Risalvato Jacquline, Zengel James, Phillips Mark, Beavis Ashley, Luo Ming, He Biao
Department of Infectious Diseases, University of Georgia College of Veterinary Medicine, Athens, GA 30601, USA.
Department of Chemistry, Georgia State University, Atlanta, GA 30302, USA.
J Gen Virol. 2025 Apr;106(4). doi: 10.1099/jgv.0.002085.
Mumps virus (MuV) is a negative-sense, single-stranded RNA virus belonging to the family . MuV causes acute infection of the parotid glands, and the infection can result in severe cases of encephalitis, meningitis and deafness in humans. The non-segmented RNA genome of MuV is encapsidated by the nucleocapsid protein (NP), which forms the ribonucleoprotein (RNP) complex that serves as a template for viral RNA synthesis. To make viral genomic RNA accessible to the viral polymerase, a conformational change within NP occurs. Recently, an atomic model of the NP of MuV was developed with cryogenic-electron microscopy (cryo-EM) using PIV5 NP crystal structure as a homology template. To examine NP's structure and function, we performed mutational analysis of MuV NP at region(s) proposed to play a role in accessing viral RNA. The MuV NP mutants containing G185P, A197Q, Q200R and groups denoted as Top (N63G, P139D, A197Q), Tip (P109E, N121G, A124R) and Bottom (G21S, E29T, P43N, R93Q, R304Q) were first tested in a minigenome system. All mutations resulted in reduced reporter gene activities with Q200R and Bottom having the most severe negative effects. Rescuing of recombinant viruses with these mutations was attempted, and only MuV mutants '185 (G185P)', '197 (A197Q)' and 'Top (N63G, P139D, A197A)' were obtained. The 'Top' MuV mutant exhibited normal growth kinetics at low multiplicities of infection (MOIs); however, at high MOIs, the virus had reduced peak litres than low MOIs. Further analysis indicates that production of defective interfering particles (DI particles or DIPs) was enhanced by the mutant virus, indicating that this region, a known alpha-helix hinge region, is important for full-length genome replication, suggesting that it plays a role in maintaining stability of viral RNA-dependent RNA-polymerase on RNP template during MuV viral RNA synthesis. Understanding the production of DI particles will lead to a better understanding of MuV pathogenesis, as well as its replication/transcription process.
腮腺炎病毒（MuV）是一种负链单链RNA病毒，属于该科。MuV可引起腮腺急性感染，这种感染可导致人类出现严重的脑炎、脑膜炎和耳聋病例。MuV的非节段RNA基因组由核衣壳蛋白（NP）包裹，核衣壳蛋白形成核糖核蛋白（RNP）复合体，作为病毒RNA合成的模板。为了使病毒基因组RNA能够被病毒聚合酶利用，NP会发生构象变化。最近，利用PIV5 NP晶体结构作为同源模板，通过低温电子显微镜（cryo-EM）构建了MuV的NP原子模型。为了研究NP的结构和功能，我们对MuV NP中提议在获取病毒RNA中起作用的区域进行了突变分析。首先在微型基因组系统中测试了含有G185P、A197Q、Q200R以及分别标记为Top（N63G、P139D、A197Q）、Tip（P109E、N121G、A124R）和Bottom（G21S、E29T、P43N、R93Q、R304Q）的MuV NP突变体。所有突变均导致报告基因活性降低，其中Q200R和Bottom的负面影响最为严重。我们尝试拯救带有这些突变的重组病毒，仅获得了MuV突变体“185（G185P）”、“197（A197Q）”和“Top（N63G、P139D、A197A）”。“Top”MuV突变体在低感染复数（MOI）下表现出正常的生长动力学；然而，在高MOI下，该病毒的峰值滴度低于低MOI时。进一步分析表明，突变病毒增强了缺陷干扰颗粒（DI颗粒或DIPs）的产生，这表明这个已知的α螺旋铰链区域对于全长基因组复制很重要，这表明它在MuV病毒RNA合成过程中，在维持病毒RNA依赖性RNA聚合酶在RNP模板上的稳定性方面发挥作用。了解DI颗粒的产生将有助于更好地理解MuV的发病机制及其复制/转录过程。
