Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USA.
Center for Phage Technology, Texas A&M University, College Station, Texas, USA.
J Virol. 2024 Nov 19;98(11):e0112824. doi: 10.1128/jvi.01128-24. Epub 2024 Oct 31.
After the ejection of viral DNA into the host cytoplasm, the temperate bacteriophage (phage) lambda integrates a cascade of expressions from various regulatory genes, coupled with DNA replication, to commit to a decision between lysis and lysogeny. Higher multiplicity of infection (MOI) greatly shifts the decision toward the lysogenic pathway. However, how the phage separates the MOI from replicated viral DNA during lysis-lysogeny decision-making is unclear. To quantitatively understand the role of viral DNA replication, we constructed a reporter system facilitating the visualization of individual copies of phage DNA throughout the phage life cycle, along with the lysis-lysogeny reporters. We showed that intracellular viral DNA diverges between the lytic and lysogenic pathways from the early phase of the infection cycle, mostly due to the synchronization and success of DNA injection, as well as the competition for replication resources, rather than the replication rate. Strikingly, we observed two distinct replication patterns during lysogenization and surprisingly heterogeneous integration kinetics, which advances our understanding of temperate phage life cycles. We revealed that the weak repression function of Cro is critical for an optimal replication rate and plays a crucial role in establishing stable lysogens.
Temperate bacteriophages, such as lambda, incorporate environmental cues including host abundance and nutrient conditions to make optimal decisions between propagation and dormancy. A higher phage-to-host ratio or multiplicity of infection (MOI) during λ infection strongly biases toward lysogeny. However, a comprehensive understanding of this decision-making process and the impact of phage replication prior to the decision is yet to be achieved. Here, we used fluorescence microscopy to quantitatively track the spatiotemporal progression of viral DNA replication in individual cells with different cell fates. The implementation of this fluorescent reporter system and quantitative analysis workflow opens a new avenue for future studies to delve deeper into various types of virus-host interactions at a high resolution.
在病毒 DNA 被喷射到宿主细胞质后，温和噬菌体（噬菌体）lambda 将一系列来自各种调控基因的表达级联整合在一起，同时进行 DNA 复制，以决定是裂解还是溶原。较高的感染复数（MOI）极大地促使决定向溶原途径倾斜。然而，在裂解-溶原决策过程中，噬菌体如何将 MOI 与复制的病毒 DNA 分开尚不清楚。为了定量理解病毒 DNA 复制的作用，我们构建了一个报告系统，该系统可以在噬菌体生命周期中可视化噬菌体 DNA 的单个拷贝，同时还构建了裂解-溶原报告系统。我们发现，在感染周期的早期，细胞内病毒 DNA 在裂解和溶原途径之间出现分歧，这主要是由于 DNA 注射的同步和成功，以及复制资源的竞争，而不是复制率。引人注目的是，我们在溶原化过程中观察到两种截然不同的复制模式，以及令人惊讶的异质整合动力学，这推进了我们对温和噬菌体生命周期的理解。我们揭示了 Cro 的弱抑制功能对于最佳复制率至关重要，并在建立稳定的溶原体方面发挥了关键作用。
温和噬菌体，如 lambda，整合了环境线索，包括宿主丰度和营养条件，以在繁殖和休眠之间做出最佳决策。在 λ 感染过程中，噬菌体与宿主的比例或感染复数（MOI）较高时，强烈偏向于溶原。然而，对于这个决策过程以及在决策之前噬菌体复制的影响，我们还没有全面的了解。在这里，我们使用荧光显微镜在具有不同细胞命运的单个细胞中定量跟踪病毒 DNA 复制的时空进展。该荧光报告系统的实施和定量分析工作流程为未来的研究开辟了新的途径，可以更深入地研究各种类型的病毒-宿主相互作用。
