Institute of Virology, Freiburg University Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany.
J Virol. 2024 Apr 16;98(4):e0197223. doi: 10.1128/jvi.01972-23. Epub 2024 Mar 12.
The coordinated packaging of the segmented genome of the influenza A virus (IAV) into virions is an essential step of the viral life cycle. This process is controlled by the interaction of packaging signals present in all eight viral RNA (vRNA) segments and the viral nucleoprotein (NP), which binds vRNA via a positively charged binding groove. However, mechanistic models of how the packaging signals and NP work together to coordinate genome packaging are missing. Here, we studied genome packaging in influenza A/SC35M virus mutants that carry mutated packaging signals as well as specific amino acid substitutions at the highly conserved lysine (K) residues 184 and 229 in the RNA-binding groove of NP. Because these lysines are acetylated and thus neutrally charged in infected host cells, we replaced them with glutamine to mimic the acetylated, neutrally charged state or arginine to mimic the non-acetylated, positively charged state. Our analysis shows that the coordinated packaging of eight vRNAs is influenced by (i) the charge state of the replacing amino acid and (ii) its location within the RNA-binding groove. Accordingly, we propose that lysine acetylation induces different charge states within the RNA-binding groove of NP, thereby supporting the activity of specific packaging signals during coordinated genome packaging.
Influenza A viruses (IAVs) have a segmented viral RNA (vRNA) genome encapsidated by multiple copies of the viral nucleoprotein (NP) and organized into eight distinct viral ribonucleoprotein complexes. Although genome segmentation contributes significantly to viral evolution and adaptation, it requires a highly sophisticated genome-packaging mechanism. How eight distinct genome complexes are incorporated into the virion is poorly understood, but previous research suggests an essential role for both vRNA packaging signals and highly conserved NP amino acids. By demonstrating that the packaging process is controlled by charge-dependent interactions of highly conserved lysine residues in NP and vRNA packaging signals, our study provides new insights into the sophisticated packaging mechanism of IAVs.
流感 A 病毒 (IAV) 分段基因组包装到病毒粒子中是病毒生命周期的一个重要步骤。这个过程由所有 8 个病毒 RNA (vRNA) 片段中的包装信号与病毒核蛋白 (NP) 相互作用控制，NP 通过带正电荷的结合沟与 vRNA 结合。然而，关于包装信号和 NP 如何协同工作以协调基因组包装的机制模型尚不清楚。在这里，我们研究了流感 A/SC35M 病毒突变体的基因组包装，这些突变体携带突变的包装信号以及 NP 中高度保守的赖氨酸 (K) 残基 184 和 229 处的特定氨基酸取代。由于这些赖氨酸在感染宿主细胞中被乙酰化，因此带中性电荷，我们用谷氨酰胺取代它们以模拟乙酰化的中性电荷状态，或用精氨酸取代它们以模拟非乙酰化的正电荷状态。我们的分析表明，八个 vRNA 的协调包装受到以下因素的影响：(i) 取代氨基酸的电荷状态和 (ii) 其在 RNA 结合沟中的位置。因此，我们提出赖氨酸乙酰化在 NP 的 RNA 结合沟中诱导不同的电荷状态，从而在协调的基因组包装过程中支持特定包装信号的活性。
流感 A 病毒 (IAVs) 具有由多个病毒核蛋白 (NP) 包裹的分段病毒 RNA (vRNA) 基因组，并组织成八个不同的病毒核糖核蛋白复合物。尽管基因组分段对病毒的进化和适应有很大贡献，但它需要一种高度复杂的基因组包装机制。八个不同的基因组复合物如何被包装到病毒粒子中还不太清楚，但之前的研究表明 vRNA 包装信号和高度保守的 NP 氨基酸都起着至关重要的作用。通过证明包装过程受 NP 和 vRNA 包装信号中高度保守的赖氨酸残基的电荷依赖相互作用控制，我们的研究为 IAV 复杂的包装机制提供了新的见解。
