Electron microscopy mapping of the DNA-binding sites of monomeric, dimeric, and multimeric KSHV RTA protein.

Molecular interactions between viral DNA and viral-encoded protein are a prerequisite for successful herpesvirus replication and production of new infectious virions. Here, we examined how the essential Kaposi's sarcoma-associated herpesvirus (KSHV) protein, RTA (replication and transcription activator), binds to viral DNA using transmission electron microscopy (TEM). Previous studies using gel-based approaches to characterize RTA binding are important for studying the predominant form(s) of RTA within a population and identifying the DNA sequences that RTA binds with high affinity. However, using TEM, we were able to examine individual protein-DNA complexes and capture the various oligomeric states of RTA when bound to DNA. Hundreds of images of individual DNA and protein molecules were collected and then quantified to map the DNA-binding positions of RTA bound to the two KSHV lytic origins of replication encoded within the KSHV genome. The relative size of RTA or RTA bound to DNA was then compared to protein standards to determine whether RTA complexed with DNA was monomeric, dimeric, or formed larger oligomeric structures. We successfully analyzed a highly heterogeneous data set and identified new DNA-binding sites for RTA that included both high- and low-frequency-binding sites. TEM micrographs provide evidence that the majority of RTA analyzed forms dimers and high order multimers when bound to KSHV origin of replication DNA sequences. Broadly, our findings expand upon our understanding of the degree to which DNA-binding locations and protein conformation are heterogeneous and how protein monomers and dimers function differently with respect to DNA-binding location specificity. IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is a human herpesvirus associated with several human cancers, typically in patients with compromised immune systems. Herpesviruses establish lifelong infections in hosts in part due to the two phases of infection: the dormant and active phases. Effective antiviral treatments to prevent the production of new viruses are needed to treat KSHV. A detailed microscopy-based investigation of the molecular interactions between viral protein and viral DNA revealed how protein-protein interactions play a role in DNA-binding specificity. This analysis will lead to a more in-depth understanding of KSHV DNA replication and serve as the basis for anti-viral therapies that disrupt and prevent the protein-DNA interactions, thereby decreasing spread to new hosts.