The family Anelloviridae includes torque teno viruses (TTVs), TT-midiviruses (TTMDV), and TT-miniviruses (TTMV), the majority originating from samples of human origin (2,37,39,41,56). The plurality of this family of singlestranded DNA (ssDNA) viruses is reflected not only in DNA sequence, but also in genome size and organization.Infections occur within the first days of life, with close to 100% of infants being infected at 1 year of age. The primary route of infection, however, remains unclear (23,38,48). The ubiquitous nature of TTV infections has hampered efforts to associate it with the pathogenesis of disease (9,15,25,41). A possible etiological association with diseases of the liver (reviewed in reference 36) and respiratory tract (3,30,31,49), hematopoietic malignancies (9,10,13,15,25,53,59), and autoimmune diseases (9, 26, 28, 54) have been reported.The presence of a variety of intragenomic rearranged TTV subviral molecules (TTV) in serum samples and the in vitro transcription of a subviral molecule constituting only 10% of the complete genome initiated the discussion whether TTVs may share similarities to the plantvirus family Geminiviridae (8, 23). Both mono-and bipartite geminiviruses associate with single-stranded DNA satellites to form disease-inducing complexes (16,36,46,47,52,55).Multiple attempts have been made to find a suitable in vitro system for the replication and propagation of TTVs. Replicative forms of its DNA have been demonstrated in bone marrow cells and in the liver (22,42,44,45). Peripheral blood acts as reservoir for TTVs (43), and replication in vivo seems to occur preferably in activated mononuclear cells (27,29,33). Although in vitro transcription has been investigated in a variety of cell lines (18-21, 35, 50), long-term replication leading to virus production has been difficult to achieve (25).More than 200 genomes of TTVs have been isolated. The isolates grouping in the genus Alphatorquevirus (ca. 3.8 kb in size) share very low DNA sequence homology and differ in their genome organizations. A short stretch (71 bp) of the intergenic region is highly conserved among all human TTV isolates (48) and is widely used to demonstrate TTV infection. We have analyzed samples from a broad spectrum of diseases for the presence of TTV DNA by applying PCR amplification of this conserved region (9, 15, 25, 54; E.-M. de Villiers and K. Gunst, unpublished results). Identification of individual TTV types, however, requires the amplification of full-length genomes. We have thus far isolated 93 full-length genomes of TTVs (ca 3.8 kb) from human samples (9, 15, 25; present study). These included samples obtained from healthy individuals and patients with leukemia and lymphoma, rheumatoid arthritis, multiple sclerosis, and kidney disease. The present study describes the in vitro replication and transcription of 12 isolates after initial transfection of the genomic DNA and followed by propagation using frozen infected cells or culture supernatant. Intragenomic rearranged subviral molecules (TTV; i.e.,...

The genome organization of the novel human papillomavirus type 108 (HPV108), isolated from a low-grade cervical lesion, deviates from those of other HPVs in lacking an E6 gene. The three related HPV types HPV103, HPV108, and HPV101 were isolated from cervicovaginal cells taken from normal genital mucosa (HPV103) and low-grade (HPV108) and high-grade cervical (HPV101) intraepithelial neoplasia (Z. Chen, M. Schiffman, R. Herrero, R. DeSalle, and R. D. Burk, Virology 360:447-453, 2007, and this report). Their unusual genome organization, against the background of considerable phylogenetic distance from the other HPV types usually associated with lesions of the genital tract, prompted us to investigate whether HPV108 E7 per se is sufficient to induce the above-mentioned clinical lesions. Expression of HPV108 E7 in organotypic keratinocyte cultures increases proliferation and apoptosis, focal nuclear polymorphism, and polychromasia. This is associated with irregular intra-and extracellular lipid accumulation and loss of the epithelial barrier. These alterations are linked to HPV108 E7 binding to pRb and inducing its decrease, an increase in PCNA expression, and BrdU incorporation, as well as increased p53 and p21 CIP1 protein levels. A delay in keratin K10 expression, increased expression of keratins K14 and K16, and loss of the corneal proteins involucrin and loricrin have also been noted. These modifications are suggestive of infection by a high-risk papillomavirus.A large number of human papillomavirus (HPV) types have been associated with benign and malignant lesions of the genital tract. High-risk papillomaviruses encode two oncoproteins, E6 and E7, which independently immortalize human keratinocytes, although their combined actions have complementary and synergistic effects (5,22,23,35,43). High-risk E7 protein binds to a multitude of cellular proteins in vitro. Probably its most important action is targeting the pocket protein pRb for degradation and thereby allowing uncontrolled cell cycle progression (30,34). More recent studies, however, have indicated that the pRb-independent functions of E7 are sufficient to induce disruption of terminal differentiation and mild hyperplasia (3). The key role of high-risk E6, on the other hand, is to inactivate the oncosuppressive protein p53 functionally by inducing its degradation through the ubiquitin-proteosome pathway (31). Expression of high-risk E7 alone stabilizes the p53 protein, leading to increased levels (12), although its transcriptional activity is disturbed (32).The genome organizations of the majority of human-pathogenic papillomaviruses are characterized by an early region containing five genes (E1, E2, E4, E6, and E7) and two genes (L1 and L2) in the late region. The HPV types of the genus Alphapapillomavirus usually harbor an E5 gene in the region between the early and late genes, whereas this gene is absent in the HPV types commonly associated with cutaneous lesions and grouped in the genera Betapapillomavirus, Gammapapillomavirus, Deltapapillomavirus, ...

The mechanism through which cutaneous papillomaviruses induce lesions is largely unknown. Ectopic expression of the DeltaNp63alpha isoform highly increased the viral promoter activity. The co-expression of c-Jun mediated and increased the DeltaNp63alpha activity by binding to the AP-1 site in an enhancer region of the HPV 20 URR. This strong activation by DeltaNp63alpha is diminished in the presence of wtp53 and abolished by the simultaneous expression of "hot-spot" mutant p53 R248W. We demonstrate that c-Jun is responsible for the viral promoter activation through its direct interaction with both DeltaNp63alpha and wtp53. The downregulation by p53 mutant R248W is accompanied by reduced protein levels of DeltaNp63alpha and phosphorylated c-Jun. The data presented in this study provide insight into a possible mechanism through which these cellular proteins may modulate a cutaneous papillomavirus genome to induce viral replication, latent infection or malignant transformation.