Biosynthesis of Human Papillomavirus from a Continuous Cell Line Upon Epithelial Differentiation

Abstract

The study of the human pathogen papillomaviruses (HPVs) has been hampered by the inability to propagate the virus in tissue culture. The addition of 12-O-tetradecanoyl phorbol-13-acetate to the media of organotypic (raft) cultures increased expression of physiological markers of keratinocyte differentiation and concomitantly induced production of virions. Capsid production was detected in differentiated suprabasal cells. Virions approximately 54 nanometers in size were observed by electron microscopy in raft tissue cross sections in the suprabasal layers. Virions purified through isopycnic gradients were found to contain type 31b DNA and exhibited an icosahedral shape similar to that of papillomaviruses found in clinical samples.

Human papillomavirus type 16 in head and neck carcinogenesis

“…We failed to demonstrate viral particles in the raft culture, which can be explained by the fact that these cells contained no episomal viral DNA. Viral particles have been identified in raft cultured cells derived from a cervical intraepithelial neoplasia type I lesion and HPV-immortalized keratinocytes containing episomal viral DNA (Meyers et al 1992;Frattini et al 1996). When a constructed chimeric HPV-18/16 plasmid DNA was introduced into primary keratinocytes and maintained episomally, the viral late functions (capsid gene expression and synthesis of virion) were active in raft cultures (Meyers et al 2002).…”

Replication and Assembly of Human Papillomaviruses

“…Once activated, the late promoter directs transcription from a heterogeneous set of start sites which are clustered around nucleotide 742 (p742) in HPV31 Meyers, 1998, 1999;Smith et al, 2007). A late promoter has been identified in HPV16 (p670), and evidence suggests that a late promoter exists in HPV18 as well (Hummel et al, 1992;Meyers et al, 1992;Grassmann et al, 1996;Parker et al, 1997). The late promoter specifically serves to produce a set of transcripts that facilitate the translation of L1 and L2 proteins.…”

“…The life cycle of HPV is strictly linked to the differentiation program of the host keratinocyte, whereby the assembly of mature virions is restricted to terminally differentiated suprabasal cells, limited in part by the differentiation-dependence of the late promoter (Bedell et al, 1991;Hummel et al, 1992;Meyers et al, 1992;Barksdale and Baker, 1993;Grassmann et al, 1996;Ozbun and Meyers, 1997;Smith et al, 2007). Initial infection by HPVs is thought to occur through micro-abrasions of the epithelial tissue, thus allowing entry of the HPV particle into cells of the basal layer (Belnap et al, 1996).…”

“…The early (E) ORFs encode the E1, E2, E4, E5, E6, and E7 proteins (Doorbar et al, 1986;Zheng and Baker, 2006;Buck et al, 2008). E1 and E2 proteins have been shown to regulate viral replication as well as the coordinated expression of the other early viral genes (Cripe et al, 1987;Gloss and Bernard, 1990;Mohr et al, 1990;Meyers et al, 1992;Conger et al, 1999). The spliced E1^E4 protein may affect the replication of viral genomes through the inhibition of the G2-to-M transition, while also interacting with cellular keratin networks, causing their collapse, thus allowing mature virions to escape from the cornified envelope (White et al, 1999;Bryan and Brown, 2000;Brown et al, 2006;Knight et al, 2006;Mach et al, 2006;Gambhira et al, 2007).…”

“…E1 and E2 are among the first early proteins that are expressed, assisting in the establishment of 20 to 100 episomal copies per basal cell (Mohr et al, 1990;Meyers et al, 1992;Conger et al, 1999;Smith et al, 2007). These 2 proteins form a complex with the viral origin of replication and recruit cellular polymerases and the necessary accessory proteins to facilitate replication (Mohr et al, 1990).…”