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

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.

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

“…HPV-31 E2 Y138E does not bind the C-terminal domain of Brd4. The E2 binding partner Brd4 has been previously shown to regulate several phases of the viral life cycle through binding of its C-terminal domain (CTD; aa 1222 to 1362) to the E2 transactivation domain (18,(23)(24)(25)(26)(27)(28). The E2 DNA binding domain has also been reported to bind a basic residue-enriched interaction domain (BID) within amino acids 524 to 579 of Brd4 and to bind to the phosphorylation-dependent interaction domain (PDID) formed by amino acids 287 to 530 (18).…”

The Promise of Proteomics in the Study of Oncogenic Viruses

“…differentiation). Quasivirus particles (recircularized viral genomes packaged in a cell line overexpressing the capsid proteins) can be used to generate papillomavirus and polyomavirus particles to study the early stages of infection, and in theory epitope tagged versions of viral proteins could be packaged in these recombinant particles to facilitate their detection and localization (33,34). More efficient methods are also being established to induce the late stages of infection.…”

Optimization of human papillomavirus-based pseudovirus techniques for efficient gene transfer

“…The 12 kb plasmid size enables copious production of viral L1/L2 proteins but cannot be encapsidated itself due to its size. The two most common cell lines utilized for viral production are HEK293 TT [9][10][11][12][13][14][15] and HEK293 TTF cell lines 16 . Both HEK293 TT and HEK293 TTF cell lines express large and small SV-40 T antigen to drive robust plasmid expression, but HEK293 TTF cells also overexpress the furin protease needed for viral capsid maturation 17 .…”