Department of Infectious Diseases, College of Veterinary Medicine, University of Georgiagrid.213876.9, Athens, Georgia, USA.
Division of Allergy and Immunology, Department of Internal Medicine, Morsani College of Medicine, University of South Floridagrid.170693.a, Tampa, Florida, USA.
J Virol. 2021 Nov 23;95(24):e0120621. doi: 10.1128/JVI.01206-21. Epub 2021 Oct 6.
Respiratory syncytial virus (RSV) is a single-stranded, negative-sense RNA virus in the family and genus that can cause severe disease in infants, immunocompromised adults, and the elderly. The RSV viral RNA-dependent RNA polymerase (vRdRp) complex is composed of the phosphoprotein (P) and the large polymerase protein (L). The P protein is constitutively phosphorylated by host kinases and has 41 serine (S) and threonine (T) residues as potential phosphorylation sites. To identify important phosphorylation residues in the P protein, we systematically and individually mutated all S and T residues to alanine (A) and analyzed their effects on genome transcription and replication by using a minigenome system. We found that the mutation of eight residues resulted in minigenome activity significantly lower than that of wild-type (WT) P. We then incorporated these mutations (T210A, S203A, T151A, S156A, T160A, S23A, T188A, and T105A) into full-length genome cDNA to rescue recombinant RSV. We were able to recover four recombinant viruses (with T151A, S156A, T160A, or S23A), suggesting that RSV-P residues T210, S203, T188, and T105 are essential for viral RNA replication. Among the four recombinant viruses rescued, rRSV-T160A caused a minor growth defect relative to its parental virus while rRSV-S156A had severely restricted replication due to decreased levels of genomic RNA. During infection, P-S156A phosphorylation was decreased, and when passaged, the S156A virus acquired a known compensatory mutation in L (L795I) that enhanced both WT-P and P-S156A minigenome activity and was able to partially rescue the S156A viral growth defect. This work demonstrates that residues T210, S203, T188, and T105 are critical for RSV replication and that S156 plays a critical role in viral RNA synthesis. RSV-P is a heavily phosphorylated protein that is required for RSV replication. In this study, we identified several residues, including P-S156, as phosphorylation sites that play critical roles in efficient viral growth and genome replication. Future studies to identify the specific kinase(s) that phosphorylates these residues can lead to kinase inhibitors and antiviral drugs for this important human pathogen.
呼吸道合胞病毒（RSV）是一种单链、负链 RNA 病毒，属于 和 科，可导致婴儿、免疫功能低下的成年人和老年人罹患重病。RSV 病毒 RNA 依赖性 RNA 聚合酶（vRdRp）复合物由磷蛋白（P）和大聚合酶蛋白（L）组成。P 蛋白被宿主激酶持续磷酸化，有 41 个丝氨酸（S）和苏氨酸（T）残基作为潜在的磷酸化位点。为了确定 P 蛋白中的重要磷酸化残基，我们系统地逐个将所有 S 和 T 残基突变为丙氨酸（A），并使用小基因系统分析它们对基因组转录和复制的影响。我们发现，8 个残基的突变导致小基因活性显著低于野生型（WT）P。然后，我们将这些突变（T210A、S203A、T151A、S156A、T160A、S23A、T188A 和 T105A）整合到全长基因组 cDNA 中以拯救重组 RSV。我们能够恢复四种重组病毒（T151A、S156A、T160A 或 S23A），这表明 RSV-P 残基 T210、S203、T188 和 T105 对于病毒 RNA 复制是必需的。在拯救的四种重组病毒中，rRSV-T160A 相对于其亲本病毒表现出轻微的生长缺陷，而 rRSV-S156A 由于基因组 RNA 水平降低而导致复制受到严重限制。在感染过程中，P-S156 的磷酸化减少，在传代过程中，S156A 病毒在 L 中获得了一个已知的补偿突变（L795I），该突变增强了 WT-P 和 P-S156A 小基因的活性，并能够部分挽救 S156A 病毒的生长缺陷。这项工作表明，残基 T210、S203、T188 和 T105 对于 RSV 复制至关重要，而 S156 在病毒 RNA 合成中起着关键作用。RSV-P 是一种高度磷酸化的蛋白质，对于 RSV 复制是必需的。在这项研究中，我们确定了几个残基，包括 P-S156，作为磷酸化位点，它们在有效病毒生长和基因组复制中发挥着关键作用。未来识别特定激酶磷酸化这些残基的研究可以开发针对这种重要人类病原体的激酶抑制剂和抗病毒药物。
