Development of Precision Analyses to Reveal "Hit&Run" Effects

对卡波西肉瘤相关疱疹病毒（KSHV）复制的遗传和分子研究主要通过测量受感染细胞群的总活性或平均活性来进行，受感染细胞群由非再活化细胞和再活化细胞的混合物组成，这些细胞在不同的复制阶段又含有KSHV。通过分离感兴趣的特定细胞群来提高分析的分辨率将更好地理解这种异质性的基础。因此，新的预防措施可能会开发的基础上，从非再活化细胞暴露于相同的再活化刺激的结果。我们最近产生了一种重组病毒，揭示了KSHV在活细胞中的复制阶段，并使用这种构建体从再活化细胞群中分离出特定的细胞组分;该方法确实增加了下游分析的动态范围。随着生物素标记KSHV，我们产生了额外的工具，以探测相互作用的蛋白质与生物素为基础的接近标记技术（迷你TurboID）在KSHV重新激活的特定阶段。此外，我们还调整了方案并生成了必要的试剂来执行CUT&RUN（使用核酸酶进行靶下切割和释放），这使我们能够从少量分离的细胞中鉴定靶分子的染色质结合位点。有了这些试剂，我们将探索病毒-宿主蛋白相互作用的动力学及其对染色质结合的影响。我们将强调病毒蛋白质的“命中和运行”的生物学后果。

Genetic and molecular studies on Kaposi's sarcoma-associated herpesvirus (KSHV) replication have been conducted primarily by measuring the total or average activity of an infected cell population, which consists of mixtures of both non-reactivating and reactivating cells that in turn contain KSHVs at various stages of replication. Increasing the resolution of analyses by isolating specific cell populations of interest will provide better understanding of the basis for this heterogeneity. Hence new preventive measures may be developed based on findings derived from non-reactivating cells exposed to the same reactivation stimuli. We recently generated a recombinant reporter-virus that reveals KSHV replication stages in live cells and used this construct to isolate specific cell fractions from reactivating cell populations; the method indeed increased the dynamic range of downstream analyses. With the reporter-KSHV, we generated additional tools to probe for interacting proteins with a biotin-based proximity labeling technique (mini-TurboID) at specific stages of KSHV reactivation. Furthermore, we also adapted protocols and generated necessary reagents to perform CUT&RUN (Cleavage Under Targets and Release Using Nuclease), which enables us to identify chromatin binding sites of target molecules from small numbers of isolated cells. With these reagents in hand, we will explore the dynamics of viral-host protein interactions and their effects on chromatin binding with precision. We will highlight the biological consequences of "HIT&RUN" by a viral protein.