Interdependence of Hemagglutinin Glycosylation and Neuraminidase as Regulators of Influenza Virus Growth: a Study by Reverse Genetics

Wagner, Ralf; Wolff, Thorsten; Herwig, Astrid; Pleschka, Stephan; Klenk, Hans‐Dieter

doi:10.1128/jvi.74.14.6316-6323.2000

Cited by 233 publications

(206 citation statements)

References 41 publications

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“…After incubation for 1 h, the cells were washed three times with PBS and then overlaid with DMEM containing 0.8 % agar (Sigma) and 2 % FBS. The infected cells were incubated for an additional 72 h, and the plaques were visualized by staining with 0.1 % crystal violet in a 10 % formaldehyde solution (Wagner et al, 2000). For each virus, 10 well-spaced plaques were measured using the GNU image manipulation program (version 2.8; www.gimp.org), as described previously (Negovetich & Webster, 2010).…”

Section: Discussionmentioning

confidence: 99%

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Role of stem glycans attached to haemagglutinin in the biological characteristics of H5N1 avian influenza virus

Zhang¹,

Chen²,

Yang³

et al. 2015

Journal of General Virology

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There are three conserved N-linked glycosites, namely, Asn10, Asn23 and Asn286, in the stem region of haemagglutinin (HA) in H5N1 avian influenza viruses (AIVs). To understand the effect of glycosylation in the stem domain of HA on the biological characteristics of H5N1 AIVs, we used site-directed mutagenesis to generate different patterns of stem glycans on the HA of A/Mallard/ Huadong/S/2005. The results indicated that these three N-glycans were dispensable for the generation of replication-competent influenza viruses. However, when N-glycans at Asn10 plus either Asn23 or Asn268 were removed, the cleavability of HA was almost completely blocked, leading to a significant decrease of the growth rates of the mutant viruses in MDCK and CEF cells in comparison with that of the WT virus. Moreover, the mutant viruses lacking these oligosaccharides, particularly the N-glycan at Asn10, revealed a significant decrease in thermostability and pH stability compared with the WT virus. Interestingly, the mutant viruses induced a lower level of neutralizing antibodies against the WT virus, and most of the mutant viruses were more sensitive to neutralizing antibodies than the WT virus. Taken together, these data strongly suggest that the HA stem glycans play a critical role in HA cleavage, replication, thermostability, pH stability, and antigenicity of H5N1 AIVs.

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Section: Discussionmentioning

confidence: 99%

“…of 0.001 in DMEM for 1 h. The unbound viruses were washed with PBS, and serum-free DMEM was added. The cells were incubated at 37 uC under 5 % CO 2 , and the virus titres in the supernatant were monitored periodically by the determination of the TCID 50 on CEF cells (Wagner et al, 2000).…”

Section: Discussionmentioning

confidence: 99%

Role of stem glycans attached to haemagglutinin in the biological characteristics of H5N1 avian influenza virus

Zhang¹,

Chen²,

Yang³

et al. 2015

Journal of General Virology

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“…NA stalk deletions have been shown to reduce the enzymatic activity of the protein [14] and, presumably, adversely affect the spread of the virus to uninfected cells. Secondly, changes in the HA gene that compensate for a shortened N stalk have been described, including increased glycosylation near the receptor binding site thereby decreasing receptor binding affinity [15,16]. Sub-lineage I displayed this hyperglycosylation, with five predicted N-glycosylations sites in the HA gene compared to only three in sub-lineage II, which did not contain the Nstalk deletion.…”

Section: Fig 2 Unrooted Phylogenetic Tree Of the H6 Ha Nucleotide Sementioning

confidence: 99%

Outbreaks of avian influenza H6N2 viruses in chickens arose by a reassortment of H6N8 and H9N2 ostrich viruses

et al. 2007

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The first recorded outbreak of avian influenza (AI) in South African chickens (low pathogenicity H6N2) occurred at Camperdown, KwaZulu/Natal Province (KZN) in June 2002. To determine the source of the outbreak, we defined the phylogenetic relationships between various H6N2 isolates, and the previously unpublished gene sequences of an H6N8 virus isolated in 1998 from ostriches in the Leeu Gamka region (A/Ostrich/South Africa/KK98/98). We demonstrated that two distinct genetic H6N2 lineages (sub-lineages I and II) circulated in the Camperdown area, which later spread to other regions. Sub-lineages I and II shared a recent common H6N2 ancestor, which arose from a reassortment event between two South African ostrich isolates A/Ostrich/South Africa/9508103/95 and (H9N2) /Ostrich/South Africa/KK98/98 (H6N8). Furthermore, the H6N2 sub-lineage I viruses had several molecular genetic markers including a 22-amino acid stalk deletion in the neuraminidase (NA) protein gene, a predicted increased Nglycosylation, and a D144 mutation of the HA protein gene, all of which are associated with the adaptation of AI viruses to chickens. The H6N2 NS1 and PB1 genes shared recent common ancestors with those of contemporary Asian HPAI H5N1 viruses. Our results suggest that ostriches are potential mixing vessels for avian influenza viruses (AIV) outbreak strains and support other reports that H6 viruses are capable of forming stable lineages in chickens.

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“…Modulation of HA and NA activities has been shown to be important for viral growth (Mitnaul et al, 2000;Wagner et al, 2000) and protein yield (Adamo et al, 2009;Jing et al, 2012). We previously showed that viruses with WT or chimeric A/VN/1203 NA (JV20 and JV24, respectively) had similar NA activity (Adamo et al, 2009).…”

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Chimeric neuraminidase and mutant PB1 gene constellation improves growth and yield of H5N1 vaccine candidate virus

Plant

2015

Journal of General Virology

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We previously showed that a mutated PB1 gene improved the growth kinetics of a H3N2 influenza reassortant. Here, we showed that the same mutations improved the growth kinetics of a virus containing the A/Vietnam/1203/2004 (H5N1) haemagglutinin and neuraminidase (NA). Total protein yield and NA activity were increased when a chimeric NA was included. These increases indicated that the synergistic effect was due to the gene constellation containing both the altered PB1 gene and the chimeric NA gene.The object of this work was to improve the genomic backbone segments used in vaccine candidate viruses whilst avoiding detrimental gene constellation effects. It is not known what attributes from each segment affect the gene constellation or reassortment, but it is desirable that vaccine seed strains containing contemporary haemagglutinin (HA) and neuraminidase (NA) segments in the context of known backbone segments can be recovered. It is also desirable that those strains replicate well and that satisfactory protein yields can be harvested. We have previously shown that five mutations to the PB1 gene of the high-growth backbone virus PR/8 improved the growth kinetics of a reassortant containing the HA and NA segments from an H3N2 virus (Plant et al., 2012). These mutations (G180E, S216G, S361R, Q621R and N654S) were not selected based on any structural or functional information. The five mutations made to the PB1 gene (PB1 m5 ) were based on sequence variations found in vaccine strain reassortants. We have also described the improved growth and protein yield of a reassortant (Table 1) and all the viruses produced large plaques on MDCK cells (Fig. 1a). The longer stalk region of the H5N1 NA has been shown to contribute to pathogenicity (Zhou et al., 2009). Here, we confirmed that removing this region reduced pathogenicity, with or without the PB1 M5 allele, by determining mean death time (MDT). Virus particles (10 6 ) were inoculated into the allantoic fluid of eight eggs, which were then incubated at 35 u C and candled at 12 h intervals. Eggs were scored as alive or dead and the time of death recorded. MDT was obtained by multiplying the number of embryos dead at each interval post-inoculation by the hours post-inoculation and dividing by the total number of embryos inoculated. The MDT range was similar for all viruses (Table 1).Optimal polymerase activity is required for efficient replication (Giesendorf et al., 1986;Li et al., 2009;Ngai et al., 2013). PB1 from a seasonal strain or with targeted mutations has been shown to improve growth or HA yield of H3 reassortants (Cobbin et al., 2013;Plant et al., 2012); however, mixed results have been observed for H5 reassortants (Abt et al., 2011;Rudneva et al., 2007;Wanitchang et al., 2010). All three polymerase segments reassort together more frequently in experimental settings than natural reassortment, suggesting gene constellation effects (Macken et al., 2006;Varich et al., 2008;Wille et al., 2013). Here, we examined the growth of H5 reassortants with the PB1 m5 allele w...

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Interdependence of Hemagglutinin Glycosylation and Neuraminidase as Regulators of Influenza Virus Growth: a Study by Reverse Genetics

Cited by 233 publications

References 41 publications

Role of stem glycans attached to haemagglutinin in the biological characteristics of H5N1 avian influenza virus

Role of stem glycans attached to haemagglutinin in the biological characteristics of H5N1 avian influenza virus

Outbreaks of avian influenza H6N2 viruses in chickens arose by a reassortment of H6N8 and H9N2 ostrich viruses

Chimeric neuraminidase and mutant PB1 gene constellation improves growth and yield of H5N1 vaccine candidate virus

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