Targeted mutagenesis of the human papillomavirus type 16 E2 transactivation domain reveals separable transcriptional activation and DNA replication functions

Sakai, Hiroyuki; Yasugi, Toshiharu; Benson, Jalen; Dowhanick, J J; Howley, Peter M.

doi:10.1128/jvi.70.3.1602-1611.1996

Cited by 112 publications

(72 citation statements)

References 60 publications

(70 reference statements)

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“…Our findings are consistent with previous observations for Y138. In an earlier study that analyzed HPV-16 E2 TAD mutants, Y138 was replaced by alanine and was found to be moderately impaired for transcriptional activation (40% to 80% of WT), DNA replication (10% to 40% of WT), and E1 binding (40% to 80% of WT), and exhibited reduced Brd4-CTD association (51,52). BPV Y138F bound E1, and although it could stimulate transcription and transient replication, it was not as active as the WT (22).…”

Section: Discussionmentioning

confidence: 99%

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

DeSmet

Jose

Isaq

et al. 2019

J Virol

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The papillomavirus (PV) E2 protein coordinates viral transcription and genome replication. Following a strategy to identify amino acids in E2 that are posttranslationally modified, we reported that tyrosine kinase fibroblast growth factor receptor 3 (FGFR3) complexes with and phosphorylates E2, which inhibits viral DNA replication. Here, we present several lines of evidence indicating that tyrosine (Y) 138 of HPV-31 E2 is a substrate of FGFR3. The active form of FGFR3 bound to and phosphorylated the region of amino acids (aa) 107 to 175 in HPV-31 E2. The E2 phenylalanine (F) mutant Y138F displayed reduced FGFR3-induced phosphotyrosine. A constitutive kinase-active FGFR3 inhibited wild-type (WT) E2-induced E1-dependent DNA replication, while the 138F mutant retained activity. The tyrosine to glutamic acid (E) mutant Y138E, which can mimic phosphotyrosine, failed to induce transient DNA replication, although it maintained the ability to bind and localize the viral DNA helicase E1 to the viral origin. The bromodomain-containing protein 4 (Brd4) binds to E2 and is necessary for initiation of viral DNA synthesis. Interestingly, the Y138E protein coimmunoprecipitated with full-length Brd4 but was defective for association with its C-terminal domain (CTD). These results imply that the activity of the FGFR3 kinase in the infected epithelial cell restricts the HPV replication program through phosphorylation of E2 at Y138, which interferes with E2 binding to the Brd4 CTD, and that this interaction is required for initiation of viral DNA synthesis. IMPORTANCE Human papillomaviruses (HPVs) are highly infectious pathogens that commonly infect the oropharynx and uterine cervix. The idea that posttranslational modifications of viral proteins coordinates viral genome replication is less explored. We recently discovered that fibroblast growth factor receptor 3 (FGFR3) phosphorylates the viral E2 protein. The current study demonstrates that FGFR3 phosphorylates E2 at tyrosine 138, which inhibits association with the C-terminal peptide of Brd4. This study illustrates a novel regulatory mechanism of virus-host interaction and provides insight into the role of Brd4 in viral replication.

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Section: Discussionmentioning

confidence: 99%

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

DeSmet

Jose

Isaq

et al. 2019

J Virol

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“…As illustrated in Figure A,C, W4H and W4H_Y6R peptides were also predicted to form hydrogen bonds with Glu39 of HPV16 E2. Glu39 of HPV16 E2 was previously reported as an essential amino acid residue for replication function of E2 . The corresponding residue of Glu39 of HPV16 E2, Glu43 of HPV18 E2, is in a favorable ionic interaction with Arg454 of HPV18 E1; and mutation of Glu43 of HPV18 E2 or Arg454 of HPV18 E1 has previously shown to result in a significant reduction of E1–E2 complex formation …”

Section: Resultsmentioning

confidence: 99%

“…Therefore, disruption of this complex could possibly inhibit viral propagation. A mutated E2, which was defective for E1 binding, has been shown to lose its DNA replication function . In the previous study, dodecapeptides that could bind to HPV16 E2 were screened from the phage display library .…”

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confidence: 99%

Design of peptides as inhibitors of human papillomavirus 16 transcriptional regulator E1–E2

et al. 2016

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Human papillomavirus 16 (HPV 16) is a DNA virus that is capable of infecting humans and causing cervical cancer. HPV16 E2 plays an important role in viral gene regulation. This work aims to predict the binding conformations and interactions between the dodecapeptides and HPV16 E2 as well as to design novel peptide inhibitors that are capable of binding to HPV16 E2 and disrupt the transcriptional regulator E1-E2 complex formation, using computational protein design techniques. Based on previously reported peptide4 (TWFWPYPYPHLP), novel peptide inhibitors were designed and five peptides that showed lower binding energy to HPV16 E2 than that of peptide4, were selected for in vitro experiments. Enzyme-linked immunosorbent (ELISA) assay showed that Y6R, W4H_Y6R, and W4H peptides bound to HPV16 E2 with higher affinity than peptide4 did. Moreover, Y6R, W4H_Y6R, and W4H peptides more effectively inhibited E1-E2 complex formation than peptide4. This work revealed important interactions between the peptides and E1-E2 complex, suggesting a strategy for development of more potent peptide inhibitors.

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“…However, the specific function(s) of E2 affected by these mutations is not known. Our assessment of the in vitro hTFIIB binding activities of HPV-16 E2 missense mutants that were previously shown to be defective for transactivation (48) has revealed no quantitative change in hTFIIB binding activity (28a). It is possible that these mutants are defective for another essential E2 function that is unrelated to TFIIB interaction.…”

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confidence: 90%

“…Full-length BPV-1 E2 and E2-TR are indicated. 13,48). However, the specific function(s) of E2 affected by these mutations is not known.…”

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confidence: 99%

Conserved interaction of the papillomavirus E2 transcriptional activator proteins with human and yeast TFIIB proteins

Benson

Lawande

Howley

1997

J Virol

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Papillomavirus early gene expression is regulated by the virus gene-encoded E2 proteins. The best-characterized E2 protein, encoded by bovine papillomavirus type 1 (BPV-1), has been shown to interact with basal transcription factor IIB (TFIIB) and the TATA binding protein basal transcription factor (N. M. Rank and P. F. Lambert, J. Virol. 69:6323-6334, 1995). We demonstrate that the potent E2 transcriptional activator protein encoded by a gene of human PV type 16 also interacts with TFIIB in vitro. Moreover, a direct comparison of domains within human TFIIB (hTFIIB) required for VP16 and BPV-1 E2 indicates that these acidic activators interact with hTFIIB in a qualitatively similar manner. Our mapping experiments identify hTFIIB interaction domains within the amino-terminal activation domain of BPV-1 E2. Finally, we demonstrate in vitro interaction between Saccharomyces cerevisiae TFIIB and BPV-1 E2, an observation that is consistent with the importance of the E2-TFIIB interaction for BPV-1 E2 transactivation in both systems.

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Targeted mutagenesis of the human papillomavirus type 16 E2 transactivation domain reveals separable transcriptional activation and DNA replication functions

Cited by 112 publications

References 60 publications

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

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

Design of peptides as inhibitors of human papillomavirus 16 transcriptional regulator E1–E2

Conserved interaction of the papillomavirus E2 transcriptional activator proteins with human and yeast TFIIB proteins

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