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Global mapping of herpesvirus-host protein complexes reveals a novel transcription strategy for late genes

Abstract

Mapping host-pathogen interactions has proven instrumental for understanding how viruses manipulate host machinery and how numerous cellular processes are regulated. DNA viruses such as herpesviruses have relatively large coding capacity and thus can target an extensive network of cellular proteins. To identify the host proteins hijacked by this pathogen, we systematically affinity tagged and purified all 89 proteins of Kaposi’s sarcoma-associated herpesvirus (KSHV) from human cells. Mass spectrometry of this material identified over 500 high-confidence virus-host interactions. KSHV causes AIDS-associated cancers and its interaction network is enriched for proteins linked to cancer and overlaps with proteins that are also targeted by HIV-1. This work revealed many new interactions between viral and host proteins. I have focused on one interaction in particular, that of a previously uncharacterized KSHV protein, ORF24, with cellular RNA polymerase II (RNAP II).

All DNA viruses encode a class of genes that are expressed only late in the infectious cycle, following replication of the viral genome. These ‘late’ genes are subject to tight regulation and must be robustly induced during a period of significant cell stress, yet in many cases have strikingly minimal promoters. For most viruses, the mechanism underlying the activation of these genes has remained largely unknown. I have shown that late gene expression in KSHV requires ORF24, which nucleates a non-canonical transcription complex. I have found that ORF24 is a modular protein that binds directly and specifically to viral late gene TATA-like boxes through its central domain, which was previously predicted to structurally mimic TATA-box-binding protein (TBP). Chromatin immunoprecipitation analyses show that ORF24 functionally replaces human TBP at these promoters. This is highly unusual, as cellular TBP canonically serves as a critical nucleation factor for the assembly of transcription pre-initiation complexes (PICs) at promoters. Through residues in its N-terminus, ORF24 then binds RNAP II to activate late gene expression. This strategy appears to be conserved in multiple related herpesviruses, and represents a novel, streamlined mechanism for transcriptional activation.

As the late gene PIC appears deviates from the canonical complex, I have worked to define the viral and cellular proteins assembled at these promoters. Foundational work in related herpesviruses has revealed a complex of six viral proteins (including ORF24) to be essential for late gene expression. We have mapped the pairwise interactions between these viral proteins and found that the four other late gene viral transactivators (vTFs) interact with ORF24 through the viral protein ORF34. The interaction between ORF24 and ORF34 is essential for the expression of late genes. Aside from ORF24 and ORF34, the precise roles of the other vTFs in late gene expression remain unknown and are an ongoing area of research.

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