Coding and non-coding elements comprise a regulatory network controlling transcription in Kaposi's sarcoma-associated herpesvirus.

Gene regulation in eukaryotes relies on many mechanisms for optimal expression, including both protein transcription factors and DNA regulatory elements. CRISPR-based screens of both protein coding genes and non-coding regions have allowed identification of these transcriptional networks in human cells. Double-stranded DNA viruses also invoke human-like regulation to control transcription of viral genes that are required at different stages of the viral lifecycle. Here, we applied CRISPR-based tools to dissect regulation of a viral gene at high resolution in the oncogenic human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV), whose compact, densely encoded genome provides unique challenges and opportunities for studying transcriptional networks. Through a combination of CRISPR-interference (CRISPRi) and Cas9 nuclease screening, we mapped a novel regulatory network comprised of coding and noncoding elements that influence expression of the essential KSHV protein ORF68 at early and late stages of the viral lifecycle. ORF68 encodes an essential protein involved in packaging the replicated viral DNA into nascent capsids. Although ORF68 expression initiates early in the viral lifecycle, we found that it is primarily required at later times. This work demonstrates the ability to exhaustively identify features controlling a given locus, essentially capturing a complete viral regulatory circuit that functions within the human nucleus to control transcription.