The Viral E8^E2C Repressor Limits Productive Replication of Human Papillomavirus 16

Abstract

Productive replication of human papillomavirus type 16 (HPV16) occurs only in differentiated keratinocyte cells. In addition to the viral E2 activator protein, HPV16 and related HPV types express transcripts coding for an E8^E2C fusion protein, which limits genome replication in undifferentiated keratinocytes. To address E8^E2C's role in productive replication of HPV16, stable keratinocyte cell lines containing wild-type (wt), E8^E2C knockout (E8؊), or E8 KWK mutant (mt) genomes, in which conserved E8 residues were inactivated, were established. Copy numbers of E8؊ and E8 KWK mt genomes and amounts of early and late viral transcripts were greatly increased compared to those for the wt in undifferentiated keratinocytes, suggesting that HPV16 E8^E2C activities are highly dependent upon the E8 part. Upon differentiation in organotypic cultures, E8 mt genomes displayed higher early viral transcript levels, but no changes in cellular differentiation or virus-induced cellular DNA replication in suprabasal cells were observed. E8 mt genomes were amplified to higher copy numbers and showed increased L1 transcripts compared to wt genomes. Furthermore, the number of cells expressing the viral late protein E4 or L1 or amplifying viral genomes was greatly increased in E8 mt cell lines. In wild-type cells, E8^E2C transcript levels did not decrease by differentiation. Our data indicate that the E8^E2C repressor limits viral transcription and replication throughout the complete life cycle of HPV16. P ersistent infections with high-risk (HR) human papillomaviruses (HPV) are a necessary risk factor for the development of cancer of the cervix uteri and are also responsible for a fraction of anal, vaginal, penile, and oropharyngeal cancers (1-3). Notably, HPV16 is an extremely powerful human carcinogen that is responsible for approximately 50% of cervical cancers and is also represented in other HR-HPV-related cancers at high frequencies (2).HPV have a double-stranded DNA genome that is covalently closed and organized in nucleosomes. HPV possess eight to nine open reading frames (E1, E2, E4 to E8, L1, and L2) that are transcribed into polycistronic messages which are further processed by alternative splicing (4). It is currently assumed that HR-HPV encode at least 10 different viral proteins.The HR-HPV replication cycle is tightly linked to the differentiation state of the infected epithelium. In undifferentiated basalcell-like cells, HR-HPV genomes are maintained in the nucleus at 10 to 100 extrachromosomal copies per cell, and only early genes, mainly transcribed from the major early viral promoter located immediately upstream of the E6 gene (P97 for HPV16), but not the late viral L1 and L2 genes are expressed (5). Replication of the viral genome in undifferentiated cells requires the viral E1 and E2 proteins (6). E1 and E2 are sequence-specific DNA binding proteins which form a complex that recognizes the viral origin of replication (6). E1 assembles into a hexamer that acts as the replicative helicase and unwinds the viral DNA...

Interferon Kappa Inhibits Human Papillomavirus 31 Transcription by Inducing Sp100 Proteins

“…We also investigated the effects of IFN-␤ on HPV16 transcription. Three different stable human keratinocyte cell lines that maintain extrachromosomal genomes (24,25) were treated with IFN-␤ (1,000 U/ml) for 6 h, and then E2 and E7 transcripts were analyzed by qPCR. Depending on the cell line, HPV16 transcripts were reduced to 60 to 80% (Fig.…”

“…HPV16 wild-type-positive cell lines have been previously described (24,25). The LKP1 and the CIN1 lesion-derived CIN612-9E cell lines contain replicating HPV31 genomes and have been described before (26,27).…”

Characterization of the Human Papillomavirus 16 E8 Promoter

“…The major HR-HPV, HPV16, -18, and -31, express a spliced mRNA that links the E8 gene to the E2 gene and has the capability to encode an E8^E2C (or E8/E2 or E8^E2) protein (7)(8)(9)(10). Genetic analyses have revealed that E8^E2C inhibits genome replication of HR-HPV16, -18, and -31 in undifferentiated cells, and there is also evidence that E8^E2C limits productive replication of HPV16 in differentiated cells (8)(9)(10)(11). The HPV16, -18, and -31 E8^E2C proteins bind to the viral origin of replication and are potent repressors of transcription and the E1/ E2-dependent replication (7,9,(11)(12)(13)(14).…”

“…Genetic analyses have revealed that E8^E2C inhibits genome replication of HR-HPV16, -18, and -31 in undifferentiated cells, and there is also evidence that E8^E2C limits productive replication of HPV16 in differentiated cells (8)(9)(10)(11). The HPV16, -18, and -31 E8^E2C proteins bind to the viral origin of replication and are potent repressors of transcription and the E1/ E2-dependent replication (7,9,(11)(12)(13)(14). Both activities require the conserved E8 part, which recruits cellular corepressor molecules (14)(15)(16).…”

“…To generate plasmid pGL16 7154/ 1268, the XhoI/NcoI fragment from pGL16 7154/868 was inserted into XhoI/NcoI-digested pGL16 863/1268. Expression plasmids pSG16 E2, E8^E2C, and E8^E2C KWK mt are based upon pSG5 (Stratagene) and have been previously described (7,11). Plasmid pSG16 E2 R37A/I73A was constructed by exchanging an in vitro-synthesized EcoRI/MscI fragment of a mutated version of HPV16 E2 (Life Technologies).…”