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Brd4 Activates Early Viral Transcription upon Human Papillomavirus 18 Infection of Primary Keratinocytes

View Article: PubMed Central - PubMed

ABSTRACT

Human papillomaviruses (HPVs) replicate in the cutaneous and mucosal epithelia, and the infectious cycle is synchronous with the differentiation program of the host keratinocytes. The virus initially infects dividing cells in the lower layers of the epithelium, where it establishes a persistent infection. The viral genome is maintained as a low-copy-number, extrachromosomal element in these proliferating cells but switches to the late stage of the life cycle in differentiated cells. The cellular chromatin adaptor protein Brd4 is involved in several stages and processes of the viral life cycle. In concert with the viral transcriptional regulator E2, Brd4 can repress transcription from the early viral promoter. Brd4 and E2 form a complex with the viral genome that associates with host chromosomes to partition the viral genome in dividing cells; Brd4 also localizes to active sites of productive HPV DNA replication. However, because of the difficulties in producing HPV viral particles, the role of Brd4 in modulating viral transcription and replication at the initial stage of infection is unclear. In this study, we have used an HPV18 quasivirus-based genome delivery system to assess the role of Brd4 in the initial infectivity of primary human keratinocytes. We show that, upon infection of primary human keratinocytes with HPV18 quasivirus, Brd4 activates viral transcription and replication. Furthermore, this activation is independent of the functional interaction between Brd4 and the HPV18 E2 protein.

No MeSH data available.


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Expression and Brd4 binding of E2 mutants. (A) Western blot analysis of protein extracts from C33A cell lines stably transfected with pMEP4 (no E2), wild-type pMEP-E2 (WT), or mutated pMEP-E2 (R41A, I77A, and R41A/I77A). E2 proteins were tagged with a FLAG epitope and detected with FLAG-M2 monoclonal antisera. α-Tubulin is provided as a control for equal cell number. (B) E2 expression was corrected to tubulin expression and normalized to WT. Raw band volume was quantified by Gene Tools software (Syngene). n = 3. Error bars represent standard errors of the means. (C) 35S-labeled in vitro-translated E2 proteins were mixed with in vitro-translated Brd4 and immunoprecipitated with the Brd4-specific antibody 2290. Immune complexes were eluted and resolved by SDS-PAGE. Shown is a representative audioradiograph. (D) E2 bands were detected by using a Typhoon phosphorimager and quantified with Gene Tools software (Syngene). Brd4-bound E2 was corrected for background binding to rabbit IgG and normalized to WT. n = 3. A paired t test was used for statistical analysis (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).
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fig5: Expression and Brd4 binding of E2 mutants. (A) Western blot analysis of protein extracts from C33A cell lines stably transfected with pMEP4 (no E2), wild-type pMEP-E2 (WT), or mutated pMEP-E2 (R41A, I77A, and R41A/I77A). E2 proteins were tagged with a FLAG epitope and detected with FLAG-M2 monoclonal antisera. α-Tubulin is provided as a control for equal cell number. (B) E2 expression was corrected to tubulin expression and normalized to WT. Raw band volume was quantified by Gene Tools software (Syngene). n = 3. Error bars represent standard errors of the means. (C) 35S-labeled in vitro-translated E2 proteins were mixed with in vitro-translated Brd4 and immunoprecipitated with the Brd4-specific antibody 2290. Immune complexes were eluted and resolved by SDS-PAGE. Shown is a representative audioradiograph. (D) E2 bands were detected by using a Typhoon phosphorimager and quantified with Gene Tools software (Syngene). Brd4-bound E2 was corrected for background binding to rabbit IgG and normalized to WT. n = 3. A paired t test was used for statistical analysis (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Mentions: Brd4 is important for viral transcription, and this is thought to be mediated primarily through interaction with the E2 protein. This interaction has been well defined, and the contact between the C-terminal tail of Brd4 and the transactivation domain of E2 has been characterized both structurally and functionally (reviewed in reference 12). Two residues on one face of the transactivation domain of E2, R37 and I73, make direct contact with the Brd4 protein (26). Replacement of these residues with alanines (R37A and I73A) abrogates the transcriptional activation or repressive function of E2 but leaves replication intact (these residues are on the opposite face of the transactivation domain from the E1-interacting region). The R37 and I73 residues are highly conserved among all papillomaviruses, and replacement of these residues severely impairs the association of E2 with Brd4 in all E2 proteins tested (reviewed in reference 12). Therefore, equivalent alanine substitutions were generated in HPV18 E2 (R41A and I77A). To ensure that the mutated proteins were stable in vivo, C-33A cells expressing FLAG-tagged versions of the E2 proteins were established (Fig. 5A and B). Western blot analysis showed that each protein was expressed at least as well as the wild-type E2 protein, with levels of the I77A and double-mutated E2 proteins being slightly elevated compared to the wild type (WT). To ensure that the mutations abrogated the E2-Brd4 interaction, the mutated E2 proteins were tested for Brd4 binding (Fig. 5C and D). As shown, alanine substitution of either R41 or I77 was sufficient to abrogate almost all binding of E2 to Brd4. Any residual binding was completely eliminated by the double amino acid substitution.


Brd4 Activates Early Viral Transcription upon Human Papillomavirus 18 Infection of Primary Keratinocytes
Expression and Brd4 binding of E2 mutants. (A) Western blot analysis of protein extracts from C33A cell lines stably transfected with pMEP4 (no E2), wild-type pMEP-E2 (WT), or mutated pMEP-E2 (R41A, I77A, and R41A/I77A). E2 proteins were tagged with a FLAG epitope and detected with FLAG-M2 monoclonal antisera. α-Tubulin is provided as a control for equal cell number. (B) E2 expression was corrected to tubulin expression and normalized to WT. Raw band volume was quantified by Gene Tools software (Syngene). n = 3. Error bars represent standard errors of the means. (C) 35S-labeled in vitro-translated E2 proteins were mixed with in vitro-translated Brd4 and immunoprecipitated with the Brd4-specific antibody 2290. Immune complexes were eluted and resolved by SDS-PAGE. Shown is a representative audioradiograph. (D) E2 bands were detected by using a Typhoon phosphorimager and quantified with Gene Tools software (Syngene). Brd4-bound E2 was corrected for background binding to rabbit IgG and normalized to WT. n = 3. A paired t test was used for statistical analysis (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).
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Related In: Results  -  Collection

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fig5: Expression and Brd4 binding of E2 mutants. (A) Western blot analysis of protein extracts from C33A cell lines stably transfected with pMEP4 (no E2), wild-type pMEP-E2 (WT), or mutated pMEP-E2 (R41A, I77A, and R41A/I77A). E2 proteins were tagged with a FLAG epitope and detected with FLAG-M2 monoclonal antisera. α-Tubulin is provided as a control for equal cell number. (B) E2 expression was corrected to tubulin expression and normalized to WT. Raw band volume was quantified by Gene Tools software (Syngene). n = 3. Error bars represent standard errors of the means. (C) 35S-labeled in vitro-translated E2 proteins were mixed with in vitro-translated Brd4 and immunoprecipitated with the Brd4-specific antibody 2290. Immune complexes were eluted and resolved by SDS-PAGE. Shown is a representative audioradiograph. (D) E2 bands were detected by using a Typhoon phosphorimager and quantified with Gene Tools software (Syngene). Brd4-bound E2 was corrected for background binding to rabbit IgG and normalized to WT. n = 3. A paired t test was used for statistical analysis (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).
Mentions: Brd4 is important for viral transcription, and this is thought to be mediated primarily through interaction with the E2 protein. This interaction has been well defined, and the contact between the C-terminal tail of Brd4 and the transactivation domain of E2 has been characterized both structurally and functionally (reviewed in reference 12). Two residues on one face of the transactivation domain of E2, R37 and I73, make direct contact with the Brd4 protein (26). Replacement of these residues with alanines (R37A and I73A) abrogates the transcriptional activation or repressive function of E2 but leaves replication intact (these residues are on the opposite face of the transactivation domain from the E1-interacting region). The R37 and I73 residues are highly conserved among all papillomaviruses, and replacement of these residues severely impairs the association of E2 with Brd4 in all E2 proteins tested (reviewed in reference 12). Therefore, equivalent alanine substitutions were generated in HPV18 E2 (R41A and I77A). To ensure that the mutated proteins were stable in vivo, C-33A cells expressing FLAG-tagged versions of the E2 proteins were established (Fig. 5A and B). Western blot analysis showed that each protein was expressed at least as well as the wild-type E2 protein, with levels of the I77A and double-mutated E2 proteins being slightly elevated compared to the wild type (WT). To ensure that the mutations abrogated the E2-Brd4 interaction, the mutated E2 proteins were tested for Brd4 binding (Fig. 5C and D). As shown, alanine substitution of either R41 or I77 was sufficient to abrogate almost all binding of E2 to Brd4. Any residual binding was completely eliminated by the double amino acid substitution.

View Article: PubMed Central - PubMed

ABSTRACT

Human papillomaviruses (HPVs) replicate in the cutaneous and mucosal epithelia, and the infectious cycle is synchronous with the differentiation program of the host keratinocytes. The virus initially infects dividing cells in the lower layers of the epithelium, where it establishes a persistent infection. The viral genome is maintained as a low-copy-number, extrachromosomal element in these proliferating cells but switches to the late stage of the life cycle in differentiated cells. The cellular chromatin adaptor protein Brd4 is involved in several stages and processes of the viral life cycle. In concert with the viral transcriptional regulator E2, Brd4 can repress transcription from the early viral promoter. Brd4 and E2 form a complex with the viral genome that associates with host chromosomes to partition the viral genome in dividing cells; Brd4 also localizes to active sites of productive HPV DNA replication. However, because of the difficulties in producing HPV viral particles, the role of Brd4 in modulating viral transcription and replication at the initial stage of infection is unclear. In this study, we have used an HPV18 quasivirus-based genome delivery system to assess the role of Brd4 in the initial infectivity of primary human keratinocytes. We show that, upon infection of primary human keratinocytes with HPV18 quasivirus, Brd4 activates viral transcription and replication. Furthermore, this activation is independent of the functional interaction between Brd4 and the HPV18 E2 protein.

No MeSH data available.


Related in: MedlinePlus