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

对卡波西肉瘤相关疱疹病毒(KSHV)复制的遗传学和分子研究主要是通过测量感染细胞群体的总或平均活性来进行的，感染细胞群体由未重新激活的细胞和重新激活的细胞组成，而这些细胞又包含处于不同复制阶段的KSHV。通过分离感兴趣的特定细胞群体来提高分析的分辨率将有助于更好地理解这种异质性的基础。因此，可以根据暴露在相同再激活刺激下的非再激活细胞的研究结果，开发新的预防措施。我们最近制造了一种重组报告病毒，揭示了KSHV在活细胞中的复制阶段，并使用这种结构从重新激活的细胞群中分离出特定的细胞部分；该方法确实增加了下游分析的动态范围。使用Report-KSHV，我们生成了额外的工具来探测在KSHV重新激活的特定阶段使用基于生物素的邻近标记技术(mini-TurboID)相互作用的蛋白质。此外，我们还修改了程序并生成了必要的试剂来执行Cut&Run(靶标下切割和使用核酸酶释放)，这使我们能够从少量分离的细胞中识别靶分子的染色质结合部位。有了这些试剂，我们将精确地探索病毒与宿主蛋白相互作用的动力学及其对染色质结合的影响。我们将重点介绍病毒蛋白“肇事逃逸”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.