Abstract
Almost one third of herpesvirus proteins are expressed with late kinetics. Many of these late proteins serve crucial structural functions such as formation of virus particles, attachment to host cells and internalization. Recently, we and others identified a group of Epstein-Barr virus early proteins that form a pre-initiation complex (vPIC) dedicated to transcription of late genes. Currently, there is a fundamental gap in understanding the role of post-translational modifications in regulating assembly and function of the complex. Here, we used mass spectrometry to map potential phosphorylation sites in BGLF3, a core component of the vPIC module that connects the BcRF1 viral TATA box binding protein to other components of the complex. We identified threonine 42 (T42) in BGLF3 as a phosphoacceptor residue. T42 is conserved in BGLF3 orthologs encoded by other gamma herpesviruses. Abolishing phosphorylation at T42 markedly reduced expression of vPIC-dependent late genes and disrupted production of new virus particles, but had no effect on early gene expression, viral DNA replication, or expression of vPIC-independent late genes. We complemented failure of BGLF3(T42A) to activate late gene expression by ectopic expression of other components of vPIC. Only BFRF2 and BVLF1 were sufficient to suppress the defect in late gene expression associated with BGLF3(T42A). These results were corroborated by the ability of wild type BGLF3 but not BGLF3(T42A) to form a trimeric complex with BFRF2 and BVLF1. Our findings suggest that phosphorylation of BGLF3 at threonine 42 serves as a new checkpoint for subsequent formation of BFRF2:BGLF3:BVLF1; a trimeric subcomplex essential for transcription of late genes. Our findings provide evidence that post-translational modifications regulate the function of the vPIC nanomachine that initiates synthesis of late transcripts in herpesviruses.
Highlights
Lytic infection is intrinsic to the pathogenesis of herpesviruses
We demonstrated that BGLF3, a component of viral pre-initiation complex (vPIC), is modified by phosphorylation during the lytic phase of the viral life cycle
Phosphorylation of BGLF3 is essential for the ability of the protein to interact with two other components of vPIC, BFRF2 and BVLF1
Summary
Lytic infection is intrinsic to the pathogenesis of herpesviruses. Virus particles are synthesized and assembled exclusively during the lytic phase. The lytic phase of oncogenic gamma herpesviruses contributes to tumor development by expanding the population of latently infected cells that possess the potential to become neoplastic. Temporal control of expression of lytic viral genes, a common theme among all herpesviruses, can be categorized into pre- and post-replication events. Pre-replication genes, referred to as early genes, are regulated in a manner similar to that of cellular genes. Post-replication genes, referred to as late genes, have unique promoter structures featuring a non-canonical TATA box element (reviewed in [1, 2]). Activation of late promoters is dependent on amplification of the viral genome. The strict dependence of late gene expression on replication of the viral genome represents one of the longstanding conundrums in the biology of DNA viruses
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