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.

Keywords: HPV transcripts; human papillomavirus DNA amplification; trans complementation.

Fig. 1.

HPV-18 major capsid protein (L1) detection. HPV-18 L1 was probed by immunohistochemistry with an L1 antibody in raft cultures containing HPV-18 WT or E6 mutant genomes (WT, F4L, F4V, YYH, or E6 null) or the E6 null mutant trans complemented by pBabe Puro HPV-18 URR-E6 or E6 mutations (E6 null + WT E6, E6 F4L, E6 F4V, or E6 YYH).

Fig. 2.

In situ analysis of HPV-18 viral DNA amplification and p53 stabilization or PCNA induction. (A) Raft cultures containing HPV-18 WT, E6 mutant genomes, or E6 null mutant in the presence of ectopic E6 WT or missense mutations were simultaneously probed for viral DNA amplification (red, Alexa Fluor 555) by DNA FISH and for p53 (green, Alexa Fluor 488) by indirect immunofluorescence. These experiments were conducted on multiple raft cultures, and they gave the same qualitative results. (B) Raft cultures of PHKs harboring HPV-18 E6 null genome were transduced with retroviruses expressing a p53 siRNA (Left and Center) or an empty vector (Right). Sections were probed for HPV-18 viral DNA amplification (red, Tyramide Cy3; Left), p53 stabilization (green, Tyramide, FITC; Left, Center, and Right), or PCNA induction (red, Alexa Fluor 555; Center and Right). Nuclei were stained with DAPI (blue).

Fig. 3.

Real-time qPCR to compare HPV-18 DNA copy numbers per cell. The average HPV-18 genomic plasmid copy numbers per diploid cell were determined by real-time qPCR in raft cultures containing HPV-18 WT or E6 mutants in five sets of independent experiments, each with a different batch of PHKs. In three of five experiments, the copy numbers of the E6 null mutant complemented by ectopic WT E6, E6 missense mutations, or empty vector in trans were conducted in parallel. The WT genome copy number averaged about 4,300 and was set as 100%. Normal PHK raft cultures were negative (0.021%) for HPV DNA.

Fig. 4.

Western blot detections of E7, E6, and p53 in raft cultures. (A) Comparison of HPV-18E7 protein levels in the lysates of raft cultures containing HPV-18 WT and E6 mutant genomes. (B) Lysates from raft cultures containing HPV-18 WT, E6 mutant genomes, or E6 null mutant complemented by WT E6 or E6 missense mutations in trans were probed for viral E6 and p53 proteins; β-actin was used as internal loading reference for all of the experiments.

Fig. 5.

RT-PCR detection of HPV-18 viral transcripts in HPV-18 WT or E6 mutants. (A) The circular HPV-18 genome of 7,857 bp was linearized in the URR. Triangles denote early (A_E) and late (A_L) polyadenylation sites. ORFs are represented by open boxes, with primers for RT-PCR indicated below. E1F2 and E1F3 primers spanned the splice junctions nucleotides 929^3465 and 929^3506, respectively. Primer sequences are shown in Table S3. (B–I) RT-PCR reactions were conducted on total RNAs extracted from the raft cultures containing HPV-18 WT or E6 mutant genomes as described in Materials and Methods. Primer pairs and PCR cycles are indicated above each panel. Primer annealing was conducted at 58 °C, except for E1F3/L2R (G) at 65 °C. The samples in each panel are indicted at the bottom. Length markers in B–D, H, and I are comprised of 1 µg 50-bp DNA ladders (Invitrogen); 1 µg 1-kb plus ladders (Invitrogen) was used in E–G. Gel images were captured using different exposures to visualize minor bands. The lengths of the PCR products are (B) band 1, 190 bp; band 2, 159 bp; (C) band 1, 584 bp; band 2, 553 bp; (D) 551 bp; (E) 1,005 bp; (F) 972 bp; (G) band 1, 1,003 bp; band 2, 931 bp. The intensity of band 1 in (G) increased relative to band 2 if the primer annealing temperature was decreased from 65 °C to 58 °C. (H) Band 1, 450 bp; band 2, 419 bp; (I) bands 1–4, 450, 419, 378, and 187 bp.