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Papillomaviruses are a family of nonenveloped DNA viruses that infect the skin or mucosa of their vertebrate hosts. The viral life cycle is closely tied to the differentiation of infected keratinocytes. Papillomavirus virions are released into the environment through a process known as desquamation, in which keratinocytes lose structural integrity prior to being shed from the surface of the skin. During this process, virions are exposed to an increasingly oxidative environment, leading to their stabilization through the formation of disulfide cross-links between neighboring molecules of the major capsid protein, L1. We used time-lapse cryo-electron microscopy and image analysis to study the maturation of HPV16 capsids assembled in mammalian cells and exposed to an oxidizing environment after cell lysis. Initially, the virion is a loosely connected procapsid that, under in vitro conditions, condenses over several hours into the more familiar 60-nm-diameter papillomavirus capsid. In this process, the procapsid shrinks by ~5% in diameter, its pentameric capsomers change in structure (most markedly in the axial region), and the interaction surfaces between adjacent capsomers are consolidated. A C175S mutant that cannot achieve normal inter-L1 disulfide cross-links shows maturation-related shrinkage but does not achieve the fully condensed 60-nm form. Pseudoatomic modeling based on a 9-Å resolution reconstruction of fully mature capsids revealed C-terminal disulfide-stabilized “suspended bridges” that form intercapsomeric cross-links. The data suggest a model in which procapsids exist in a range of dynamic intermediates that can be locked into increasingly mature configurations by disulfide cross-linking, possibly through a Brownian ratchet mechanism., IMPORTANCE Human papillomaviruses (HPVs) cause nearly all cases of cervical cancer, a major fraction of cancers of the penis, vagina/vulva, anus, and tonsils, and genital and nongenital warts. HPV types associated with a high risk of cancer, such as HPV16, are generally transmitted via sexual contact. The nonenveloped virion of HPVs shows a high degree of stability, allowing the virus to persist in an infectious form in environmental fomites. In this study, we used cryo-electron microscopy to elucidate the structure of the HPV16 capsid at different stages of maturation. The fully mature capsid adopts a rigid, highly regular structure stabilized by intermolecular disulfide bonds. The availability of a pseudoatomic model of the fully mature HPV16 virion should help guide understanding of antibody responses elicited by HPV capsid-based vaccines.

The viral DNA sequences in mouse C127 cells transformed by bovine papillomavirus type 1 (BPV-1) virions, by full-length linear BPV-1 DNA, or by a defined transforming subgenomic DNA segment of BPV-1 were examined by reassociation kinetics and blot hybridization. In all cases, the transformed cells contained multiple copies of BPV-1 DNA, present exclusively as supercoiled or nicked circular extrachromosomal molecules or as a slowly migrating complex of circular viral DNA molecules. In the transformed cell lines established from cells transfected with full-length linear BPV-1 DNA, there was recircularization of the input DNA which in some cases resulted in the loss of the restriction site used in the linearization of the DNA. In the transformed cell lines established with the defined subgenomic segment there was circularization of the DNA accompanied by the acquisition of new sequences or duplication and rearrangement of the BPV-1 sequences. In contrast to other well-studied virus transformation systems, no integration of the BPV-1 genome into the host chromosome could be detected under conditions sensitive enough to detect 0.1-0.2 viral genome equivalent. It was concluded that maintenance of transformation may be mediated by nonintegrated viral DNA.

We have studied tumorigenic transformation of mouse tissue culture cells by bovine papillomavirus (BPV) as a model of papillomavirus-induced cell proliferation. When BPV or its 7.9-kb full-length viral DNA genome induces focal transformation of mouse calls, the viral DNA is maintained in the transformed cells as multiple extrachromosomal copies. The transforming capacity was initially localized to a 69% subgenomic fragment of the viral DNA genome. We have further characterized the BPV DNA sequences that can encode the transforming function by generating and analyzing the transforming activity of a series of BPV DNA deletion mutants. The results indicated that two discontinuous segments of the viral DNA are required for transformation. One segment, near the 5' end of the 69% transforming fragment, probably represents a control element of the viral DNA. The second segment, which lies within the 3' end of the 69% fragment, encodes transforming sequences of the viral DNA. A retroviral control element (the long terminal repeat DNA. A retroviral control element (the long terminal repeat DNA of Harvey murine sarcoma virus) will activate the 2.3-kb segment at the 3' end of the 69% fragment to transform the mouse cells.

The effect of mouse L-cell interferon on bovine papillomavirus type 1 (BPV-1) transformation of murine cells was examined. Mouse interferon reduced the level of BPV-1-induced transformation of mouse C127 cells by 95%. Long-term treatment of established BPV-1-transformed mouse cell clones with mouse L-cell interferon led to a decrease in the average number of the plasmid viral genomes present in these cells to 1/3 to 1/8. Although revertant lines could not be isolated from these lines in the absence of treatment with interferon, flat revertants were easily selected from two independent clonal transformed lines carried for 60 generations in the continued presence of 200 units of interferon per ml. These flat revertants had the biological characteristics of nontransformed C127 cells and could be retransformed by BPV-1. Southern blot hybridization failed to detect BPV-1 DNA in any of eight independent revertant lines examined under conditions that could detect 0.2 copies per cell. We conclude that interferon treatment has resulted in a selective reduction of the amount of extrachromosomal BPV-1 DNA in transformed cells and has cured some treated cells completely of their viral DNA.

The papillomaviruses, which are widely distributed in nature, typically induce benign skin tumours (warts) in their natural hosts (reviewed in ref. 1). These viruses are members of the papovavirus group, as are the polyomaviruses2. In contrast to polyomaviruses such as SV40, polyoma and BK (reviewed in ref. 3), the papillomaviruses have not been extensively studied, primarily because of the lack of a suitable tissue culture system. In general, the proliferative skin changes induced by the papillomaviruses are limited to the epidermal cells, and the viruses have a very narrow host range. However, some bovine papilloma viruses (BPV) are exceptional in that they induce fibropapillomas in their natural host (cows); these BPV can also induce cellular transformation in tissue culture4–6 and fibroblastic tumours in heterologous animals, including hamsters7, mice5 and horses8,9. BPV DNA from virions can also transform rodent cells and is tumorigenic in hamsters10. Recently, a tissue culture focus assay11 in mouse cells (NIH 3T3 and C127) has been described for those BPV which are the aetiological agents of fibropapillomas in cows. The virally transformed cells, which did not contain infectious virus, grew in soft agar and were tumorigenic in nude (nu/nu) mice. We12 have recently molecularly cloned the entire genomes of BPV-1 and BPV-2 (ref. 13) in Escherichia coli K12 using the certified plasmid vector pBR322 (ref. 14). We now demonstrate that these cloned DNAs can transform NIH 3T3 and C127 cells. Further, a molecularly cloned fragment which contains 69% of the BPV-1 genome can also induce cellular transformation.

Mouse L cell interferon reduced the level of bovine papillomavirus (BPV-1) induced transformation of mouse C127 cells approximately 20-fold. Continued treatment of established BPV-1 transformed mouse cells with mouse L cell interferon lead to a reduction in the copy number of the plasmid viral genomes present in these transformed cells. Flat revertants were selected from two independent transformed lines carried 60 generations in the presence of 200 units/ml of interferon. These flat revertants had the biologic characteristics of their untransformed parent C127 cells which included low cell saturation density and the inability to form colonies in soft agar. The revertants could be retransformed by BPV and each of 8 different revertant lines examined was found to be “cured” of their viral DNA sequences as determined by Southern blot hybridization.

A quantitative tissue culture assay for bovine papilloma virus (BPV) has been developed. Extracts of bovine papillomas which contained either BPV-1 or BPV-2 induced distinct foci in two mouse cell lines (NIH 3T3 and C127). Focus formation followed single-hit kinetics, suggesting that a single particle was required for focus induction. Preincubation of extracts with rabbit anti-BPV-1 serum neutralized their infectivity. The morphologically transformed cells contained BPV DNA sequences, grew in low serum and in soft agar, and were tumorigenic for nude mice.

† We describe a family with typical epidermodysplasia verruciformis (EV) in which only male members are affected. Whereas none of the index patient's ten children have EV, four of eight grandsons born to his daughters have inherited the disorder. All are infected with human papillomavirus (HPV) 3 and HPV 8. The inheritance in this kindred most likely results from an X-linked recessive genetic defect. Since other kindreds have been described with autosomal inheritance, this novel inheritance pattern suggests that the persistent high clinical susceptibility to HPV infection characteristic of EV may result from defects in either of at least two different genetic loci, one of which may be located on the X chromosome. (Arch Dermatol1985;121:864-868)