Agu Ilechukwu, José Ivy, Ram Abhineet, Oberbauer Daniel, Albeck John, Díaz Muñoz Samuel L
Department of Microbiology and Molecular Genetics, University of California, Davis, One Shields Ave, Davis CA 95616.
Department of Molecular and Cellular Biology, University of California, Davis, One Shields Ave, Davis CA 95616.
bioRxiv. 2024 Jul 3:2024.07.03.601932. doi: 10.1101/2024.07.03.601932.
RNA viruses produce abundant defective viral genomes during replication, setting the stage for interactions between viral genomes that alter the course of pathogenesis. Harnessing these interactions to develop antivirals has become a recent goal of intense research focus. Despite decades of research, the mechanisms that regulate the production and interactions of Influenza A defective viral genomes are still unclear. The role of the host is essentially unexplored; specifically, it remains unknown whether host metabolism can influence the formation of defective viral genomes and the particles that house them. To address this question, we manipulated host cell anabolic signaling activity and monitored the production of defective viral genomes and particles by A/H1N1 and A/H3N2 strains, using a combination of single-cell immunofluorescence quantification, third-generation long-read sequencing, and the cluster-forming assay, a method we developed to titer defective and fully-infectious particles simultaneously. Here we show that alpelisib (Piqray), a highly selective inhibitor of mammalian Class 1a phosphoinositide-3 kinase (PI3K) receptors, significantly changed the proportion of defective particles and viral genomes (specifically deletion-containing viral genomes) in a strain-specific manner, under conditions that minimize multiple cycles of replication. Alpelisib pre-treatment of cells led to an increase in defective particles in the A/H3N2 strain, while the A/H1N1 strain showed a decrease in total viral particles. In the same infections, we found that defective viral genomes of polymerase and antigenic segments increased in the A/H1N1 strain, while the total particles decreased suggesting defective interference. We also found that the average deletion size in polymerase complex viral genomes increased in both the A/H3N2 and A/H1N1 strains. The A/H1N1 strain, additionally showed a dose-dependent increase in total number of defective viral genomes. In sum, we provide evidence that host cell metabolism can increase the production of defective viral genomes and particles at an early stage of infection, shifting the makeup of the infection and potential interactions among virions. Given that Influenza A defective viral genomes can inhibit pathogenesis, our study presents a new line of investigation into metabolic states associated with less severe flu infection and the potential induction of these states with metabolic drugs.
RNA病毒在复制过程中会产生大量有缺陷的病毒基因组，为病毒基因组之间的相互作用奠定了基础，而这种相互作用会改变发病机制的进程。利用这些相互作用来开发抗病毒药物已成为近期研究的一个重要目标。尽管经过了数十年的研究，但甲型流感病毒有缺陷的病毒基因组的产生和相互作用的调控机制仍不清楚。宿主的作用基本上未被探索；具体而言，宿主代谢是否会影响有缺陷的病毒基因组及其所含颗粒的形成仍然未知。为了解决这个问题，我们通过单细胞免疫荧光定量、第三代长读长测序以及我们开发的同时滴定有缺陷和完全感染性颗粒的簇形成试验相结合的方法，操纵宿主细胞的合成代谢信号活性，并监测A/H1N1和A/H3N2毒株有缺陷的病毒基因组和颗粒的产生情况。在此我们表明，在将复制循环次数降至最低的条件下，哺乳动物1a类磷酸肌醇-3激酶（PI3K）受体的高度选择性抑制剂阿培利司（Piqray）以毒株特异性方式显著改变了有缺陷颗粒和病毒基因组（特别是含缺失的病毒基因组）的比例。对细胞进行阿培利司预处理导致A/H3N2毒株中有缺陷颗粒增加，而A/H1N1毒株中总病毒颗粒减少。在相同的感染中，我们发现A/H1N1毒株中聚合酶和抗原片段的有缺陷病毒基因组增加，而总颗粒减少，提示存在缺陷干扰。我们还发现，A/H3N2和A/H1N1毒株中聚合酶复合体病毒基因组的平均缺失大小均增加。此外，A/H1N1毒株中，有缺陷病毒基因组的总数呈剂量依赖性增加。总之，我们提供的证据表明，宿主细胞代谢可在感染早期增加有缺陷病毒基因组和颗粒的产生，改变感染的构成以及病毒粒子之间的潜在相互作用。鉴于甲型流感病毒有缺陷的病毒基因组可抑制发病机制，我们的研究为与较轻流感感染相关的代谢状态以及用代谢药物诱导这些状态的可能性提出了一条新的研究路线。
