. Differences with a p value of less than 0.05 were regarded to be statistically significant.
Rescue of the mutant viruses
The overlapping glycosylation site at 10/11 in HA was modified by changing the rSY amino acid sequence NNST to NNAT, NNSA or NNAA, and the respective mutants were named rSY-Δ10, rSY-Δ11 and rSY-Δ10/11. All rescued viruses were confirmed by sequence analysis and were without spontaneous mutations in the first generation. However, after passage in SPF chicken embryonic egg or CEF for three generations, rSY-Δ11 reverted from NNSA to NNST and rSY-Δ10/11 from NNAA to NNAT, while no reversion was found for NNAT in rSY-Δ10. Thus, all mutant viruses of the first generations were used for further experimentation except when indicated.
Western blot analysis
Thermal stability of the recombinant viruses
pH stability of the recombinant viruses
Pathogenicity in chickens
Determination of intravenous pathogenicity indexes for the mutant viruses
Pathogenicity in mice
Distribution of the mutant viruses in mice organs
Virus replication in experimentally infected mice [number of virus-positive mice/number tested mice (mean titer ± SD)]
3/3 (3.3 ± 1)
3/3 (4.0 ± 0.5)
2/3 (0.8 ± 0)
3/3 (2.9 ± 1.3)
3/3 (4.2 ± 0.6)
1/3 (0.5 ± 0)
2/3 (0.5 ± 0.2)
2/3 (0.8 ± 0.3)
2/3 (1 ± 0.5)
1/3 (0.3 ± 0)
1/3 (1.5 ± 0)
There are a variety of potential glycosylation sites distributed among SY-H5N1 subtype AIV hemagglutinin. Among them, 10/11NNST is highly conserved. A previous study showed that 10/11NNST is an overlapping glycosylation site, the double deletion of which hinders HA0 cleavage , and that 10N might be a real glycosylation site based on Western blot analysis of viruses mutated from NNST to NPST and NNAA. In this study, three mutant viruses were constructed to delete the 10N, 11N and 10/11NN glycosylation site by substituting NNST with NNAT, NNSA and NNAA, respectively. Although all rescued viruses were confirmed by sequence analysis and were without spontaneous mutations in the first generation, Western blot analysis showed that HA1 patterns from the first generations of mutant viruses rSY-Δ11 or rSY-Δ10/11 were not consistent with that from their fifth generation viruses. Further sequence analysis of each generation of the mutant viruses revealed that, since the mutants rSY-Δ11 or rSY-Δ10/11were passaged in chicken embryonic egg or CEF for three generations, the NNSA or NNAA sequences of mutant viruses had been reverted to NNST or NNAT, respectively, which allowed the mutant viruses to form a glycosylation site at 11N again. In a previous study, the fifth generations of mutant viruses (NPST, NNSA, NNAA) were used to determine the HA1 patterns by Western blot analysis, this may be the reason that the reverted viruses were used and no change was found between the 11N mutant (NNSA) and the wild-type virus rSY . Since the deletion of 11N glycosylation site reverted quickly, the H5 subtype influenza virus survival may depend on this glycosylation site. This may explain why this overlapping glycosylation site is highly conserved. Our Western blot analysis shows that once the 11-glycosylation site was removed, HA0 cleavage was hindered. Only HA0 was detectable at the beginning, but this cleavage of HA0 still happened during late infection, which resulted in a low virus titer in the early stage of infection in CEF. Also, we found that HA0 cleavage correlated with virus growth. When HA0 can be cleaved, the virus titer is relative high. We also found that the molecular masses of HA1 from the mutant viruses rSY-Δ11 and rSY-Δ10/11 were lower than that of the mutant virus rSY-Δ10 and the wild-type virus rSY. These data indicate that 11N, rather than 10N, is the real glycosylation site, which was consistent with other studies suggesting that NST is more likely to form a glycosylation site among NNST combination [12, 28–30]. We also speculated that steric hindrance may be the main cause of cleavage delay in rSY-Δ11 and rSY-Δ10/11, but this hypothesis remains to be tested.
One of the main functions of stem glycosylation sites is to maintain the structural stability of the virus. Thus, disrupted viral stability due to glycosylation site removal likely accounted for heat and pH sensitivity in rSY-Δ11 and rSY-Δ10/11. However, if 10N could not form a glycosylation site, why is rSY-Δ10 still unstable? It is generally believed that the N-glycosylation site has a fixed amino acid sequence motif: NXS/T(X 〈〉 P). Different X in NXS/T may have different glycosylation stability. In this study, substituting NNST with NNAT did not affect the 10N-glycosylation since 10N is not a glycosylation site, while substituting NST with NAT may affect the 11N glycosylation site  or changed the HA structure, which also resulted in lower stability of virus to heat and low-pH.
Head glycosylation of HA may contribute to virulence and antigenicity of influenza viruses, and influenza viruses have variety patterns of head glycosylation [31–34]. However, the role of overlapping stem glycosylation on virulence remains unknown. Although the IPVI of mutant viruses rSY-Δ11 and rSY-Δ10/11 in chickens were lower than that of mutant virus rSY-Δ10 and rSY, both mutant viruses were still highly pathogenic to chickens. In mice, rSY-Δ11 and rSY-Δ10/11 mutants showed a significant reduction in virulence compared to wild-type virus rSY and mutant virus rSY-Δ10, presenting a relative limited and low-titer virus distribution among organs, less body weight loss, and lower mortality. The attenuation of virulence of the mutant viruses in chickens and mice may be attributable to the delay of HA0 cleavage .
In conclusion, we successfully rescued three modified viruses at the overlapping glycosylation site in the stem of HA, and found that the 11N glycosylation site of SY H5N1 virus was the true glycosylation site, which was critical for HA cleavage and viral virulence. This study facilitates an improved understanding of the role of overlapping glycosylation sites.
The authors declare that they have no competing interests.
DP and YY participated in the design of the study. YY, XZ, YQ, XW performed the experiment. YS, SC, and TQ analyzed the data and drafted the manuscript. DP and XL planned the experiments and helped write the manuscript. All authors read and approved the final manuscript.
This study was partially supported by the Important National Science & Technology Specific Projects (2016YFD0500202), the National Natural Science Foundation of China (Nos. 31372450, 31402229), the Agricultural Science & Technology Independent Innovation Fund of Jiangsu Province [CX(15)1065], and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.
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