Center for Molecular Immunology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences (CAS), Beijing, P. R. China.
PLoS One. 2011 Feb 24;6(2):e16546. doi: 10.1371/journal.pone.0016546.
Heat shock protein 70 (Hsp70) was identified as a cellular interaction partner of the influenza virus ribonucleoprotein (RNP) complex. The biological significance of the interaction between Hsp70 and RNP has not been fully investigated.
Here we demonstrated that Hsp70 was involved in the regulation of influenza A viral transcription and replication. It was found that Hsp70 was associated with viral RNP by directly interacting with the PB1 and PB2 subunits, and the ATPase domain of Hsp70 was required for the association. Immunofluorescence analysis showed that Hsp70 was translocated from the cytoplasm into the nucleus in infected cells. Then we found that Hsp70 negatively regulated the expression of viral proteins in infected cells. Real-time PCR analysis revealed that the transcription and replication of all eight viral segments were significantly reduced in Hsp70 overexpressed cells and greatly increased as Hsp70 was knocked down by RNA interference. Luciferase assay showed that overexpression of Hsp70 could inhibit the viral RNP activity on both vRNA and cRNA promoters. Biochemical analysis demonstrated that Hsp70 interfered with the integrity of RNP. Furthermore, delivered Hsp70 could inhibit the replication of influenza A virus in mice.
Our study indicated that Hsp70 interacted with PB1 and PB2 of RNP and could interfere with the integrity of RNP and block the virus replication in vitro and in vivo possibly through disrupting the binding of viral polymerase with viral RNA.
热休克蛋白 70（Hsp70）被鉴定为流感病毒核糖核蛋白（RNP）复合物的细胞相互作用伙伴。Hsp70 与 RNP 之间相互作用的生物学意义尚未得到充分研究。
在这里，我们证明 Hsp70 参与了甲型流感病毒转录和复制的调节。发现 Hsp70 通过与 PB1 和 PB2 亚基直接相互作用与病毒 RNP 相关联，并且 Hsp70 的 ATP 酶结构域是关联所必需的。免疫荧光分析表明 Hsp70 在感染细胞中从细胞质易位到细胞核。然后我们发现 Hsp70 负调控感染细胞中病毒蛋白的表达。实时 PCR 分析显示，在 Hsp70 过表达细胞中，所有八个病毒片段的转录和复制均显著降低，而在 Hsp70 通过 RNA 干扰被敲低时则大大增加。荧光素酶测定显示 Hsp70 过表达可抑制 vRNA 和 cRNA 启动子上的病毒 RNP 活性。生化分析表明 Hsp70 干扰了 RNP 的完整性。此外，递送的 Hsp70 可抑制小鼠体内甲型流感病毒的复制。
我们的研究表明 Hsp70 与 RNP 的 PB1 和 PB2 相互作用，可通过破坏病毒聚合酶与病毒 RNA 的结合来干扰 RNP 的完整性并阻断体外和体内的病毒复制。
