Interdependence of hemagglutinin glycosylation and neuraminidase as regulators of influenza virus growth: a study by reverse genetics
Interdependence of hemagglutinin glycosylation and neuraminidase as regulators of influenza virus growth: a study by reverse genetics
复制标题
DOI：
10.1128/jvi.74.14.6316-6323.2000
复制
发表时间：
2000-07-01
影响因子：
5.4
通讯作者：
Klenk, HD
中科院分区：
文献类型：
--
作者：
Wagner, R;Wolff, T;Klenk, HD
禽疫病毒A/FPV/Rostock/34(H7N1)的血凝素(HA)携带两个N-连接的寡糖，分别与Asn123和Asn149紧密结合在受体结合口袋附近。在以前的研究中，从猴病毒40载体中表达了缺乏一个糖基化位点(突变体G1和G2)或两个糖基化位点(突变体G1，2)的HA突变体，我们发现这些多糖调节受体结合亲和力(M，Ohuchi，R.Ohuchi，A.Feldmann和H.D.Klenk，J.Virol，71：8377-8384,1997)。我们现在已经使用基于RNA聚合酶I的反向遗传学系统产生的重组病毒来研究这些突变对病毒生长的影响。用流感病毒A/WSN/33株的两个重组体作为辅助病毒，获得了仅神经氨酸酶(NA)不同的两个系列HA突变病毒，对N1 NA病毒的研究表明，Asn149寡糖的缺失(突变体G2)或两个寡糖的缺失(突变体G1，2)对病毒在MDCK细胞中的生长有显著影响。从感染细胞中缺乏这两种寡糖的病毒的生长被抑制，并且在培养液中的病毒产量减少了约20倍。同样，有一个斑块大小的减少，这在G1，2和不那么明显的G2是明显的，这些影响可能归因于从宿主细胞释放突变后代病毒的高度受损。相比之下，对于含有N2NA的重组病毒，这些限制就不那么明显了。N1重组子的神经氨酸酶活性低于N2重组子，表明N2NA能够部分阻断突变体HA与受体的高亲和力结合。这些结果表明，HA分子受体结合位点两侧的N-糖链是流感病毒生长的有效调节因子，Asn149位的糖链占主导地位，Asn123位的糖链作用较弱。此外，我们在这里表明，HA和NA的活性需要高度平衡，才能进行有效的流感病毒感染。
The hemagglutinin (HA) of fowl plague virus A/FPV/Rostock/34 (H7N1) carries two N-linked oligosaccharides attached to Asn123 and Asn149 in close vicinity to the receptor-binding pocket. In previous studies in which HA mutants lacking either one (mutants G1 and G2) or both (mutant G1,2) glycosylation sites had been expressed from a simian virus 40 vector, we showed that these glycans regulate receptor binding affinity (M, Ohuchi, R. Ohuchi, A. Feldmann, and H. D. Klenk, J. Virol, 71:8377-8384, 1997). We have now investigated the effect of these mutations on virus growth using recombinant viruses generated by an RNA polymerase I-based reverse genetics system. Two reassortants of influenza virus strain A/WSN/33 were used as helper viruses to obtain two series of HA mutant viruses differing only in the neuraminidase (NA), Studies using N1 NA viruses revealed that loss of the oligosaccharide from Asn149 (mutant G2) or loss of both oligosaccharides (mutant G1,2) has a pronounced effect on virus growth in MDCK cells. Growth of virus lacking both oligosaccharides from infected cells was retarded, and virus yields in the medium were decreased about 20-fold. Likewise, there was a reduction in plaque size that was distinct with G1,2 and less pronounced,vith G2, These effects could be attributed to a highly impaired release of mutant progeny viruses from host cells. In contrast, with recombinant viruses containing N2 NA, these restrictions were much less apparent. N1 recombinants showed lower neuraminidase activity than N2 recombinants, indicating that N2 NA is able to partly overrule the high-affinity binding of mutant HA to the receptor. These results demonstrate that N-glycans flanking the receptor binding site of the HA molecule are potent regulators of influenza virus growth, with the glycan at Asn149 being dominant and that at Asn123 being less effective. In addition, we show here that HA and NA activities need to be highly balanced in order to allow productive influenza virus infection.