Table_1_Genetic Variability of Human Cytomegalovirus Clinical Isolates Correlates With Altered Expression of Natural Killer Cell-Activating Ligands and IFN-γ.pdf

Human cytomegalovirus (HCMV) infection often leads to systemic disease in immunodeficient patients and congenitally infected children. Despite its clinical significance, the exact mechanisms contributing to HCMV pathogenesis and clinical outcomes have yet to be determined. One of such mechanisms involves HCMV-mediated NK cell immune response, which favors viral immune evasion by hindering NK cell-mediated cytolysis. This process appears to be dependent on the extent of HCMV genetic variation as high levels of variability in viral genes involved in immune escape have an impact on viral pathogenesis. However, the link between viral genome variations and their functional effects has so far remained elusive. Thus, here we sought to determine whether inter-host genetic variability of HCMV influences its ability to modulate NK cell responses to infection. For this purpose, five HCMV clinical isolates from a previously characterized cohort of pediatric patients with confirmed HCMV congenital infection were evaluated by next-generation sequencing (NGS) for genetic polymorphisms, phylogenetic relationships, and multiple-strain infection. We report variable levels of genetic characteristics among the selected clinical strains, with moderate variations in genome regions associated with modulation of NK cell functions. Remarkably, we show that different HCMV clinical strains differentially modulate the expression of several ligands for the NK cell-activating receptors NKG2D, DNAM-1/CD226, and NKp30. Specifically, the DNAM-1/CD226 ligand PVR/CD155 appears to be predominantly upregulated by fast-replicating (“aggressive”) HCMV isolates. On the other hand, the NGK2D ligands ULBP2/5/6 are downregulated regardless of the strain used, while other NK cell ligands (i.e., MICA, MICB, ULBP3, Nectin-2/CD112, and B7-H6) are not significantly modulated. Furthermore, we show that IFN-γ; production by NK cells co-cultured with HCMV-infected fibroblasts is directly proportional to the aggressiveness of the HCMV clinical isolates employed. Interestingly, loss of NK cell-modulating genes directed against NK cell ligands appears to be a common feature among the “aggressive” HCMV strains, which also share several gene variants across their genomes. Overall, even though further studies based on a higher number of patients would offer a more definitive scenario, our findings provide novel mechanistic insights into the impact of HCMV genetic variability on NK cell-mediated immune responses.

人巨细胞病毒（HCMV）感染常导致免疫缺陷患者和先天性感染儿童出现全身性疾病。尽管其临床意义显著，但促成HCMV致病机制和临床结局的确切机制尚待明确。其中一种机制涉及HCMV介导的自然杀伤细胞（NK）免疫反应，该反应通过阻碍NK细胞介导的细胞溶解，有利于病毒免疫逃逸。这一过程似乎取决于HCMV遗传变异的程度，因为参与免疫逃逸的病毒基因的高变异性对病毒致病性产生影响。然而，病毒基因组变异与其功能效应之间的联系迄今为止仍难以捉摸。因此，本研究旨在确定宿主间HCMV遗传变异是否影响其调节感染时NK细胞反应的能力。为此，我们从先前确定的具有确认HCMV先天性感染病史的儿科患者队列中选取了五个HCMV临床分离株，通过下一代测序（NGS）对其遗传多态性、系统发育关系和多菌株感染进行了评估。我们发现所选临床菌株中遗传特征的变异水平不一，与调节NK细胞功能的基因组区域存在适度变异。值得注意的是，我们表明不同的HCMV临床菌株以不同的方式调节NK细胞活化受体NKG2D、DNAM-1/CD226和NKp30的多种配体的表达。具体而言，DNAM-1/CD226配体PVR/CD155似乎主要由快速复制（“侵袭性”）HCMV分离株过度表达。另一方面，无论使用何种菌株，NKG2D配体ULBP2/5/6均下调，而其他NK细胞配体（例如MICA、MICB、ULBP3、Nectin-2/CD112和B7-H6）则没有显著调节。此外，我们发现与HCMV感染成纤维细胞共培养的NK细胞产生的IFN-γ产量与所使用的HCMV临床分离株的侵袭性呈直接比例。有趣的是，针对NK细胞配体的NK细胞调节基因的丢失似乎是“侵袭性”HCMV菌株的共有特征，这些菌株在其基因组中也共享多个基因变异。总的来说，尽管基于更多患者的高水平研究将提供更为确切的情景，但我们的发现为HCMV遗传变异对NK细胞介导的免疫反应的影响提供了新的机制见解。