Understanding how viral and host factors interact and how perturbations impact infection is the basis for designing antiviral interventions. Here we define the functional contribution of each viral and host factor involved in human cytomegalovirus infection in primary human fibroblasts through pooled CRISPR interference and nuclease screening. To determine how genetic perturbation of critical host and viral factors alters the timing, course and progression of infection, we applied Perturb-seq to record the transcriptomes of tens of thousands of CRISPR-modified single cells and found that, normally, most cells follow a stereotypical transcriptional trajectory. Perturbing critical host factors does not change the stereotypical transcriptional trajectory per se but can stall, delay or accelerate progression along the trajectory, allowing one to pinpoint the stage of infection at which host factors act. Conversely, perturbation of viral factors can create distinct, abortive trajectories. Our results reveal the roles of host and viral factors and provide a roadmap for the dissection of host–pathogen interactions.

了解病毒和宿主因素如何相互作用以及扰动如何影响感染是设计抗病毒干预措施的基础。在这里，我们通过合并的 CRISPR 干扰和核酸酶筛选，定义了原代人成纤维细胞中人巨细胞病毒感染所涉及的每种病毒和宿主因子的功能贡献。为了确定关键宿主和病毒因子的遗传扰动如何改变感染的时间、过程和进展，我们应用 Perturb-seq 记录数万个 CRISPR 修饰的单细胞的转录组，发现通常情况下，大多数细胞遵循刻板的转录轨迹。扰乱关键宿主因素本身不会改变刻板的转录轨迹，但会延缓、延迟或加速沿轨迹的进展，允许人们查明宿主因素起作用的感染阶段。相反，病毒因子的扰动会产生截然不同的流产轨迹。我们的结果揭示了宿主和病毒因子的作用，并为剖析宿主-病原体相互作用提供了路线图。