Cottontail rabbit papillomavirus E8 protein is essential for wart formation and provides new insights into viral pathogenesis.

The cottontail rabbit papillomavirus (CRPV) a and b subtypes display a conserved E8 open reading frame encoding a 50-amino-acid hydrophobic protein, with structural similarities to the E5 transmembrane oncoprotein of genital human PVs (HPVs). CRPV E8 has been reported to play a role in papilloma growth but not to be essential in papilloma formation. Here we report that the knockout of E8 start codon almost prevented wart induction upon biobalistic inoculation of viral DNA onto rabbit skin.

Variation in the nucleotide sequence of cottontail rabbit papillomavirus a and b subtypes affects wart regression and malignant transformation and level of viral replication in domestic rabbits.

We previously reported the partial characterization of two cottontail rabbit papillomavirus (CRPV) subtypes with strikingly divergent E6 and E7 oncoproteins. We report now the complete nucleotide sequences of these subtypes, referred to as CRPVa4 (7,868 nucleotides) and CRPVb (7,867 nucleotides). The CRPVa4 and CRPVb genomes differed at 238 (3%) nucleotide positions, whereas CRPVa4 and the prototype CRPV differed by only 5 nucleotides.

Human papillomavirus vaccines.

Genital human papillomavirus (HPV) infections are the viral sexually transmitted diseases most frequently diagnosed that include anogenital condylomas and squamous intra-$bepithelial lesions, among which the precursors of invasive carcinomas of the uterine cervix. In animal PV models, vaccination against L1 and/or L2 viral capsid proteins provides an efficient protection against infection, involving virus type-specific neutralizing antibodies. Vaccination against non-structural E1, E2, E6 or E7 viral proteins does not prevent infection, unless administered altogether, but tends to stimulate regression, warranting the design of therapeutic vaccines.

A cottontail rabbit papillomavirus strain (CRPVb) with strikingly divergent E6 and E7 oncoproteins: An insight in the evolution of papillomaviruses.

We previously observed that warts induced by an isolate of cottontail rabbit papillomavirus (CRPV) showed incomplete instead of systemic regression in some domestic rabbits. We report that the viral isolate contained, as a major component, a CRPV strain (CRPVb) showing an unexpectedly high divergence in the E6 and E7 open reading frames (ORFs), compared to the prototype CRPVa present in the isolate as a minor component. The E6 and E7 oncoproteins of CRPVa and -b disclosed only 87.

HLA control in the progression of human papillomavirus infections.

Cellular immunity is likely to be of major importance for the clearance of inapparent or overt infections caused by human papillomaviruses (HPVs). The highly polymorphic class I or class II human leukocyte antigen (HLA) molecules present HPV-derived peptides to cytotoxic (CD8+) or helper (CD4+) T lymphocytes bearing specific receptors and condition the immune responsiveness to HPV infections. Recent data point to a role of an altered expression of HLA molecules in the persistence of HPV-induced cervical premalignant lesions and their progression towards invasive carcinoma.

Progressive growth of human papillomavirus type 16-transformed keratinocytes is associated with an increased release of soluble tumour necrosis factor (TNF) receptor.

Analysis of conditioned media generated by weakly and highly tumorigenic SKv-1 keratinocyte lines harbouring integrated human papillomavirus type 16 (HPV16) DNA sequences revealed a factor inhibiting TNF-alpha and TNF-beta cytotoxic activity. This inhibitory activity was specifically blocked by htr-9 monoclonal antibody (MAb) recognising 55/60 kDa type I TNF receptor suggesting that it is related to a soluble form of this particular receptor (sTNF-RI). The presence of sTNF-RI was confirmed by Western blot analysis of SKv-1 cell-conditioned medium showing a band of 31.

Papillomavirus L1 capsids agglutinate mouse erythrocytes through a proteinaceous receptor.

Virus-like particles (VLPs) composed of L1 derived from bovine papillomavirus type 1 (BPV-1), several human papillomavirus types, or cottontail rabbit papillomavirus (CRPV) agglutinated mouse but not human or rat erythrocytes. Treatment of mouse erythrocytes with trypsin prevented hemagglutination (HA) by BPV-1. Sera from rabbits immunized with native CRPV VLPs, which protect against experimental CRPV infection, exhibited high titers of antibodies that inhibited CRPV VLP HA activity, while sera from rabbits immunized with denatured CRPV VLPs or native BPV VLPs, which do not protect against CRPV infection, were not inhibitory.

Immunization with viruslike particles from cottontail rabbit papillomavirus (CRPV) can protect against experimental CRPV infection.

We tested the ability of vaccination with virus-like particles (VLPs) to protect domestic rabbits against papillomas induced by the cottontail rabbit papillomavirus (CRPV). A recombinant baculovirus system that expressed only the L1 major papillomavirus structural protein or L1 plus the minor L2 protein was used in insect cells as the source of VLPs. Groups of 10 rabbits were immunized with native or denatured VLPs from CRPV or type 1 bovine papillomavirus by using Freund's adjuvant.

Analysis of the nucleotide sequence variation of the antigen-binding domain of DR alpha and DQ alpha molecules as related to the evolution of papillomavirus-induced warts in rabbits.

We previously found that regression of skin warts induced by the Shope cottontail rabbit papillomavirus in New Zealand White rabbits, as well as malignant conversion of persistent warts, are linked to a restriction fragment length polymorphism of the major histocompatibility complex class II DR alpha and DQ alpha genes. To find out whether this immunogenetic control could be connected with the antigen binding and presentation function of the alpha 1 domain of class II molecules, we have sequenced the exon 2 of the four DR alpha EcoRI and six of the seven DQ alpha PvuII restriction fragment length polymorphism alleles identified, and deduced the encoded amino acid sequences. We found no amino acid polymorphism among DR alpha alleles, indicating that the alpha 1 domain of the DR alpha chain does not condition wart regression or cancer development.

A mouse model for studying epidermodysplasia-verruciformis-associated carcinogenesis.

Epidermodysplasia verruciformis (EV) is considered as a model of genetic cancer due to unusual susceptibility to EV-specific human papillomaviruses (HPVs). We established an in vivo experimental system for long-term propagation of EV tissue which should facilitate the study of tumor progression in EV. Skin fragments from benign and early pre-malignant lesions of 6 EV patients were grafted under the kidney capsule of athymic mice.

Increased tumorigenicity of human keratinocytes harboring human papillomavirus type 16 is associated with resistance to endogenous tumor necrosis factor-alpha-mediated growth limitation.

The aim of this study was to evaluate the relationship between tumorigenicity of cell sublines derived from weakly tumorigenic SKv-e and SKv-l keratinocytes harboring human papillomavirus type 16 (HPV16) and their susceptibility to autocrine growth limitation mediated by tumor necrosis factor-alpha (TNF-alpha). These sublines displayed different in vitro proliferative potential which correlated with tumorigenicity in nu/nu mice. Recombinant TNF-alpha inhibited in vitro growth of weakly tumorigenic parental SKv cell lines while it did not affect proliferation of their respective highly tumorigenic sublines.

Cytopathic effect in human papillomavirus type 1-induced inclusion warts: in vitro analysis of the contribution of two forms of the viral E4 protein.

Myrmecia warts induced by human papillomavirus type 1 (HPV1) are characterized by abundant eosinophilic inclusions associated with HPV1 E4 gene products. The major HPV1 E4 proteins are a 17-kilodalton (kDa) E1-E4 fusion protein and a 16-kDa species lacking the five E1 amino acids and a few E4 residues. To study the contribution of E4 proteins to the formation of myrmecia inclusions, we used a previously designed transient expression system in the rabbit VX2-R keratinocyte line.

Linkage of regression and malignant conversion of rabbit viral papillomas to MHC class II genes.

Human papillomaviruses associated with cutaneous and anogenital cancers induce intraepithelial precursor lesions which may regress spontaneously or progress into invasive carcinomas. Cell-mediated immune responses are probably involved in regression of precancerous lesions and the polymorphism of the genes responsible may thus have a key role in the variability of the host response. Skin warts and cancers induced in rabbits by Shope papillomavirus provide a model to test this hypothesis.

Human papillomavirus type 1 E4 proteins differing by their N-terminal ends have distinct cellular localizations when transiently expressed in vitro.

Two major human papillomavirus type 1 (HPV-1) E4 proteins are found in large amounts in productively infected differentiating wart cells, a 17-kDa protein translated from an E1-E4 transcript and a processed 16-kDa protein lacking the E1 amino acids at least. The functions of the E4 proteins are still unknown. We have designed an in vitro system allowing the transient expression of three forms of HPV-1 E4 proteins: the 17-kDa E1-E4 protein, an E4 protein without the five E1 amino acids (E4-3200), and E4 protein initiated at the E4 ATG located upstream of the splice acceptor site (E4-3181).

Expression of the human papillomavirus type 16 genome in SK-v cells, a line derived from a vulvar intraepithelial neoplasia.

The SK-v cells, established from a premalignant vulvar lesion, contain human papillomavirus type 16 (HPV-16) sequences integrated at a single cellular site and derive from a cell clone present in vivo. Transcription of the HPV-16 genome in SK-v cells was analysed by cDNA heteroduplex mapping and sequencing, and by RNase mapping. Viral sequences were shown to be transcribed into virus-cell fusion messengers.

The L2 open reading frame of human papillomavirus type 1a encodes a minor structural protein carrying type-specific antigens.

The proteins encoded by the open reading frames of papillomavirus genomes and the minor polypeptides detected in purified virions are still poorly defined. We show here by its expression in Escherichia coli that the open reading frame L2 of human papillomavirus type 1a codes for a minor structural protein of Mr 76,000. Antisera raised against a truncated L2-beta-galactosidase fusion protein in which the conserved N-terminal region of L2 is missing are type specific for human papillomavirus type 1 virions and are reactive at high dilutions.

A new method for studying epidermalization in vitro.

A new method for studying epidermalization in vitro is described. It consists of inserting a punch biopsy that serves as a source of epidermis into dermal equivalent freshly made up, with fibroblasts mixed in a collagen matrix. Fibroblasts cling to collagen fibrils and contract the matrix, leading in 3 days to a resistant dermal equivalent holding the punch biopsy firmly in place.

Multiple cutaneous papillomas and carcinomas that develop spontaneously in a mouse mutant, the repeated epilation heterozygote Er/+.

After a chance observation that multiple cutaneous papillomas and squamous cell carcinomas occurred in 2 adult mice heterozygous for the repeated epilation gene Er, we surveyed a panel of 10 +/+ (wild type) and 30 Er/+ (heterozygous) mice from birth to over 2 years of age. Homozygous Er/Er mice could not be included since their defect is lethal at birth. Whereas no cutaneous tumors developed in the +/+ mice, 20 of the Er/+ mice, males and females, had developed 1-5 cutaneous papillomas and at least 1 cutaneous invasive squamous cell carcinoma by 2 years of age.

Two Shope papillomavirus-associated VX2 carcinoma cell lines with different levels of keratinocyte differentiation and transplantability.

Two cell lines, named VX2T and VX2R, were isolated from the transplantable VX2 carcinoma, a wholly anaplastic tumor established from a carcinoma induced by the Shope cottontail rabbit papillomavirus (CRPV) (J.G. Kidd and P.

A type-II DNA topoisomerase and a catenating protein from the transplantable VX2 carcinoma.

It has recently been suggested that topoisomerases could be important targets for several DNA intercalating drugs used in cancer therapy. This prompted us to purify and characterize a type II topoisomerase in a highly tumorigenic transplantable rabbit tumor isolated from a skin carcinoma associated with cottontail rabbit papillomavirus. We have found that the decatenating activity present in tumor cells was 40-100 times higher than that in the rabbit liver, while no activity could be found in skin extracts.