Neuraminidase Is Essential for Fowl Plague Virus Hemagglutinin to Show Hemagglutinating Activity

The arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) contains four glycoproteins, GP 2a , GP 3 , GP 4 and GP 5 , the functions of which are still largely unresolved. In this study, the significance of the N-glycosylation of the GP 2a and GP 5 proteins of PRRSV strain LV was investigated. Both glycoproteins contain two predicted N-glycosylation sites that are highly conserved between North American-type and European-type PRRSV. Using site-directed mutagenesis, single and double mutant full-length PRRSV cDNA clones were generated. After analysing the expression of the mutant proteins and the actual use of the four putative glycosylation sites in the wild-type proteins, the production of mutant virus particles and their infectivities were investigated. The results showed that the N-linked glycans normally present on the GP 2a protein are not essential for particle formation, as is the oligosaccharide attached to N53 of the GP 5 protein. In contrast, the oligosaccharide linked to N46 of the GP 5 protein is strongly required for virus particle production. The specific infectivities of the mutant viruses were investigated by comparing their infectivity-per-particle ratios with that of wild-type virus. The results showed that the lack of either one or both of the N-linked oligosaccharides on GP 2a or of the oligosaccharide attached to N53 of GP 5 did not significantly affect the infectivities of the viruses. In contrast, the two recombinant viruses lacking the oligosaccharide bound to N46 exhibited a significantly reduced specific infectivity compared with the wild-type virus. The implications of the differential requirements of the modifications of GP 2a and GP 5 for PRRSV assembly and infectivity are discussed.

Section: Discussionmentioning

confidence: 91%

Significance of the oligosaccharides of the porcine reproductive and respiratory syndrome virus glycoproteins GP2a and GP5 for infectious virus production

Wissink

Kroese

Maneschijn-Bonsing

et al. 2004

“…Amino acid sequence analysis has revealed that there is considerable variation in both the number and location of potential glycosylation sites among different HA subtypes and even among variants from a single subtype (Chen et al, 2012;Wang et al, 2009). In contrast, the oligosaccharides attached to the upper globular domain at variable sites of the HA molecule have been shown to modulate antigenic properties (Skehel et al, 1984;Wang et al, 2009Wang et al, , 2010bZhang et al, 2015), receptor binding (Gao et al, 2009;Liao et al, 2010;Matrosovich et al, 1999;Ohuchi et al, 1997a;Wang et al, 2010b), and resistance to collectins (Hartshorn et al, 2008;Reading et al, 2009;Tate et al, 2011). The conserved glycosylation sites are located within the stem region of the HA molecule, which has been assumed to provide the main forces that stabilize the HA trimer (Liao et al, 2010;Wiley & Skehel, 1987).…”

Section: Introductionmentioning

confidence: 99%

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

Zhang¹,

Chen²,

Yang³

et al. 2015

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.

“…An increase in the receptor-binding activity of HA, as observed after coexpression of NA from simian virus 40 vectors (Ohuchi et al, 1995), could not be clearly recognized. The low receptor-binding activity in the vaccinia virus system may be explained by the relatively high cytopathogenic effect of the vaccinia virus vector and by shedding of HA1 into the medium when using this expression system (Roberts et al, 1993).…”

mentioning

confidence: 88%

“…This concept was further supported by the observation that an NA-minus mutant of influenza virus is still infectious but produces progeny virus only in the presence of exogenously added bacterial NA (Liu & Air, 1993). Furthermore, removal of terminal neuraminic acid by viral NA is important for full receptor binding and fusion activity of haemagglutinin (HA) (Ohuchi et al, 1995). Influenza NAs of subtype N9 and also fowl plague virus (FPV) N1 NA display a second biological activity with unknown function which results in binding of erythrocytes (Laver et al, 1984 ;Hausmann et al, 1995).…”

Section: Introductionmentioning

confidence: 98%

Biosynthesis, intracellular transport and enzymatic activity of an avian influenza A virus neuraminidase: role of unpaired cysteines and individual oligosaccharides.

Hausmann

Kretzschmar

Garten

et al. 1997

Intracellular transport, glycosylation, tetramerization and enzymatic activity of the neuraminidase (NA) of fowl plague virus (FPV) were analysed in vertebrate cells after expression from a vaccinia virus vector. Tetramerization occurred with a halftime of 15 min, whereas passage through the medial Golgi apparatus and transport to the plasma membrane occurred with half-times of 2 and 3 h, respectively, suggesting a step in NA maturation beyond tetramerization that limits the rate of transport to the medial Golgi. NA transport rates were about fourfold slower than those of haemagglutinin (HA). Slow transport and processing of FPV NA was not altered by coexpression of FPV HA, nor was the transport rate of HA influenced by NA. The slow transport kinetics of NA were also observed in FPVinfected CV-1 cells. As deduced from the coding sequence, FPV NA has the shortest stalk of all naturally occurring NAs described to date and