Cataldo Katie M, Roche Kathryn L, Monti Christopher E, Dash Ranjan K, Murphy Eain A, Terhune Scott S
bioRxiv. 2024 Oct 23:2024.10.21.619499. doi: 10.1101/2024.10.21.619499.
Human cytomegalovirus (HCMV) is a betaherpesvirus capable of infecting numerous cell types and persisting throughout an infected individual's life. Disease usually occurs in individuals with compromised or underdeveloped immune systems. Several antivirals exist but have limitations relating to toxicity and resistance. HCMV replication involves upregulation of host proteasomal activities which play important roles in the temporal stages of replication. Here, we defined the impact on replication kinetics of the proteasome inhibitor, bortezomib. We demonstrate that bortezomib significantly reduces levels of viral genomes and infectious virions produced from a population of cells. Inhibition reduced expression of viral proteins that are influenced by genome synthesis. When added prior to 24 hpi, we observe decreases in PCNA and Cdk1 while increases in p21 whose regulations contribute to efficient replication. This response synergized with an antiviral, maribavir. Since some replication occurred, we tested the hypothesis that a subset of infected cells might break through inhibition. Initially, we simulated bortezomib activities using a mechanistic computational model of late-lytic replication. Upon reducing MOI , we observed near identical simulated results compared to experimental data. Next, we analyzed replication using live-cell imaging. This revealed treated cultures do contain a population of cells with fully developed late-stage cytoplasmic assembly compartments but at significantly lower numbers. We refer to this as the effective MOI. Overall, our studies support a hypothesis in which 20S proteasome inhibition disrupts HCMV replication by reducing the MOI to an effective MOI, defined by a fraction of infected cells capable of progressing to fulminant infection.
HCMV infection and reactivation continues to contribute to morbidity and mortality around the world. Antiviral compounds are available but have limitations. Here, we have defined the impact of the proteasome inhibitor bortezomib on HCMV replication. Proteasomal activities play a critical role in temporal changes required for replication. We demonstrate that disrupting these activities inhibits viral replication while likely supporting increased antiviral activity of the anti-HCMV agent, maribavir. Using a combination of live cell imaging and computational tools, we discover that a subset of infected cells progresses to fulminant infection which we define as the effective MOI, and this subset would otherwise be missed when analyzing the average of the population.
人巨细胞病毒（HCMV）是一种β疱疹病毒，能够感染多种细胞类型，并在受感染个体的一生中持续存在。疾病通常发生在免疫系统受损或发育不全的个体中。现有几种抗病毒药物，但在毒性和耐药性方面存在局限性。HCMV复制涉及宿主蛋白酶体活性的上调，这在复制的时间阶段中起重要作用。在此，我们确定了蛋白酶体抑制剂硼替佐米对复制动力学的影响。我们证明硼替佐米显著降低了一群细胞产生的病毒基因组和感染性病毒粒子的水平。抑制作用降低了受基因组合成影响的病毒蛋白的表达。在感染后24小时之前添加时，我们观察到增殖细胞核抗原（PCNA）和细胞周期蛋白依赖性激酶1（Cdk1）减少，而p21增加，其调节有助于有效复制。这种反应与抗病毒药物马立巴韦协同作用。由于发生了一些复制，我们测试了一个假设，即一部分受感染细胞可能突破抑制。最初，我们使用晚期裂解复制的机械计算模型模拟硼替佐米的活性。降低感染复数（MOI）后，我们观察到与实验数据相比几乎相同的模拟结果。接下来，我们使用活细胞成像分析复制。这表明处理过的培养物确实含有一群具有完全发育的晚期细胞质装配区室的细胞，但数量明显较少。我们将此称为有效MOI。总体而言，我们的研究支持一种假设，即20S蛋白酶体抑制通过将MOI降低到有效MOI来破坏HCMV复制，有效MOI由能够发展为暴发性感染的受感染细胞部分定义。
HCMV感染和再激活继续在世界各地导致发病和死亡。有抗病毒化合物可用，但有局限性。在此，我们确定了蛋白酶体抑制剂硼替佐米对HCMV复制的影响。蛋白酶体活性在复制所需的时间变化中起关键作用。我们证明破坏这些活性会抑制病毒复制，同时可能支持抗HCMV药物马立巴韦的抗病毒活性增加。通过结合活细胞成像和计算工具，我们发现一部分受感染细胞会发展为暴发性感染，我们将其定义为有效MOI，否则在分析群体平均值时会遗漏这一部分。
