Sialidase activity of influenza A virus in an endocytic pathway enhances viral replication
Sialidase activity of influenza A virus in an endocytic pathway enhances viral replication
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DOI：
10.1128/jvi.79.18.11705-11715.2005
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发表时间：
2005-09-01
影响因子：
5.4
通讯作者：
Suzuki, Y
中科院分区：
文献类型：
--
作者：
Suzuki, T;Takahashi, T;Suzuki, Y
与大多数流行毒株不同，1957年和1968年流感大流行毒株的N_2神经氨酸酶(NA)基因具有禽样低pH稳定性的唾液酸酶活性。我们从A/WSN/33(H1N1)的遗传背景中产生了四种反向遗传病毒，其中包括1968流行株(H3N2)和流行性(H2)毒株的亲本N2NA或其对应的N2Nas，其中唾液酸酶活性的低pH稳定性通过一两个氨基酸残基的替换而改变。我们发现，携带低pH稳定唾液酸酶的转基因病毒(WSN/STRATE-NAS)在Madin-Darby犬肾(MDCK)细胞中的复制能力是携带低pH不稳定唾液酸酶的转基因病毒(WSN/STRISTER-NAS)的25~80倍。病毒内化90min后，用5-bromo-4-chloro-indole-3-yl-ct-N-acetyineuraminic酸和固红紫LB进行原位荧光检测，检测到WsN/STRATE-NAS的酶活性。用4-胍基-2，4-二脱氧-N-乙酰神经氨酸(Zanamivir)预先抑制WSN/STRATE-NAS细胞内吞途径上的唾液酸酶活性，导致子代病毒数量显著减少。相反，在相同条件下，不稳定的WSN/Nas的酶活性在细胞中检测不到，其复制对扎那米韦的预处理没有影响。转染型病毒感染细胞的血液吸附分析表明，唾液酸酶的低pH稳定性对高尔基地区血凝素中唾液酸的去除过程没有影响。此外，经鼻接种WSN/STRISTER-NAS的小鼠在第3天从肺中恢复了高滴度的病毒，但WSN/STRISTER-NAS从小鼠的肺中被清除。这些结果表明，晚期内切体/溶酶体运输中的唾液酸酶活性增强了甲型流感病毒的复制。
N2 neuraminidase (NA) genes of the 1957 and 1968 pandemic influenza virus strains possessed avian-like low-pH stability of sialidase activity, unlike most epidemic strains. We generated four reverse-genetics viruses from a genetic background of A/WSN/33 (H1N1) that included parental N2 NAs of 1968 pandemic (H3N2) and epidemic (H2N2) strains or their counterpart N2 NAs in which the low-pH stability of the sialidase activity was changed by substitutions of one or two amino acid residues. We found that the transfectant viruses bearing low-pH-stable sialidase (WSN/Stable-NAs) showed 25- to 80-times-greater ability to replicate in Madin-Darby canine kidney (MDCK) cells than did the transfectant viruses bearing low-pH-unstable sialidase (WSN/ Unstable-NAs). Enzymatic activities of WSN/Stable-NAs were detected in endosomes of MDCK cells after 90 min of virus internalization by in situ fluorescent detection with 5-bromo-4-chloro-indole-3-yl-ct-N-acetyineuraminic acid and Fast Red Violet LB. Inhibition of sialidase activity of WSN/Stable-NAs on the endocytic pathway by pretreatment with 4-guanidino-2,4-dideoxy-N-acetylneuraminic acid (zanamivir) resulted in a significant decrease in progeny viruses. In contrast, the enzymatic activities of WSN/Unstable-NAs, the replication of which had no effect on pretreatment with zanamivir, were undetectable in cells under the same conditions. Hemadsorption assays of transfectant-virus-infected cells revealed that the low-pH stability of the sialidase had no effect on the process of removal of sialic acid from hemagglutinin in the Golgi regions. Moreover, high titers of viruses were recovered from the lungs of mice infected with WSN/Stable-NAs on day 3 after intranasal inoculation, but WSN/Unstable-NAs were cleared from the lungs of the mice. These results indicate that sialidase activity in late endosome/lysosome traffic enhances influenza A virus replication.