Brd4 Is Displaced from HPV Replication Factories as They Expand and Amplify Viral DNA

2013-11-21 14:18:13

PLOS Pathogens; 2013 Nov 21; DOI: 10.1371/journal.ppat.1003777

Nozomi Sakakibara, Dan Chen, Moon Kyoo Jang, Dong Wook Kang, Hans F. Luecke, Shwu-Yuan Wu,
Cheng-Ming Chiang, Alison A. McBride


Replication foci are generated by many viruses to concentrate and localize viral DNA synthesis to specific regions of the cell. Expression of the HPV16 E1 and E2 replication proteins in keratinocytes results in nuclear foci that recruit proteins associated with the host DNA damage response. We show that the Brd4 protein localizes to these foci and is essential for their formation. However, when E1 and E2 begin amplifying viral DNA, Brd4 is displaced from the foci and cellular factors associated with DNA synthesis and homologous recombination are recruited. Differentiated HPV-infected keratinocytes form similar nuclear foci that contain amplifying viral DNA. We compare the different foci and show that, while they have many characteristics in common, there is a switch between early Brd4-dependent foci and mature Brd4-independent replication foci. However, HPV genomes encoding mutated E2 proteins that are unable to bind Brd4 can replicate and amplify the viral genome. We propose that, while E1, E2 and Brd4 might bind host chromatin at early stages of infection, there is a temporal and functional switch at later stages and increased E1 and E2 levels promote viral DNA amplification, displacement of Brd4 and growth of a replication factory. The concomitant DNA damage response recruits proteins required for DNA synthesis and repair, which could then be utilized for viral DNA replication. Hence, while Brd4 can enhance replication by concentrating viral processes in specific regions of the host nucleus, this interaction is not absolutely essential for HPV replication.

Author Summary

Papillomaviruses have a remarkable infection cycle that depends on the development of a stratified epithelium. The virus infects the lower, dividing layers of the epithelium and viral genomes replicate at low copy number, and are maintained in these cells, for long periods of time. As infected cells differentiate and move to the surface of the epithelium, they switch on high level viral DNA replication, synthesize capsid proteins and form new viral particles. Viral replication takes place in nuclear foci and is dependent on the E1 and E2 replication proteins. Brd4 is a cellular chromatin binding protein that interacts with E2 and is important for transcriptional regulation of papillomaviruses. In this study we examine the role of Brd4 at different stages in the formation of viral replication foci. In the absence of viral DNA replication, Brd4 links the viral proteins to host chromatin. However, when viral genomes begin to amplify to high levels, Brd4 is displaced from nuclear foci and is not required for replication.


Papillomaviruses have a complex life cycle in which viral DNA replication is tightly linked to host cell differentiation. Papillomaviruses initially infect the basal cells of the host epithelium and establish a long-term persistent infection in which the viral genome is maintained as an extrachromosomal replicon in the dividing cells. As the infected cells differentiate, replication switches to a vegetative mode to produce large numbers of progeny genomes. There are thought to be three different modes of replication in this life cycle. The first is the initial burst of amplificational replication that occurs when a virion genome is delivered to the dividing host cell and becomes established as a low copy number extrachromosomal plasmid. The second phase is when these established genomes are replicated and partitioned along with host cellular DNA in proliferating cells. For many papillomaviruses, the E2 replication protein maintains and partitions the genomes by tethering them to mitotic chromosomes, often in complex with the chromatin binding protein, Brd4. Finally, the third phase occurs when persistently infected cells differentiate and the cells begin to express high levels of the E1 and E2 replication proteins and large numbers of progeny viral genomes are synthesized.

We, and others, have previously shown that expression of HPV E1 and E2 proteins results in the formation of nuclear foci that recruit DNA damage response proteins and show evidence of DNA synthesis. The replication process and/or replication proteins of many viruses activate a DNA damage response, but many viruses exploit this response to enhance their own replication. We have proposed that the HPV E1/E2 foci represent viral replication foci and have hypothesized that HPV replication induces a cellular DNA damage response, but the virus takes advantage of this to recruit components required for DNA synthesis to specific nuclear foci in differentiated cells. Studies using keratinocytes naturally infected with HPV31 demonstrate that the ATM DNA damage response enhances differentiation dependent amplification of the viral genome and this occurs in replication centers that recruit DNA damage markers as well as markers of DNA repair.

In our previous study we defined the functions of the E1 and E2 proteins that are required for the formation of nuclear foci and induction of the DNA damage response. The origin specific binding and ATPase functions of the E1 protein were necessary for induction of the ATM/ATR pathways as well as for the formation of foci. Therefore, the foci are not simply due to interaction and colocalization of the E1 and E2 proteins in the nucleus, since they require specific activities of the replication initiator helicase. The E2 protein was not required for the induction of the DNA damage response, but it was necessary for the formation of the nuclear foci. As expected, a mutation that disrupts the interaction of the E1 and E2 proteins abrogated the nuclear E1–E2 foci, while the DNA binding function of E2 was not required. However, a mutation (R37A/I73A) that specifically inactivated the transcriptional regulatory function of E2 also abrogated the formation of nuclear E1–E2 foci. These residues are crucial for interaction of the E2 protein with the host Brd4 protein and this led us to examine the role of Brd4 in the formation of papillomavirus replication foci, as well as in the processes of papillomavirus DNA replication.

In this study we examine the relationship and requirement of Brd4 within viral replication foci. We find that Brd4 is required for the formation of small early foci formed by the E1 and E2 proteins. However, when the genome begins to amplify in the replication foci there is a temporal switch of E2 function and Brd4 is not essential for the replication function of E2 and is not required for the amplification of these foci.

All papillomavirus E2 proteins bind to the cellular Brd4 protein to regulate viral transcription, but only the E2 proteins from a subset of viruses bind Brd4 with high affinity and stabilize the interaction of Brd4 with chromatin. These E2 proteins also readily bind to mitotic chromosomes and this is thought to be important for viral genome tethering and partitioning. E2 proteins from the alpha genus of papillomaviruses (a group of viruses that infect primarily the mucosal epithelium and contain the oncogenic HPVs) do not bind tightly to host chromatin and are not easily detected on mitotic chromosomes. In this study we also show that in the presence of the homologous E1 protein, the oncogenic, alpha-HPV16 E2 protein binds tightly to regions of host chromatin enriched for Brd4.

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