Amino acid residues in the carboxy-terminal region of cottontail rabbit papillomavirus E6 influence spontaneous regression of cutaneous papillomas

Abstract

Previous studies have identified two different strains of cottontail rabbit papillomavirus (CRPV) that differ by approximately 5% in base pair sequence and that perform quite differently when used to challenge New Zealand White (NZW) rabbit skin. One strain caused persistent lesions (progressor strain), and the other induced papillomas that spontaneously regressed (regressor strain) at high frequencies (J. Salmon, M. Nonnenmacher, S. Caze, P. Flamant, O. Croissant, G. Orth, and F. Breitburd, J. Virol. 74:10766-10777, 2000; J. Salmon, N. Ramoz, P. Cassonnet, G. Orth, and F. Breitburd, Virology 235:228-234, 1997). We generated a panel of CRPV genomes that contained chimeric and mutant progressor and regressor strain E6 genes and assessed the outcome upon infection of both outbred and EIII/JC inbred NZW rabbits. The carboxy-terminal 77-amino-acid region of the regressor CRPV strain E6, which contained 15 amino acid residues that are different from those of the equivalent region of the persistent CRPV strain E6, played a dominant role in the conversion of the persistent CRPV strain to one showing high rates of spontaneous regressions. In addition, a single amino acid change (G252E) in the E6 protein of the CRPV progressor strain led to high frequencies of spontaneous regressions in inbred rabbits. These observations imply that small changes in the amino acid sequences of papillomavirus proteins can dramatically impact the outcome of natural host immune responses to these viral infections. The data imply that intrastrain differences between separate isolates of a single papillomavirus type (such as human papillomavirus type 16) may contribute to a collective variability in host immune responses in outbred human populations.

FIG. 1.

Design of chimeric CRPV E6 genes used in this study (constructs C to E). All CRPV chimeric E6 genes were placed into the H.CRPVp genome after removal of the whole E6p gene from the introduced SacII site to the EcoRI site. All constructs were sequenced prior to infection of rabbit skin.

FIG. 2.

Amino acid alignment between H.CRPVp and H.CRPVr E6 proteins over the carboxy-terminal 77 amino acids (encoded by the sequence between the AvrII site and the end of E6 gene). There are 12 aa differences (shaded) between the two E6 proteins over this region. In addition, the H.CRPVr E6 has three fewer amino acids at the C terminus than the H.CRPVp E6 because of a mutation that introduces an early stop codon in the associated gene.

FIG. 3.

Papilloma growth curves for E6 mutants in outbred rabbits. (A) Six sites on two rabbits were challenged with control and H.CRPV-NE6r constructs. Papillomas induced by H.CRPV-NE6r were smaller than those induced by wild-type constructs (P < 0.05, t test). SE, standard error. (B) Six sites on two rabbits were challenged with control and H.CRPVp-E6EFRdel constructs. There was no difference in papilloma growth rates for these two constructs (P > 0.05, t test).

FIG. 4.

Papilloma growth curve following infection with CRPV genomes encoding single amino acid changes in H.CRPVp E6. Six sites on two rabbits were challenged with each mutant. No significant difference between the sizes of papillomas produced by these constructs and the sizes of those produced by the control H.CRPVp genome were observed (P > 0.05, t test). SE, standard error.

FIG. 5.

Papilloma growth curves following infection with H.CRPVp-CE6r genomes encoding single or double amino acid changes in CE6r. Eight sites on four rabbits were challenged with each mutant CRPV. Sites challenged with H.CRPVp-CE6rG258D+S259P and H.CRPVp-CE6rR233S+E252G induced bigger papillomas (P < 0.05, t test) than sites challenged with control H.CRPVp-CE6r, whereas there was no significant difference in papilloma size for sites infected with H.CRPVp-CE6rE252G. SE, standard error.

FIG. 6.

Growth rates of papillomas induced by H.CRPVp-CE6r and H.CRPVp-E6 control genomes delivered onto inbred rabbit skin. Eight sites on four rabbits were challenged with two constructs. H.CRPVp-CE6rR233S+E252G and H.CRPVp-CE6rG258D+S259P+D265N induced papillomas significantly bigger than those induced by the control genomes (P < 0.05, t test), whereas no significant difference in papilloma size was found at sites challenged with H.CRPVp-CE6r R233S and H.CRPVp-CE6r E252G(P > 0.05, t test). SE, standard error.

FIG. 7.

Outgrowth and regression of H.CRPVp-E6 G252E-induced papillomas. (A) Six sites on two outbred rabbits were challenged with H.CRPVp-E6G252E and H.CRPVp wild-type constructs. Papillomas induced by the mutant construct were larger than those induced by the control construct in outbred rabbits (P < 0.05, t test). SE, standard error. (B) Twenty-four sites on four inbred rabbits were challenged with mutant H.CRPVp-E6G252E and wild-type constructs, respectively. Twenty-two of 24 sites regressed at the end of this experiment. The two remaining papillomas were smaller than those at sites challenged with control H.CRPVp (P < 0.01, t test).