Evidence Supporting a Role for TopBP1 and Brd4 in the Initiation but Not Continuation of Human Papillomavirus 16 E1/E2-Mediated DNA Replication

Gauson, Elaine J.; Donaldson, Mary; Dornan, Edward S.; Wang, Xu; Bristol, Molly L.; Bodily, Jason M.; Morgan, Iain M.

doi:10.1128/jvi.00335-15

Cited by 66 publications

(108 citation statements)

References 61 publications

Supporting

Mentioning

103

Contrasting

Order By: Relevance

“…The replication competence of the R37A/I73A mutant in RTS3b cells seen here differs from observations in C33a cells reported by Wang et al (36). However, a recent publication demonstrated that the failure of the HPV16 E2 R37A mutant to replicate the viral origin together with E1 in C33a cells can be overcome by greatly increasing the amount of expression vector (37). Thus, the replication phenotype of R37A/I73A may depend on E2 protein levels.…”

Section: Resultscontrasting

confidence: 56%

Characterization of the Human Papillomavirus 16 E8 Promoter

Straub¹,

Fertey²,

Dreer³

et al. 2015

J Virol

View full text Add to dashboard Cite

Persistent infections with certain human papillomaviruses (HPV) such as HPV16 are a necessary risk factor for the development of anogenital and oropharyngeal cancers. HPV16 genomes replicate as low-copy-number plasmids in the nucleus of undifferentiated keratinocytes, which requires the viral E1 and E2 replication proteins. The HPV16 E8^E2C (or E8^E2) protein limits genome replication by repressing both viral transcription and the E1/E2-dependent DNA replication. How E8^E2C expression is regulated is not understood. Previous transcript analyses indicated that the spliced E8^E2C RNA is initiated at a promoter located in the E1 region upstream of the E8 gene. Deletion and mutational analyses of the E8 promoter region identify two conserved elements that are required for basal promoter activity in HPV-negative keratinocytes. In contrast, the transcriptional enhancer in the upstream regulatory region of HPV16 does not modulate basal E8 promoter activity. Cotransfection studies indicate that E8^E2C inhibits, whereas E2 weakly activates, the E8 promoter. Interestingly, the cotransfection of E1 and E2 induces the E8 promoter much more strongly than the major early promoter, and this is partially dependent upon binding of E2 to Brd4. Mutation of E8 promoter elements in the context of HPV16 genomes results in an increased genome copy number and elevated levels of viral early and late transcripts. In summary, the promoter responsible for the expression of E8^E2C is both positively and negatively regulated by viral and cellular factors, and this regulatory circuit may be crucial to maintain a low but constant copy number of HPV16 genomes in undifferentiated cells. IMPORTANCEHPV16 replicates in differentiating epithelia and can cause cancer. How HPV16 maintains its genome in undifferentiated cells at a low but constant level is not well understood but may be relevant for the immunological escape of HPV16 in the basal layers of the infected epithelium. This study demonstrates that the expression of the viral E8^E2C protein, which is a potent inhibitor of viral replication in undifferentiated cells, is driven by a separate promoter. The E8 promoter is both positively and negatively regulated by viral proteins and thus most likely acts as a sensor and modulator of viral copy number.

show abstract

Section: Resultscontrasting

confidence: 56%

Characterization of the Human Papillomavirus 16 E8 Promoter

Straub¹,

Fertey²,

Dreer³

et al. 2015

J Virol

View full text Add to dashboard Cite

show abstract

“…Lanes 1 and 2 demonstrates that at 10ng and 1000ng of E2 wt expression plasmid respectively there is a 9 and 25 fold increase in transcription. The response to E2 wt expression plasmids is never linear, i.e., 100 times more plasmid never gives 100 times more activation, so these results are in line with those from other cell lines (Donaldson et al, 2012; Gauson et al, 2015). In lanes 3 and 4 it is clear that E2 −Brd4 is severely compromised in the ability to activate transcription, 10ng and 1000 ng give reporter levels of 0.75 and 1.56 respectively relative to no E2.…”

Section: Resultssupporting

confidence: 83%

“…There has also been a proposed role for the interaction between E2 and Brd4 regulating the DNA replication properties of E2 (Baxter and McBride, 2005; Gauson et al, 2015; Sakai et al, 1996; Wang et al, 2013). A recent report demonstrates co-localization of E2 with Brd4 on fragile sites of chromatin in C33a cells and proposed that this co-localization was involved in regulating E1-E2 mediated DNA replication in areas where viral genome breakage would promote viral integration, very often found in HPV related cancers (Jang et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

Failure to interact with Brd4 alters the ability of HPV16 E2 to regulate host genome expression and cellular movement

et al. 2016

Self Cite

View full text Add to dashboard Cite

The E2 protein of the carcinogen human papillomavirus 16 (HPV16) regulates replication and transcription of the viral genome in association with viral and cellular proteins. Our previous work demonstrated that E2 can regulate transcription from the host genome. E2 can activate transcription from adjacent promoters when located upstream using E2 DNA binding sequences and this activation is dependent upon the cellular protein Brd4; this report demonstrates that a Brd4 binding E2 mutant alters host genome expression differently from wild type E2. Of particular note is that highly down regulated genes are mostly not affected by failure to interact with Brd4 suggesting that the E2-Brd4 interaction is more responsible for the transcriptional activation of host genes rather than repression. Therefore failure to interact efficiently with Brd4, or altered levels of Brd4, would alter the ability of E2 to regulate the host genome and could contribute to determining the outcome of infection.

show abstract

“…To test this directly, we compared levels of SR proteins in normal foreskin keratinocytes (NFKs) stably transfected with wild-type HPV genomes or with genomes containing an inactivating point mutation in the transactivation domain of E2. HPV16 genomes containing such a mutant cannot be maintained episomally in keratinocytes (39). However, keratinocytes containing HPV31 genomes with this mutation are available (mutant E2: I73L) (30).…”

Section: Resultsmentioning

confidence: 99%

Human Papillomavirus E2 Regulates SRSF3 (SRp20) To Promote Capsid Protein Expression in Infected Differentiated Keratinocytes

Klymenko

Hernandez-Lopez

MacDonald

et al. 2016

J Virol

View full text Add to dashboard Cite

The human papillomavirus (HPV) life cycle is tightly linked to differentiation of the infected epithelial cell, suggesting a sophisticated interplay between host cell metabolism and virus replication. Previously, we demonstrated in differentiated keratinocytes in vitro and in vivo that HPV type 16 (HPV16) infection caused increased levels of the cellular SR splicing factors (SRSFs) SRSF1 (ASF/SF2), SRSF2 (SC35), and SRSF3 (SRp20). Moreover, the viral E2 transcription and replication factor that is expressed at high levels in differentiating keratinocytes could bind and control activity of the SRSF1 gene promoter. Here, we show that the E2 proteins of HPV16 and HPV31 control the expression of SRSFs 1, 2, and 3 in a differentiation-dependent manner. E2 has the greatest transactivation effect on expression of SRSF3. Small interfering RNA depletion experiments in two different models of the HPV16 life cycle (W12E and NIKS16) and one model of the HPV31 life cycle (CIN612-9E) revealed that only SRSF3 contributed significantly to regulation of late events in the virus life cycle. Increased levels of SRSF3 are required for L1 mRNA and capsid protein expression. Capsid protein expression was regulated specifically by SRSF3 and appeared independent of other SRSFs. Taken together, these data suggest a significant role of the HPV E2 protein in regulating late events in the HPV life cycle through transcriptional regulation of SRSF3 expression. IMPORTANCEHuman papillomavirus replication is accomplished in concert with differentiation of the infected epithelium. Virus capsid protein expression is confined to the upper epithelial layers so as to avoid immune detection. In this study, we demonstrate that the viral E2 transcription factor activates the promoter of the cellular SRSF3 RNA processing factor. SRSF3 is required for expression of the E4^L1 mRNA and so controls expression of the HPV L1 capsid protein. Thus, we reveal a new dimension of virus-host interaction crucial for production of infectious virus. SRSF proteins are known drug targets. Therefore, this study provides an excellent basis for developing strategies to regulate capsid protein production in the infected epithelium and the production of new virions.

show abstract

Evidence Supporting a Role for TopBP1 and Brd4 in the Initiation but Not Continuation of Human Papillomavirus 16 E1/E2-Mediated DNA Replication

Cited by 66 publications

References 61 publications

Characterization of the Human Papillomavirus 16 E8 Promoter

Characterization of the Human Papillomavirus 16 E8 Promoter

Failure to interact with Brd4 alters the ability of HPV16 E2 to regulate host genome expression and cellular movement

Human Papillomavirus E2 Regulates SRSF3 (SRp20) To Promote Capsid Protein Expression in Infected Differentiated Keratinocytes

Contact Info

Product

Resources

About