1918年大流行性流感A型病毒的神经氨酸苷酶中发现的低pH稳定性增强了病毒的复制

The Low pH Stability Discovered in Neuraminidase of 1918 Pandemic Influenza A Virus Enhances Virus Replication

　

　

　

1918年大流行性流感A型病毒的神经氨酸苷酶中发现的低pH稳定性增强了病毒的复制
 
1918年至1919年，“西班牙”大流行性流感A型病毒在全球范围内造成2000多万人死亡，是有记载的历史上最严重的病原体之一。

　

　

通过反向遗传学重建的1918年流感病毒的特性表明，PB1、血凝素(HA)和神经氨酸酶(NA)基因参与了1918年流感病毒的复制和毒性。

　

然而，NA基因的功能仍不清楚。这里我们证明了在1918年的流感病毒NA中发现的类似于禽流感的唾液酸酶活性的低ph稳定性有助于病毒的复制效率。

　

　

通过对比其他人类NI NAs序列和唾液酸酶活动的嵌合结构，我们发现1918大流感病毒中的NA435位置苏氨酸（Thr)或455位置甘氨酸(Gly)的移除与1918年大流感病毒有关的NA活性的低ph值稳定性有关。

　

这两种氨基酸都位于或靠近对在低ph条件下稳定的原生四聚体氨基酸很重要的位置，就像1957年和1968年出现的大流行病毒的N2 NAs。

　

Figure 1

Low-pH stability of sialidase activity of the 1918 virus NA.

A, Low-pH stabilities of N1 NA sialidase activities. N1 NA-expressing cells transfected with the NA genes of the 1918 virus (1918 and 1918 L256F), human H1N1 viruses (WSN33, USSR77, Tex91 and Kaw02) or duck H4N1 virus (Duck80) were incubated at pH 4.0 (closed column), 5.0 (hatched column) and 6.0 (open column) and sialidase activities were measured. Sialidase activities were expressed as a percentage of each activity at pH 6.0. B, Quantitation of NA expression on the cell surface. After fixation of cells with paraformaldehyde, NA expression on the cell surface was detected by rabbit anti-N1 NA polyclonal antibody and analyzed with a flowcytometer. Mean fluorescent intensity (MFI) was expressed as that relative to mock. C, Sialidase activity at NA expression levels of (A) under pH 6.0. Sialidase activities were expressed as a percentage of that of the 1918 virus NA.

　

图一：1918年NA病毒唾液酸酶活性的低ph稳定性。
N1 NA唾液酸酶活性的低ph稳定性。将转染1918年病毒(1918年和1918年L256F)、人H1N1病毒(WSN33、USSR77、Tex91和Kaw02)或鸭H4N1病毒(Duck80)的NA基因的N1表达细胞在pH 4.0(闭柱)、5.0(孵化柱)和6.0(开柱)下孵育，测定唾液酸酶活性。在ph6.0时，唾液酸酶活性以每个活性的百分比表示。B，细胞表面NA表达的定量。多聚甲醛固定细胞后，用兔抗n1 NA多克隆抗体检测细胞表面NA的表达，流式细胞仪分析。平均荧光强度(MFI)表示为相对于mock的。在pH 6.0下，NA表达水平(A)的唾液酸酶活性。Sialidase活性表达为1918年NA病毒的百分比。
两个反向遗传学病毒产生的遗传背景/网络/ 33(H1N1),包括low-pH-unstable N1 NA从苏联/ 92/77(H1N1)及其对应的N1 NA中唾液酸酶活动转化为low-pH-stable属性的删除和替换435年和455年两个氨基酸残基的位置与唾液酸酶活动的低ph值稳定在1918年NA。

　

　

　

　

Figure S1

 

Effect of Ca2+ on Low-pH stability of sialidase activity of the 1918 virus NA. 293T cells genetically expressing each N1 NA from USSR77 and 1918 viruses were incubated with 10 mM acetate buffer (pH 4.0, 5.0, and 6.0) containing 5 μM CaCl2 at 37°C for 30 min and sialidase activities were measured. Sialidase activities were expressed as a percentage of each activity at pH 6.0. As a control, 10 mM acetate buffer (pH 4.0, 5.0, and 6.0) without CaCl2 was used.

　

图S1
 
Ca2+对1918年NA病毒唾液酸酶低ph稳定性的影响。293 t细胞基因表达的N1 NA USSR77和1918年病毒的潜伏在10mol醋酸缓冲(pH值4.0,5.0,和6.0)包含5μM氯化钙在37°C 30分钟和唾液酸酶测定的活动。在ph6.0时，唾液酸酶活性以每个活性的百分比表示。以10mm醋酸盐缓冲液(pH 4.0, 5.0, 6.0)为对照，不使用CaCl2。
与低ph -不稳定的N1病毒相比，包含“西班牙流感”样低ph -稳定NA的突变病毒表现出了显著的复制。
我们的结果表明，1918年流感病毒NA中唾液酸酶活性的类似于禽流感的低ph稳定性有助于病毒的复制效率。

　

　

　

The Low pH Stability Discovered in Neuraminidase of 1918 Pandemic Influenza A Virus Enhances Virus Replication

　

The “Spanish” pandemic influenza A virus, which killed more than 20 million worldwide in 1918-19, is one of the serious pathogens in recorded history.

　

Characterization of the 1918 pandemic virus reconstructed by reverse genetics showed that PB1, hemagglutinin (HA), and neuraminidase (NA) genes contributed to the viral replication and virulence of the 1918 pandemic influenza virus.

　

However, the function of the NA gene has remained unknown. Here we show that the avian-like low-pH stability of sialidase activity discovered in the 1918 pandemic virus NA contributes to the viral replication efficiency.

　

We found that deletion of Thr at position 435 or deletion of Gly at position 455 in the 1918 pandemic virus NA was related to the low-pH stability of the sialidase activity in the 1918 pandemic virus NA by comparison with the sequences of other human N1 NAs and sialidase activity of chimeric constructs.

　

Both amino acids were located in or near the amino acid resides that were important for stabilization of the native tetramer structure in a low-pH condition like the N2 NAs of pandemic viruses that emerged in 1957 and 1968.

　

Figure 1

Low-pH stability of sialidase activity of the 1918 virus NA.

A, Low-pH stabilities of N1 NA sialidase activities. N1 NA-expressing cells transfected with the NA genes of the 1918 virus (1918 and 1918 L256F), human H1N1 viruses (WSN33, USSR77, Tex91 and Kaw02) or duck H4N1 virus (Duck80) were incubated at pH 4.0 (closed column), 5.0 (hatched column) and 6.0 (open column) and sialidase activities were measured. Sialidase activities were expressed as a percentage of each activity at pH 6.0. B, Quantitation of NA expression on the cell surface. After fixation of cells with paraformaldehyde, NA expression on the cell surface was detected by rabbit anti-N1 NA polyclonal antibody and analyzed with a flowcytometer. Mean fluorescent intensity (MFI) was expressed as that relative to mock. C, Sialidase activity at NA expression levels of (A) under pH 6.0. Sialidase activities were expressed as a percentage of that of the 1918 virus NA.

　

　

　

Two reverse-genetic viruses were generated from a genetic background of A/WSN/33 (H1N1) that included low-pH-unstable N1 NA from A/USSR/92/77 (H1N1) and its counterpart N1 NA in which sialidase activity was converted to a low-pH-stable property by a deletion and substitutions of two amino acid residues at position 435 and 455 related to the low-pH stability of the sialidase activity in 1918 NA.

　

　

Figure S1

 

Effect of Ca2+ on Low-pH stability of sialidase activity of the 1918 virus NA. 293T cells genetically expressing each N1 NA from USSR77 and 1918 viruses were incubated with 10 mM acetate buffer (pH 4.0, 5.0, and 6.0) containing 5 μM CaCl2 at 37°C for 30 min and sialidase activities were measured. Sialidase activities were expressed as a percentage of each activity at pH 6.0. As a control, 10 mM acetate buffer (pH 4.0, 5.0, and 6.0) without CaCl2 was used.

　

　

　

The mutant virus that included “Spanish Flu”-like low-pH-stable NA showed remarkable replication in comparison with the mutant virus that included low-pH-unstable N1 NA.

　

Our results suggest that the avian-like low-pH stability of sialidase activity in the 1918 pandemic virus NA contributes to the viral replication efficiency.

　

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0015556

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3000343/