HPV-18 E6 mutants reveal p53 modulation of viral DNA amplification in organotypic cultures

Abstract

Human papillomaviruses (HPVs) amplify in differentiated strata of a squamous epithelium. The HPV E7 protein destabilizes the p130/retinoblastoma susceptibility protein family of tumor suppressors and reactivates S-phase reentry, thereby facilitating viral DNA amplification. The high-risk HPV E6 protein destabilizes the p53 tumor suppressor and many other host proteins. However, the critical E6 targets relevant to viral DNA amplification have not been identified, because functionally significant E6 mutants are not stably maintained in transfected cells. Using Cre-loxP recombination, which efficiently generates HPV genomic plasmids in transfected primary human keratinocytes, we have recapitulated a highly productive infection of HPV-18 in organotypic epithelial cultures. By using this system, we now report the characterization of four HPV-18 E6 mutations. An E6 null mutant accumulated high levels of p53 and amplified very poorly. p53 siRNA or ectopic WT E6 partially restored amplification, whereas three missense E6 mutations that did not effectively destabilize p53 complemented the null mutant poorly. Unexpectedly, in cis, two of the missense mutants amplified, albeit to a lower extent than the WT and only in cells with undetectable p53. These observations and others implicate p53 and additional host proteins in regulating viral DNA amplification and also suggest an inhibitory effect of E6 overexpression. We show that high levels of viral DNA amplification are critical for late protein expression and report several previously undescribed viral RNAs, including bicistronic transcripts predicted to encode E5 and L2 or an alternative form of E1^E4 and L1.human papillomavirus DNA amplification | trans complementation | HPV transcripts P apillomaviruses are small DNA viruses with a protein capsid harboring a double-stranded circular genome of ∼7,900 bp. Dozens of human papillomavirus (HPV) types are tropic for the anogenital tract (1). HPV-16 and -18 and closely related genotypes are high-risk (HR) types and at a low frequency, can cause cancers, in which the viral E6 and E7 oncogenes are invariably overexpressed. HPV-6 and -11 are low-risk (LR) types and induce benign anogenital warts and laryngeal papillomas (review in ref. 2). Typically, viral DNA is maintained as low copy nuclear plasmids in basal and parabasal keratinocytes, and vegetative amplification depends on squamous differentiation (review in ref.3). Because viral DNA replication requires the host DNA replication machinery, the role of the HPV E7 protein is to promote S-phase reentry in differentiated cells that have withdrawn from the cell cycle. It does so by destabilizing the p130 protein (4, 5), a pocket protein related to the retinoblastoma susceptibility protein, a major tumor suppressor. The HR but not the LR HPV E7 protein also destabilizes retinoblastoma susceptibility protein (6). Both HR and LR HPV E6 proteins inactivate the transcription regulatory activities of another major tumor suppressor, p53 (7-10), abrogating its control over cell cycle ar...

Serine/Arginine-Rich Splicing Factor 3 and Heterogeneous Nuclear Ribonucleoprotein A1 Regulate Alternative RNA Splicing and Gene Expression of Human Papillomavirus 18 through Two Functionally Distinguishable cis Elements

“…The coding potentials for HPV18 viral proteins are shown on the right of each transcript. #, mRNAs reported only in transiently transfected U2OS cells (25) but not in HPV18-infected keratinocytes (24,26).…”

“…The individual ORFs are shown above the linearized HPV18 genome, along with various RNA splicing isoforms below. The transcription map originated from the Zheng laboratory (24) and has been updated to include less abundant RNA isoforms from two recent publications (25,26), with additional support from the current study. Exons (thick lines) and introns (thin lines) are illustrated for each RNA species derived from alternative promoter usage and alternative RNA splicing, with splice site positions numbered by nucleotide positions in the virus genome.…”

“…Productive HPV18 infection in keratinocyte-derived raft tissues leads to the generation of more than 15 isoforms of viral early and late RNAs through alternative RNA splicing by selection of alternative splice sites ( Fig. 1) (24,26,27). With other, weak splice sites recently identified in HPV18-transfected U2OS cells (25), the updated HPV18 transcription map ( Fig.…”

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

“…It is now well established that the reentry of infected cells into the cell cycle in suprabasal layers is a prerequisite for HPV genome amplification and late protein expression. In the context of replicating HR-HPV genomes, the combined activities of E4, E5, E6, and E7 are required to achieve this, and mutations in one of these genes result in decreased or absent viral DNA amplification and late gene expression levels (18)(19)(20)(21)(22)(23)(24). Based on their essential replication functions in undifferentiated cells, E1 and E2 are very likely required for differentiation-dependent genome amplification.…”

“…The knockout of E7 in HPV16 or HPV18 leads to a loss of genome amplification and L1 expression in differentiated cells, which is most likely due to the inability to induce DNA replication in suprabasal cells (18,19). Similarly, the knockout of E6 in the context of the HPV18 genome resulted in a lack of genome amplification in differentiated cells, and this correlated with increased p53 protein levels and the absence of DNA replication in suprabasal cells (20). The knockout of E5 in HPV16 and -31 decreases but does not abolish late gene expression, and this correlates with reduced numbers of suprabasal cells in which DNA replication is induced (21,22).…”