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. 2017 Jan 3;91(2):e01854-16.
doi: 10.1128/JVI.01854-16. Print 2017 Jan 15.

Phosphorylation of the Bovine Papillomavirus E2 Protein on Tyrosine Regulates Its Transcription and Replication Functions

Sara P Culleton  1 , Sriramana Kanginakudru  2 , Marsha DeSmet  2 , Timra Gilson  2 , Fang Xie  2   3 , Shwu-Yuan Wu  4 , Cheng-Ming Chiang  4   5 , Guihong Qi  6 , Mu Wang  6 , Elliot J Androphy  7   2
Affiliations

Phosphorylation of the Bovine Papillomavirus E2 Protein on Tyrosine Regulates Its Transcription and Replication Functions

Sara P Culleton et al. J Virol. .

Abstract

Papillomaviruses are small, double-stranded DNA viruses that encode the E2 protein, which controls transcription, replication, and genome maintenance in infected cells. Posttranslational modifications (PTMs) affecting E2 function and stability have been demonstrated for multiple types of papillomaviruses. Here we describe the first phosphorylation event involving a conserved tyrosine (Y) in the bovine papillomavirus 1 (BPV-1) E2 protein at amino acid 102. While its phosphodeficient phenylalanine (F) mutant activated both transcription and replication in luciferase reporter assays, a mutant that may act as a phosphomimetic, with a Y102-to-glutamate (E) mutation, lost both activities. The E2 Y102F protein interacted with cellular E2-binding factors and the viral helicase E1; however, in contrast, the Y102E mutant associated with only a subset and was unable to bind to E1. While the Y102F mutant fully supported transient viral DNA replication, BPV genomes encoding this mutation as well as Y102E were not maintained as stable episomes in murine C127 cells. These data imply that phosphorylation at Y102 disrupts the helical fold of the N-terminal region of E2 and its interaction with key cellular and viral proteins. We hypothesize that the resulting inhibition of viral transcription and replication in basal epithelial cells prevents the development of a lytic infection.

Importance: Papillomaviruses (PVs) are small, double-stranded DNA viruses that are responsible for cervical, oropharyngeal, and various genitourinary cancers. Although vaccines against the major oncogenic human PVs are available, there is no effective treatment for existing infections. One approach to better understand the viral replicative cycle, and potential therapies to target it, is to examine the posttranslational modification of viral proteins and its effect on function. Here we have discovered that the bovine papillomavirus 1 (BPV-1) transcription and replication regulator E2 is phosphorylated at residue Y102. While a phosphodeficient mutant at this site was fully functional, a phosphomimetic mutant displayed impaired transcription and replication activity as well as a lack of an association with certain E2-binding proteins. This study highlights the influence of posttranslational modifications on viral protein function and provides additional insight into the complex interplay between papillomaviruses and their hosts.

Keywords: papillomavirus; papillomavirus E2; tyrosine phosphorylation; viral replication.

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Figures

FIG 1
FIG 1
Location of Y102 in the TAD and mutagenesis. (A) A ribbon diagram of the BPV-1 E2 TAD crystal structure reported under PDB accession number 2JEU (60) (dark blue) overlapping the HPV-16 E2 TAD/Brd4 cocrystal structure reported under PDB accession number 2NNU (38) (light blue and purple, respectively) was created by using the UCSF Chimera package. The Y102 side chain, depicted in a green ball-and-stick model, is hydrogen bonded to E74, depicted in a wire frame model. N, amino terminus; C, carboxyl terminus. (B) Site-directed mutagenesis converts Y102 to phenylalanine (F) or glutamate (E) in pCG-E2. One microgram of WT and mutant constructs (YF and YE), or mRFP-GFP as negative control (−), was expressed in HEK293TT cells. The immunoblot was probed for BPV-1 E2 with mouse monoclonal antibody B201. E2, full-length BPV-1 E2; *, nonspecific band.
FIG 2
FIG 2
Y102E abrogates E2 transcriptional activation. (A) C33a cells were transfected in triplicate with 50 ng/well of the pCG-E2 wild type or mutants (Y102F and Y102E) or mRFP-GFP (−) as well as 75 ng/well pGL2-E2BS-Luc. Cells were lysed on-plate for luminescence detection using the PHERAstar system. RLU, relative light units; **, P < 0.01; NS, not significant. Values are expressed as means ± SEM. (B) One microgram each of the WT and mutant constructs (YF and YE) or mRFP-GFP (−) was expressed in C33a cells and probed for BPV-1 E2 with B201 antibodies. E2, full-length BPV-1 E2; *, nonspecific band. (C) CV-1 cells were transfected with 10 ng/well E2, pCG-E2R (E2R [aa 162 to 410]), or mRFP-GFP plus wells with 10 times the amount of Y102E (100 ng/well) (10×YE). **, P < 0.01. Values are expressed as means ± SEM. (D) CV-1 cells were transfected and prepared for Western blotting with the inclusion of samples with 5 times the amount of Y102E (5×YE), 10 times the amount of Y102E (10×YE), or E2R. The blot was probed for BPV-1 E2 with B201. E2, full-length BPV-1 E2; *, nonspecific band.
FIG 3
FIG 3
Y102E does not bind the Brd4 CTM but associates with an internal E2-binding region. (A) Domains of the Brd4 chromatin modulator protein (39). BD1, bromodomain 1; BID, basic interacting domain; ET, extraterminal domain. (B) HEK293TT cells were transfected with mRFP-GFP (−), the BPV-1 E2 WT and mutants (YF and YE), and FLAG-tagged full-length Brd4 (pVL-F:hBrd4) or pCI. Immunoprecipitates were collected with anti-FLAG mouse antibody M2 conjugated to agarose beads. Samples were probed by Western blotting for FLAG-Brd4 and BPV-1 E2 using M2 and B201, respectively. E2, full-length BPV-1 E2; *, nonspecific band. (C and D) GST-tagged CTM (C) and GST-BID (D). Complexes were collected with glutathione beads. Western blots were probed for GST with rabbit polyclonal antibody SD8 and for E2 with B201. *, nonspecific band.
FIG 4
FIG 4
Y102 mutants associate with Gps2 and Tax1BP1. (A) HEK293TT cells were transfected with 1 μg each mRFP-GFP (−), WT BPV-1 E2 and mutants (YF and YE) plus E2R, and hemagglutinin-tagged Gps2 (HA-Gps2) (pHA:AMF1) or pCI. Western blots were probed for HA-Gps2 and BPV-1 E2 by using mouse monoclonal anti-HA antibodies HA-7 and B201, respectively. (B) Samples were prepared as described above for panel A by using p3XFLAG-CMV-7.1 Tax1BP1. Western blots were probed with FLAG-M2 and B201.
FIG 5
FIG 5
Y102E fails to stimulate transient BPV-1 replication. (A) C33a cells transfected in 8 replicate wells with 10 ng/well of the pCG-E2 WT or mutants (Y102F and Y102E), E2R, or mRFP-GFP (−) as well as 10 ng/well pCG-E1, 2.5 ng/well pFLORI-BPV1 (firefly luciferase reporter), and 0.5 ng/well pRL (Renilla luciferase reporter). Lysates were processed for luminescence. F/R, firefly output (in relative light units) divided by the Renilla output; **, P < 0.01; ****, P < 0.00001. Values are expressed as means ± SEM. (B) Replication assay completed as described above for panel A but with firefly and renilla luciferase levels being graphed separately. (C) Replication assay as described above for panel A. Each group had E1 transfected with either WT E2 or increasing amounts of Y102E. Fluc, firefly luciferase; Rluc, renilla luciferase (D) HEK293TT cells were transfected with 3 μg each mRFP-GFP (−), the BPV-1 E2 WT and mutants (YF and YE), and BPV-1 E1 or pCI. Lysates were immunoprecipitated with Sepharose A beads and BPV E2 antibody II-I. Western blots were probed for E1 and E2 by using rabbit anti-E1 peptide antibody 502-2 and antibody B201, respectively. *, nonspecific band.
FIG 6
FIG 6
Y102 mutants localize to nuclei. CV-1 and A3 (positive control for E2 expression [+]) cells were cultured on 18-mm glass coverslips. CV-1 cells were transfected with 100 ng pCG-E1 and 100 ng pEGFP-C1 (eGFP [−]), WT E2, the Y102F or Y102E mutant, or E2R. Forty-eight hours after CV-1 cell transfection, cells were fixed and permeabilized, followed by incubation with B201. Cells were reacted with Alexa Fluor 594 anti-mouse antibody, washed, and mounted onto glass slides by using ProLong Gold with DAPI. Green, eGFP; red, BPV-1 E2; blue, DNA. Bar, 10 μm. All images were taken in all three channels (green, red, and blue) at a ×1,000 magnification.
FIG 7
FIG 7
C127 cell lines do not maintain Y102F and Y102E episomes. (A) Total DNA from transformed C127 cells containing Y102E, Y102F, or wild-type BPV genomes was subjected to Southern blotting using a BPV LCR-specific probe. The number below each genome represents an individual clonal cell line. (B) A 677-bp PCR amplicon of the LCR region of the same cell lines represented in panel A demonstrates that each clonal population contains BPV DNA.
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References

    1. Kocjan BJ, Bzhalava D, Forslund O, Dillner J, Poljak M. 21 May 2015. Molecular methods for identification and characterization of novel papillomaviruses. Clin Microbiol Infect doi:10.1016/j.cmi.2015.05.011. - DOI - PubMed
    1. Thomas M, Boiron M, Tanzer J, Levy JP, Bernard J. 1964. In vitro transformation of mice cells by bovine papilloma virus. Nature 202:709–710. doi:10.1038/202709a0. - DOI - PubMed
    1. Law MF, Lowy DR, Dvoretzky I, Howley PM. 1981. Mouse cells transformed by bovine papillomavirus contain only extrachromosomal viral DNA sequences. Proc Natl Acad Sci U S A 78:2727–2731. doi:10.1073/pnas.78.5.2727. - DOI - PMC - PubMed
    1. Chesters PM, McCance DJ. 1985. Human papillomavirus type 16 recombinant DNA is maintained as an autonomously replicating episome in monkey kidney cells. J Gen Virol 66(Part 3):615–620. - PubMed
    1. Botchan M, Berg L, Reynolds J, Lusky M. 1986. The bovine papillomavirus replicon. Ciba Found Symp 120:53–67. - PubMed

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