Date Published: August 28, 2019
Publisher: Public Library of Science
Author(s): Jinlin Li, Ann Walsh, TuKiet T. Lam, Henri-Jacques Delecluse, Ayman El-Guindy, Zhen Lin.
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.
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. Lytic gene products also encode and upregulate expression of inflammatory cytokines, anti-apoptotic proteins, signaling molecules, and immunoevasins that promote cell proliferation and suppress immune recognition.
Phosphorylation represents one of the most important post-translational modifications that regulates the activity, interaction, localization, and stability of proteins. Recently, we and others identified a viral pre-initiation complex dedicated to transcription of beta and gamma herpesvirus late genes. Previous studies mapped a number of important protein interactions that contribute to our current understanding of the overall organization of vPIC. However, none of these studies addressed the role of post-translational modifications, particularly phosphorylation, in regulating assembly or function of vPIC in transcription of late genes. Here, we studied phosphorylation of BGLF3, a protein that is indispensable for transcription of late genes and serves as a core protein connecting BcRF1, the vTBP, to other components of the vPIC module. We report the following novel findings: 1) BGLF3 is phosphorylated in vivo at threonine 42, a site conserved in other gamma herpesviruses, during the late phase of lytic infection. 2) Phosphorylation of BGLF3 at T42 is crucial for the role of the protein in transcription of late genes. 3) BFRF2 and BVLF1 suppress the defect observed in transcription of late genes due to lack of phosphorylation of BGLF3 at threonine 42. 4) As a result of phosphorylation of BGLF3 at threonine 42, a novel trimeric subcomplex forms that includes BFRF2, BGLF3 and BVLF1. 5) Phosphorylation of BGLF3 does not regulate its interaction with BcRF1 (S1 Fig) suggesting that lack of phosphorylation does not impact recruitment of BGLF3 to late promoters but abolishes binding of BVLF1 to BGLF3 and hence formation of a functional vPIC. Our findings demonstrate that phosphorylation at T42 is crucial for BGLF3 to interact functionally and physically with BFRF2 and BVLF1 (Fig 9). These findings demonstrate that post-translational modifications regulate the function of vPIC in transcription of late genes.