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.
Having a research tool to nondestructively identify the stage of KSHV replication in an infected cell would not only allow us to effectively isolate cells of interest from heterogeneous cell populations, but also enable more precise sample selection for advanced single cell analysis. We are establishing a method to increase the resolution of genetic and proteomics approaches, and study dynamic protein-protein interactions to identify druggable targets of KSHV-associated malignancies. This study will develop new research tools and also deepen our understanding of KSHV gene regulation, which may lead to new therapeutic approaches to inhibit KSHV replication.