Cox Justin B, Nukui Masatoshi, Murphy Eain A
Microbiology and Immunology Department, SUNY Upstate Medical University, Syracuse, New York, USA.
J Virol. 2025 May 20;99(5):e0048125. doi: 10.1128/jvi.00481-25. Epub 2025 Apr 17.
Post-translational modifications (PTMs) are key regulators of various processes important for cell survival. These modifications are critical for dealing with stress conditions, such as those observed in disease states, and during infections with various pathogens. We previously reported that during infection of primary dermal fibroblasts, multiple human cytomegalovirus (HCMV)-encoded proteins were post-translationally modified by the addition of a nitric oxide group to cysteine residues, a modification called protein-S-nitrosylation. For example, tegument protein pp71 is nitrosylated, diminishing its ability to inhibit STING, a protein necessary for DNA virus immune response. Herein, we report that an additional HCMV tegument protein, pp65, responsible for the inhibition of cGAS is also modified by protein-S-nitrosylation on two cysteine residues. Utilizing site-directed mutagenesis to generate recombinant viruses that encode a pp65 that cannot be protein-S-nitrosylated, we evaluated the impact of this PTM on viral replication and how the virus impacts the cGAS/STING pathway. We report that the nitrosylation of pp65 negatively impacts its ability to block cGAS enzymatic functions. pp65 protein-S-nitrosylation mutants demonstrated a decrease in cGAS/STING-induced IRF3 and TBK1 phosphorylation. Additionally, we observed a reduction in IFN-β1 secretion in NuFF-1 cells expressing a nitrosylation-resistant pp65. We report that HCMV expressing a protein-S-nitrosylation-deficient pp65 is resistant to the activation of cGAS in the infection of primary dermal fibroblasts. Our work suggests that nitrosylation of viral proteins may serve as a broadly neutralizing mechanism in HCMV infection.
Post-translational modifications (PTM) are utilized by host cells to limit an invading pathogen's ability to establish a productive infection. A potent PTM, called protein-S-nitrosylation, has anti-bacterial and anti-viral properties. Increasing protein-S-nitrosylation with the addition of nitric oxide donor compounds reduced HCMV replication in fibroblasts and epithelial cells. We previously reported that protein-S-nitrosylation of HCMV pp71 limits its ability to inhibit STING. Herein, we report that the protein-S-nitrosylation of HCMV pp65 impacts its ability to limit cGAS activity, an additional protein important in regulating interferon response. Therapeutically, patients provided nitric oxide by inhalation reduced viral replication in coronavirus disease 2019, influenza, and even impacted bacterial growth within patients' lungs. It is thought that an increase in free nitric oxide increases the frequency of nitrosylated proteins. Understanding how protein-S-nitrosylation regulates a common DNA virus like HCMV will provide insights into the development of broadly neutralizing therapeutics in drug-resistant viral infections.
翻译后修饰（PTM）是细胞存活所必需的各种重要过程的关键调节因子。这些修饰对于应对应激条件至关重要，例如在疾病状态以及感染各种病原体期间所观察到的应激条件。我们之前报道过，在原代表皮成纤维细胞感染期间，多种人类巨细胞病毒（HCMV）编码的蛋白质通过在半胱氨酸残基上添加一氧化氮基团进行翻译后修饰，这种修饰称为蛋白质S-亚硝基化。例如，衣壳蛋白pp71被亚硝基化，从而削弱了其抑制STING的能力，STING是DNA病毒免疫反应所必需的一种蛋白质。在此，我们报道另一种HCMV衣壳蛋白pp65，它负责抑制cGAS，也在两个半胱氨酸残基上被蛋白质S-亚硝基化修饰。利用定点诱变产生编码不能进行蛋白质S-亚硝基化的pp65的重组病毒，我们评估了这种PTM对病毒复制的影响以及病毒如何影响cGAS/STING途径。我们报道pp65的亚硝基化对其阻断cGAS酶功能的能力产生负面影响。pp65蛋白质S-亚硝基化突变体显示cGAS/STING诱导的IRF3和TBK1磷酸化减少。此外，我们在表达抗亚硝基化pp65的NuFF-1细胞中观察到IFN-β1分泌减少。我们报道表达蛋白质S-亚硝基化缺陷型pp65的HCMV在原代表皮成纤维细胞感染中对cGAS的激活具有抗性。我们的工作表明病毒蛋白的亚硝基化可能是HCMV感染中的一种广泛中和机制。
宿主细胞利用翻译后修饰（PTM）来限制入侵病原体建立有效感染的能力。一种名为蛋白质S-亚硝基化的有效PTM具有抗菌和抗病毒特性。添加一氧化氮供体化合物增加蛋白质S-亚硝基化可减少成纤维细胞和上皮细胞中的HCMV复制。我们之前报道过HCMV pp71的蛋白质S-亚硝基化限制了其抑制STING的能力。在此，我们报道HCMV pp65的蛋白质S-亚硝基化影响其限制cGAS活性的能力，cGAS是调节干扰素反应中另一种重要蛋白质。在治疗方面，通过吸入一氧化氮为患者提供一氧化氮可减少2019冠状病毒病、流感中的病毒复制，甚至影响患者肺部细菌的生长。据认为，游离一氧化氮的增加会提高亚硝基化蛋白质出现的频率。了解蛋白质S-亚硝基化如何调节像HCMV这样常见的DNA病毒，将为耐药病毒感染中广泛中和疗法的开发提供见解。
