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. 2001 Aug;75(16):7602-11.
doi: 10.1128/JVI.75.16.7602-7611.2001.

Human papillomavirus E6E7-mediated adenovirus cell killing: selectivity of mutant adenovirus replication in organotypic cultures of human keratinocytes

C Balagué  1 , F Noya, R Alemany, L T Chow, D T Curiel
Affiliations

Human papillomavirus E6E7-mediated adenovirus cell killing: selectivity of mutant adenovirus replication in organotypic cultures of human keratinocytes

C Balagué et al. J Virol. 2001 Aug.

Abstract

Replication-competent adenoviruses are being investigated as potential anticancer agents. Exclusive virus replication in cancer cells has been proposed as a safety trait to be considered in the design of oncolytic adenoviruses. From this perspective, we have investigated several adenovirus mutants for their potential to conditionally replicate and promote the killing of cells expressing human papillomavirus (HPV) E6 and E7 oncoproteins, which are present in a high percentage of anogenital cancers. For this purpose, we have employed an organotypic model of human stratified squamous epithelium derived from primary keratinocytes that have been engineered to express HPV-18 oncoproteins stably. We show that, whereas wild-type adenovirus promotes a widespread cytopathic effect in all infected cells, E1A- and E1A/E1B-deleted adenoviruses cause no deleterious effect regardless of the coexpression of HPV18 E6E7. An adenovirus deleted in the CR2 domain of E1A, necessary for binding to the pRB family of pocket proteins, shows no selectivity of replication as it efficiently kills all normal and E6E7-expressing keratinocytes. Finally, an adenovirus mutant deleted in the CR1 and CR2 domains of E1A exhibits preferential replication and cell killing in HPV E6E7-expressing cultures. We conclude that the organotypic keratinocyte culture represents a distinct model to evaluate adenovirus selectivity and that, based on this model, further modifications of the adenovirus genome are required to restrict adenovirus replication to tumor cells.

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Figures

FIG. 1
FIG. 1
(A) Structure of the E1A regions of the adenovirus used in this study (for clarity, the E1A 13S-derived polypeptide is shown). (B) Immunoprecipitation of E1A and E1A-bound cellular proteins. SiHa cells were mock infected or infected with CB016 or Adwt (20 PFU/cell), and E1A was immunoprecipitated in nondenaturing conditions. E1A immunoblotting reveals a truncated set of E1A protein species for CB016 as opposed to Adwt; coprecipitation of pRB, p300, and p107 by CB016 E1A is abrogated.
FIG. 2
FIG. 2
Effects of adenovirus infection in raft cultures of normal PHK and HPV18 E6E7- and E7-expressing keratinocytes. H-E staining of day 10 raft culture sections is shown. Untransduced keratinocytes and keratinocytes acutely transduced with retrovirus expressing HPV18 E6E7 or E7 from the native HPV18 URR were infected with the indicated adenoviruses at an MOI of 1 prior to being transferred to dermal equivalents (see Materials and Methods) and allowed to stratify for 10 days. Adβgal-infected cultures were stained with X-Gal prior to H-E staining. Blue X-Gal staining is indicated by arrowheads. Magnification, ×200 (except Adwt [×400]).
FIG. 3
FIG. 3
Immunofluorescence analysis of adenovirus early and late proteins in day 10 CB016-infected normal (PHK) and HPV18 E6E7- and HPV18 E7-transduced raft cultures (same experiment as in Fig. 2). BrdU and hexon are revealed by green fluorescence, while E1A is revealed by red fluorescence. BrdU and E1A were detected by double immunofluorescence on the same section. Hexon immunofluorescence corresponds to the same field of a serial section. Magnification, ×200.
FIG. 4
FIG. 4
Quantification of adenoviral genomes in CB016-infected raft cultures. The numbers of adenovirus genome copies are expressed per nanogram of cellular genomic DNA. The error bars represent the standard deviation of the assay.
FIG. 5
FIG. 5
Time course of adenovirus infection in CB016-infected normal (PHK) and HPV18 E6E7-transduced raft cultures. (A) H-E staining of day 8, day 11, and day 14 uninfected control culture sections. (B) H-E staining of day 8, day 11, and day 14 CB016-infected culture sections. Arrowheads point to representative cells showing ballooning degeneration secondary to adenovirus replication. Magnification, ×200.
FIG. 6
FIG. 6
Time course of adenovirus infection in CB016-infected normal (PHK) and HPV18 E6E7-transduced raft cultures (same experiment as in Fig. 5). Immunofluorescence staining for BrdU, E1A, and hexon was performed as for Fig. 3. Colocalization of BrdU (green) and E1A (red) in the same cell yields a yellow fluorescence signal. Magnification, ×200.
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