Katoh Hiroshi, Kubota Toru, Nakatsu Yuichiro, Tahara Maino, Kidokoro Minoru, Takeda Makoto
Department of Virology III, National Institute of Infectious Diseases, Tokyo, Japan
Department of Virology III, National Institute of Infectious Diseases, Tokyo, Japan.
J Virol. 2017 Feb 28;91(6). doi: 10.1128/JVI.02220-16. Print 2017 Mar 15.
Paramyxoviral RNAs are synthesized by a viral RNA-dependent RNA polymerase (RdRp) consisting of the large (L) protein and its cofactor phosphoprotein (P protein). The L protein is a multifunctional protein that catalyzes RNA synthesis, mRNA capping, and mRNA polyadenylation. Growing evidence shows that the stability of several paramyxovirus L proteins is regulated by heat shock protein 90 (Hsp90). In this study, we demonstrated that Hsp90 activity was important for mumps virus (MuV) replication. The Hsp90 activity was required for L-protein stability and activity because an Hsp90-specific inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), destabilized the MuV L protein and suppressed viral RNA synthesis. However, once the L protein formed a mature polymerase complex with the P protein, Hsp90 activity was no longer required for the stability and activity of the L protein. When the Hsp90 activity was inhibited, the MuV L protein was degraded through the CHIP (C terminus of Hsp70-interacting protein)-mediated proteasomal pathway. High concentrations of 17-AAG showed strong cytotoxicity to certain cell types, but combined use of an Hsp70 inhibitor, VER155008, potentiated degradation of the L protein, allowing a sufficient reduction of 17-AAG concentration to block MuV replication with minimum cytotoxicity. Regulation of the L protein by Hsp90 and Hsp70 chaperones was also demonstrated for another paramyxovirus, the measles virus. Collectively, our data show that the Hsp90/Hsp70 chaperone machinery assists in the maturation of the paramyxovirus L protein and thereby in the formation of a mature RdRp complex and efficient viral replication. Heat shock protein 90 (Hsp90) is nearly universally required for viral protein homeostasis. Here, we report that Hsp90 activity is required for efficient propagation of mumps virus (MuV). Hsp90 functions in the maintenance of the catalytic subunit of viral polymerase, the large (L) protein, prior to formation of a mature polymerase complex with the polymerase cofactor of L, phosphoprotein. Hsp70 collaborates with Hsp90 to regulate biogenesis of the MuV L protein. The functions of these chaperones on the viral polymerase may be common among paramyxoviruses because the L protein of measles virus is also similarly regulated. Our data provide important insights into the molecular mechanisms of paramyxovirus polymerase maturation as well as a basis for the development of novel antiviral drugs.
副粘病毒RNA由一种病毒RNA依赖性RNA聚合酶（RdRp）合成，该聚合酶由大（L）蛋白及其辅因子磷蛋白（P蛋白）组成。L蛋白是一种多功能蛋白，可催化RNA合成、mRNA加帽和mRNA聚腺苷酸化。越来越多的证据表明，几种副粘病毒L蛋白的稳定性受热休克蛋白90（Hsp90）调节。在本研究中，我们证明Hsp90活性对腮腺炎病毒（MuV）复制很重要。Hsp90活性是L蛋白稳定性和活性所必需的，因为一种Hsp90特异性抑制剂17-烯丙基氨基-17-去甲氧基格尔德霉素（17-AAG）会破坏MuV L蛋白的稳定性并抑制病毒RNA合成。然而，一旦L蛋白与P蛋白形成成熟的聚合酶复合物，Hsp90活性对于L蛋白的稳定性和活性就不再是必需的。当Hsp90活性受到抑制时，MuV L蛋白通过CHIP（Hsp70相互作用蛋白的C末端）介导的蛋白酶体途径被降解。高浓度的17-AAG对某些细胞类型具有很强的细胞毒性，但联合使用Hsp70抑制剂VER155008可增强L蛋白的降解，从而使17-AAG浓度充分降低以阻断MuV复制，同时使细胞毒性最小化。对于另一种副粘病毒麻疹病毒，也证明了Hsp90和Hsp70伴侣蛋白对L蛋白的调节作用。总体而言，我们的数据表明，Hsp90/Hsp70伴侣蛋白机制有助于副粘病毒L蛋白的成熟，从而有助于形成成熟的RdRp复合物和高效的病毒复制。热休克蛋白90（Hsp90）几乎是病毒蛋白稳态普遍需要的。在此，我们报告Hsp90活性是腮腺炎病毒（MuV）高效繁殖所必需的。在L蛋白的聚合酶辅因子磷蛋白形成成熟的聚合酶复合物之前，Hsp90在维持病毒聚合酶的催化亚基大（L）蛋白方面发挥作用。Hsp70与Hsp90协作调节MuV L蛋白的生物合成。这些伴侣蛋白对病毒聚合酶的功能在副粘病毒中可能是常见的，因为麻疹病毒的L蛋白也受到类似的调节。我们的数据为副粘病毒聚合酶成熟的分子机制提供了重要见解，也为新型抗病毒药物的开发提供了基础。
