The E8∧E2 Gene Product of Human Papillomavirus Type 16 Represses Early Transcription and Replication but Is Dispensable for Viral Plasmid Persistence in Keratinocytes

High-risk (HR) oncogenic mucosal human papillomavirus (HPV) types are the major cause of most carcinomas of the uterine cervix, as well as many other anogenital tumors, and are found in 20 to 30% of cancers of the head and neck (HNC) (36). While many HR HPVs share genomic organization and conserved sequence homologies, they vary significantly in their prevalences in vivo. HPV-16 is the most prevalent HR HPV, as it is present in nearly 50% of cervical and anogenital carcinomas and in more than 90% of HPV-associated HNC (15, 46). In contrast, the sequence-related HPV-31 is significantly less prevalent than HPV-16 in cervical carcinomas and is rarely detected in HNC. Mechanisms determining HPV type-specific variations in viral persistence and malignant progression, however, are poorly understood. Papillomaviruses (PVs) replicate as extrachromosomal double-stranded DNA plasmids after infection of the basal keratinocyte host, stably persisting in low copy numbers (10, 19, 25). Early PV gene expression and plasmid amplification in the initial stages of HPV infection appear to be tightly regulated (18, 21, 30) through both transcriptional and posttranscriptional mechanisms (reviewed in references 43 and 60). Both the E1 and E2 proteins are required for PV replication (6, 14, 18), and as shown for HPV-31, transcripts encoding these factors from the plasmid genome are detected early after infection, followed by the production of mRNA encoding other early viral gene products (34). This temporal, regulated expression of limiting levels of these transcription and replication modulators early in HPV infection suggests that these viral gene products are critical to the establishment phase of the viral life cycle. The precise mechanisms that limit HPV early gene expression or initial plasmid amplification and modulate the establishment of a stable viral copy number, however, have not been completely defined. The full-length E2 and spliced E8∧E2 isoforms are conserved in PVs, with E8∧E2 transcripts identified in bovine papillomavirus type 1 (BPV-1) (26, 27), HPV-11 (40), cottontail rabbit papillomavirus (CRPV) (23), HPV-31 (52), and HPV-16 (11, 45). As in BPV (1, 13, 16, 58), E2 gene products expressed from a variety of HPVs modulate early gene transcription (5, 47, 48, 53, 56) and initial plasmid amplification (4, 21, 29, 35, 37) by distinct E2 structural domains, as shown in HPV-16 (41) and HPV-31 (51). The HPV E2 protein isoforms exert their transcriptional and replication effects by interacting with defined cis binding sites which are also conserved in mucosal and cutaneous HPVs (42) as well as in animal PVs. E8∧E2 interferes with E2-dependent transcriptional activation by the full-length E2 proteins via competitive binding to conserved E2 binding sites in BPV-1 (T. Haugen, unpublished data) and HPV-31 (54). Similarly, HPV E8∧E2 products can also inhibit plasmid replication (2, 61). This study uses a newly developed complementation assay for HPV-16 replication to define the structure of the HPV-16 E8∧E2 (16-E8∧E2) cistron, its regulation by cellular and viral trans-acting factors, and the role of the 16-E8∧E2 gene product in modulating critical early events in HPV-16 infection. These results demonstrate that 16-E8∧E2 inhibits initial HPV-16 plasmid amplification primarily by repressing E1 replicase expression. Furthermore, in contrast to E8∧E2 function in HPV-31, 16-E8∧E2 is not required for plasmid persistence and maintenance.