Phosphorylation of a Conserved Tyrosine in the Papillomavirus E2 Protein Regulates Brd4 Binding and Viral Replication

DeSmet, Marsha; Jose, Leny; Isaq, Nasro A.; Androphy, Elliot J.

doi:10.1128/jvi.01801-18

Cited by 15 publications

(24 citation statements)

References 60 publications

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“…Several serine/threonine phosphorylation sites have been identified in the papillomavirus protein E2 (3,5,6,28,29), but only two tyrosine phosphorylation sites have been examined: Y102 and Y138 (7,30). Phosphorylation at Y102 was originally identified in BPV-1 E2 through mass spectrometric analysis (7) and subsequently confirmed in the current work for HPV-31 E2.…”

Section: Discussionsupporting

confidence: 51%

Human Papillomavirus 31 Tyrosine 102 Regulates Interaction with E2 Binding Partners and Episomal Maintenance

Gilson

Culleton

Xie

et al. 2020

J Virol

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Several serine and threonine residues of the papilloma virus early E2 protein have been found to be phosphorylated. By contrast, only one E2 tyrosine phosphorylation site in BPV-1 (tyrosine 102) and one HPV-16/31 (tyrosine 138) site have been characterized. Between BPV-1 and HPV-31 E2, 8 of the 11 tyrosines are conserved in the N-terminal domain, suggesting that phosphorylation of tyrosines has an essential role in E2 biology. In this paper we examine the effect of Y102 phosphorylation on HPV-31 E2 biology. Y102 proteins mutated either to the potential phospho-mimetic glutamic acid (Y102E) or to the non-phosphorylated homologue phenylalanine (Y102F) remain nuclear; however, Y102E is more associated with the nuclear matrix fraction. This is consistent with the inability of Y102E to bind TopBP1. Both BPV-1 and HPV-31 Y102E are similar in that neither bind the C-terminus of Brd4, but in all other aspects, the mutant behaves differently between the two families of papillomaviruses. BPV-1 Y102E was unable to bind E1 and did not replicate in a transient in-vitro assay, while HPV-31 Y102E binds E1 and replicated albeit at lower levels than wild type. To examine effect of E2 mutations under more native-like infection conditions, a neomycin selectable marker was inserted into L1/L2 of HPV-31 genome, creating HPV-31neo. This genome was maintained in every cell line tested for at least 50 days post-transfection/infection. Y102E in both transfection and infection conditions was unable to maintain high episome copy numbers in epithelial cell lines. IMPORTANCE Post-translational modifications by phosphorylation can change protein activities, binding partners, or localization. Tyrosine 102 is conserved between delta papillomavirus BPV-1 and alpha papillomavirus HPV-31 E2. We characterized mutations of HPV-31 E2 for interactions with relevant cellular binding partners and replication in the context of the viral genome.

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Section: Discussionsupporting

confidence: 51%

Human Papillomavirus 31 Tyrosine 102 Regulates Interaction with E2 Binding Partners and Episomal Maintenance

Gilson

Culleton

Xie

et al. 2020

J Virol

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“…HPV-31 genomes with the E2 Y138E phosphomimetic mutation were unable to maintain episomes in keratinocytes. We previously reported that the HPV-31 Y138E E2 protein did not bind to the Brd4 CTM but coimmunoprecipitated with full-length Brd4 (16). The Brd4 CTM has been shown to contact amino acids R37 and I73 within the TAD of E2 (24, 29-32).…”

Section: Resultsmentioning

confidence: 99%

“…Our previous studies demonstrated that HPV-31 E2 with tyrosine 138 mutated to glutamate (Y138E) bound E1 and was capable of binding the ori yet was inactive for induction of E1-dependent transient DNA replication. Y138E did not form nuclear replication foci, and it bound to full-length Brd4 but did not associate with its CTM (16). Our transient-transfection DNA replication experiments utilized cytomegalovirus (CMV) promoter-driven E1 and E2 expression vectors with the HPV origin.…”

Section: Discussionmentioning

confidence: 99%

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Phosphorylation of the Human Papillomavirus E2 Protein at Tyrosine 138 Regulates Episomal Replication

Jose

Androphy

DeSmet

2020

J Virol

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The papillomavirus (PV) E2 protein is a critical regulator of viral transcription and genome replication. We previously reported that tyrosine (Y) 138 of HPV-31 E2 is phosphorylated by the fibroblast growth factor receptor 3 (FGFR3) kinase. In this study, we generated quasiviruses containing G418-selectable HPV-31 genomes with phosphodeficient phenylalanine mutant E2 Y138F and phosphomimetic glutamic acid mutant Y138E. We observed significantly fewer early viral transcripts immediately after infection with these Y138 mutant genomes even though E2 occupancy at the viral origin was equivalent to that of wild-type E2. Keratinocytes infected with Y138F quasiviruses formed stable colonies, and the genomes were maintained as episomes, while those infected with Y138E quasiviruses did not. We previously reported that the HPV-31 E2 Y138 mutation to glutamic acid did not bind to the Brd4 C-terminal motif (CTM). Here, we demonstrate that HPV-16 E2 Y138E bound to full-length Brd4 but not to the Brd4 CTM. We conclude that association of E2 with the Brd4 CTM is necessary for viral genome replication and suggest that this interaction can be regulated by phosphorylation of E2 Y138. IMPORTANCE Papillomavirus (PV) is a double-stranded DNA tumor virus infecting the cutaneous and mucosal epithelium. The PV E2 protein associates with a number of cellular factors to mediate replication of the HPV genome. Fibroblast growth factor receptor 3 (FGFR3) regulates HPV replication through phosphorylation of tyrosine 138 in the HPV E2 protein. Employing a quasivirus infection model and selection for G418 resistant genomes, we demonstrated that Y138 is a critical residue for Brd4 association and that inability to complex with Brd4 does not support episomal replication.

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“…Plasmids, siRNA, and Antibodies. The plasmids used were FLAG-SMC6/pcDNA3 (GenScript, Piscataway, NJ, USA), HPV-31 FLAG-E2/pcDNA3, HPV-31 FLAG-E2 mt (residues 205-372; [ 33 ]), HPV-31 V5-E2/pcDNA3, HPV-31 HA-E1/pcDNA3, and pcDNA3. The siRNA depletion of SMC5/6 subunits used siScramble DsiRNA (IDT; 51-01-19-08), siSMC6 (IDT; hs.Ri.SMC6.13.2), and siNSE3 (IDT; hs.Ri.NDNL2.13.1).…”

Section: Methodsmentioning

confidence: 99%

The SMC5/6 Complex Represses the Replicative Program of High-Risk Human Papillomavirus Type 31

Gibson

Androphy

2020

Pathogens

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The multi-subunit structural maintenance of chromosomes (SMC) 5/6 complex includes SMC6 and non-SMC element (NSE)3. SMC5/6 is essential for homologous recombination DNA repair and functions as an antiviral factor during hepatitis B (HBV) and herpes simplex-1 (HSV-1) viral infections. Intriguingly, SMC5/6 has been found to associate with high-risk human papillomavirus (HPV) E2 regulatory proteins, but the functions of this interaction and its role during HPV infection remain unclear. Here, we further characterize SMC5/6 interactions with HPV-31 E2 and its role in the HPV life cycle. Co-immunoprecipitation (co-IP) revealed that SMC6 interactions with HPV-31 E2 require the E2 transactivation domain, implying that SMC5/6 interacts with full-length E2. Using chromatin immunoprecipitation, we found that SMC6 is present on HPV-31 episomes at E2 binding sites. Th depletion of SMC6 and NSE3 increased viral replication and transcription in keratinocytes maintaining episomal HPV-31, indicating that SMC5/6 restricts the viral replicative program. SMC6 interactions with E2 were reduced in the presence of HPV-31 E1, suggesting that SMC6 and E1 compete for E2 binding. Our findings demonstrate SMC5/6 functions as a repressor of the viral replicative program and this may involve inhibiting the initiation of viral replication.

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Phosphorylation of a Conserved Tyrosine in the Papillomavirus E2 Protein Regulates Brd4 Binding and Viral Replication

Cited by 15 publications

References 60 publications

Human Papillomavirus 31 Tyrosine 102 Regulates Interaction with E2 Binding Partners and Episomal Maintenance

Human Papillomavirus 31 Tyrosine 102 Regulates Interaction with E2 Binding Partners and Episomal Maintenance

Phosphorylation of the Human Papillomavirus E2 Protein at Tyrosine 138 Regulates Episomal Replication

The SMC5/6 Complex Represses the Replicative Program of High-Risk Human Papillomavirus Type 31

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