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Human papillomaviruses (HPVs) are the most studied members of the family designated Papillomaviridae, DNA viruses sharing an unenveloped icosahedral structure as well as the basic organization and replication strategy of the circular, double-stranded, 8-kb DNA genome. To date more than 80 genotypes have been completely characterized, and a number of partially characterized isolates, probably representing novel genotypes, are presently under investigation (43). HPVs can be divided into 3 of the 7 supergroups described for all papillomaviruses (6); supergroup A is associated with genital disease, and a subset of its members is found in the vast majority of genital cancers (1, 43).

HPV infection is extremely frequent throughout the world. Most of these infections are transient (15), although the virus can persist in a very limited number of infected cells, and do not reach clinical observation. In a small proportion of non-immune-suppressed subjects and in the majority of immune-suppressed individuals HPV infection can persist for years in clinically evident lesions. During the last few years, different molecular strategies have been employed for detecting and typing novel HPVs, mostly by using PCR amplification and general primers for the conserved L1 or E1 viral regions. The widespread use of these methods (14, 28, 32, 40) is leading to the discovery of a rising number of HPV genotypes (4, 7, 11, 12, 18, 20-22) whose roles as human pathogens remain to be established.

Recently, we were interested in identifying unknown HPV genotypes infecting patients with gynecologic or dermatologic lesions and adopted a molecular method that allowed the identification of potentially novel HPVs involved in human lesions. In the present report we describe the detection of six putative novel HPV sequences and the molecular cloning, sequencing, and biologic characterization of one isolate that turned out to be a new HPV genotype.

Clinical samples and molecular methods. Cytologic or bioptic samples from patients examined in different Italian clinical centers (mainly gynecologic centers and, in a few cases, dermatologic centers) were tested for HPV DNA during the period of January 1991 to May 2000 (5,115 clinical samples in total, mostly collected after clinical evidence of lesions or pathological Pap-test findings). Cytologic specimens were obtained by rubbing a dry swab over the epithelium suspected for HPV infection. Cells were recovered by mixing and squeezing the swab in a microcentrifuge tube containing 1.0 ml of transport medium (10 mM Tris-buffered saline, pH 8.0, with antibiotics and amphotericin B to inhibit microbial growth during transportation and storage). In a minority of cases (about 10%), samples were obtained by biopsy taken within the suspected lesion. Bioptic specimens were directly immersed in the transport medium. Upon arrival at the laboratory, they were finely minced with a scalpel and subsequently processed as the cytologic specimens. The cytologic material was washed twice in a microcentrifuge tube in Tris-buffered saline, and the final pellet was resuspended in a variable volume of lysis buffer that was approximately 10 times the volume of the pellet. The lysis buffer contained Tris (10 mM, pH 8.0), Tween 20 and Nonidet P40 (0.5% each), and proteinase K (200 µg/ml). The tubes were subsequently incubated overnight at 56°C. The degenerate primers used in this study were derived from a consensus sequence in the L1 gene and were the following: sense (MY11), GCA CAG GGT (T/A)CA TAA (T/C)AA TGC, modified from Resnick and Manos (22, 26); antisense (GP6+), AAC TGT AAA TCA (A/T)AT TC(T/C) TC, modified from Snijders et al. (33, 34). These primers were used in a 50-cycle reaction at 35°C annealing temperature. The amplified fragment ranged from 173 to 206 bp, depending on HPV type, and was always clearly distinguishable from nonspecific products on a 10% polyacrylamide gel. Twelve microliters of the amplified product were used in separate restriction reactions and were directly incubated in PCR tubes with 5 U of RsaI or 5 U of Tru91. After 4 h of reaction the digested amplified products were resolved on a 20% polyacrylamide gel. The restriction pattern allowed type definition in 68% of the cases. The remaining cases were mixed infections (14.5%) and ambiguous or untyped digests (further analyzed by sequencing the amplified product).

Detection of putative novel HPV genotypes. Out of 1,248 HPV DNA-positive clinical samples, 31 (2.48%) yielded L1 sequences that did not match (<85% similarity to the amplified fragment) any of the previously described sequences from the Los Alamos HPV database or the GenBank and EBI databases. These 31 sequences could be grouped into six prototypes; five were deposited in the European Molecular Biology Laboratory (EMBL) nucleic acids data bank in March 1997 and were designated HANHD25 (EMBL accession No. Y12223), HAN2294 (accession no. Y12217), HAN2500 (accession no. Y12215), HAN1353 (accession no. Y12220), and HAN1112 (accession no. Y12219), whereas HANOA464 was submitted in May 2000 (EMBL accession no. AJ277788). Clinical information was available for the 4 subjects infected with the HAN2294 virus, which was detected in 3 human immunodeficiency virus type 1 (HIV-1)-positive subjects (2 with less than 200 CD4⁺ T lymphocytes per microliter of blood at the time of first HPV detection) and 1 HIV-1-negative subject. In all 4 cases, HPV detection followed atypical cell finding (koilocytic atypia) in the Pap test. In one of the HIV-1-positive patients, colposcopy showed grade 1 transformation zone atypia. The patient underwent physical treatment, but the infection persisted for 5 years without signs of clinical progression of the HPV infection until PCR results became negative after HIV suppression and an increase in CD4⁺ T cells by combination antiretroviral therapy. The HAN2500 virus was associated with condylomas in 2 HIV-1-positive and in 1 HIV-1-negative subject. The HAN1353 virus was detected in 1 severely immunosuppressed (CD4⁺ T cells, <200) HIV-1-positive and in 5 HIV-1-negative subjects, the former with an apparently normal Pap test, while one of the latter group had evidence of koilocytosis. No clinical information was available for the 3 HIV-negative subjects infected with HANHD25, HANOA464, and HAN1112, who had low-grade atypias at the Pap test.

Sequencing of the complete genome of HAN2294. In order to characterize the complete genome of the HAN2294 HPV genotype, a conserved region in the E1 gene, roughly opposite the L1 gene in the circular genome, was amplified by using the following general primers: sense, AAT TCC AAA AGC CA(T/A) TTT TGG (T/C)T; antisense, TGG AAA TCT TTT TTT (A/T)(A/C)A AGG (as synthesized). The amplified product was subsequently sequenced and compared to the available E1 HPV sequences in order to exclude a similarity exceeding 85% to known types or isolated sequences. To sequence the complete genome, the regions between the E1 and L1 genes were amplified by long PCR. Two primer pairs were synthesized facing opposite directions within the E1 and L1 amplified products. The primers that encompassed the L1, LCR, E6, and E7 genes were synthesized as TCG CAG TAC CAA TTT TAC TAT TAG TGC TG and CGT AAA TAC TTT AAA CTG TCA TCT GCC TC. For the E1, E4, and L2 regions, the sequences of the primers were TGG ATG GCA ATA CCA TGA GCA TAG ACA G and AAA CTT TGT GGG GTC ATA TTC AGT GGT TG. Long PCR was performed by using the enzyme mixture (Finnzymes) and the buffer as sold by the manufacturer. The MgCl concentration was adjusted at 4 mM, and the reactions were carried out for 20 cycles as follows: denaturation for 30 s at 94°C, annealing for 50 s at 50°C, and polymerization for 180 s (increased by 10 s per cycle) at 68°C. The two amplified products, 3,772 and 4,420 bp, respectively, were visible after agarose gel electrophoresis and were cloned into an appropriate cloning vector (PCR T Easy vector; Promega Corp., Madison, Wis.). After screening the transformed colonies for the presence of inserts, the positive recombinant clones were grown and plasmid preparations were obtained to check the integrity of the HPV inserts. Unfortunately, all inserts bore random deletions, and in order to clone the complete genome, multiple clones had to be drawn. Figure 2 shows the final map of the clones that were used for the definition of a new HPV type by the Reference Center for Human Papillomavirus.

Phylogenetic analysis, open reading frames (ORFs), and promoter features of candHPV87. The partial L1 sequence segment encompassed by the common primers MY11/09 is the most widely used for HPV PCR amplification and therefore accounts for most of the novel HPV sequences. We performed an alignment of this region (by using the ClustalW 1.7 program, followed by phylogenetic analysis by DNADIST and NEIGHBOR in the Phylogeny Inference Package, version 3.572; Phylip) (10) including all the sequences of typed and novel HPVs available in public databases, in order to produce an updated phylogeny of HPVs. All untyped sequences from putative novel HPVs were included for the alignment, whereas for each typed HPV only the prototype was included. Figure 1 shows the phylogenetic trees of supergroup A (mucosal) HPVs. The shaded sequences represent the putative novel HPVs described in this work. In panel A (relative to the MY region), three of these sequences (HANOA464, HAN1112, and candHPV87) cluster in the A3 group together with other sequences yet to be defined, such as HPVXS4 (which seems to be a candHPV87 strain), CP8304 or L1AE7, CP6108 or L1AE6, HPGA6053, and HPA012757. HAN2500 clusters in the A8 group together with AF070938 (same HPV type). HANHD25 clusters in the A10 group, while HAN1353, together with JC9710 (same type), and CP8304 and L1AE7 (both belonging to the same type) apparently are in a group as-yet undefined. The complete candHPV87 genome was also aligned with 48 complete supergroup A HPV genomes available in data banks. Figure 1b shows the phylogenetic relationships of the complete HPV genomes. candHPV87 clusters in group A3, which also includes HPV61, HPV62, HPV72, HPV83, and HPV84, sharing maximum similarity (18% divergence) with HPV 84 and satisfying the criteria for a new HPV type (8). After this paper was submitted for publication, a novel A3 HPV type (designated candHPV86) has been described; candHPV86 is closely related to candHPV87 (86% homology) (36).

View larger version (69K): [in this window] [in a new window]   FIG. 1.   Phylogenetic trees of Supergroup A (mucosal) HPVs. (a) Partial L1 region encompassed by primers MY11/09, with group division at ancestor branching. The sequences described in this work are printed in boldface. (b) Complete genomes of all available HPV sequences, with group divisions.

ORF analysis of the candHPV87 genome. ORF analysis was performed using the DNASTAR package (DNAstar Inc., Madison, Wis.). The ORFs found in the candHPV87 genome are illustrated in Fig. 2. On the whole, the coding region organization resembles very closely that of HPV84 (37), its closest relative. Some typical HPV features were identified. The putative E6 protein contains the typical zinc-binding domain constituted by two C-X₂-C-X₂₉-C-X₂-C motifs separated by 36 residues (1). The same motif is also present once in the E7 protein together with the CR1 and CR2 domains (25) HGQTPTIKDIIISE and DSSEEEDN, respectively, whereas the retinoblastoma protein (pRB)-binding domain appears different from that of other HPVs in that the (G/D)LXCXE core is HIHCDE. Features of the E2 polypeptide include a nuclear localization signal, KGCWKKQGR, and the typical DNA-binding helix, GDPNRLKCFRYR, conserved in papillomaviruses (24). The L2 protein shows the typical TTPAVLDI motif of most mucosal HPVs (27, 30). The most striking difference from most papillomaviruses consists in the loss of the E5 coding region due to a point mutation just downstream of the starting ATG codon, introducing a stop codon. The presence of this stop codon was confirmed by directly sequencing the amplified product including this region from all the available clinical samples from the same patient and from two other subjects bearing the same HPV type (performed in different sessions along with samples from HPV negative subjects as controls). An additional point mutation further downstream also introduces a stop codon after 44 amino acids, leading to a shorter polypeptide, and might have been selected before the complete ablation of the ORF. Although not unique to candHPV87, the absence of a functional E5 region in this type is intriguing, since the gene, apart from the stop codons, is very conserved compared to that of HPV61, where it should be expressed as a functional polypeptide. Since there is no overlapping reading frame in this silenced ORF, the fact that this sequence has not been subject to extensive genetic drift, as would be expected in nonfunctional sequences, suggests that it could represent a fairly recent evolutionary step.

View larger version (33K): [in this window] [in a new window]   FIG. 2.   Genome organization and PCR clones of candHPV87. ORFs are represented as thick arrows, and numbers show nucleotide positions of start and stop codons; clones are represented as thin arrows. Clones L1-E1 16 and L1-E1 37 were derived from the E1-L1 amplified product, E1-L1 11/2 and L1atg were derived from the E1 L1 amplified product; clone E11-E2as was subsequently derived from an independent PCR product in order to complete the E1-E4 region and clone MY 16 was derived from a MY11-MY09 PCR product in order to complete the genome in the L1 gene.

Properties of the E6 and E7 genes of candHPV87. Since no functional E5 gene was detected in the candHPV87 genome, E6 and E7 remain the principal viral genes that can directly interact with cell components to regulate cell functions. These genes have been found to play a crucial role in cell cycle regulation and to be responsible for cell transformation in some HPVs and other animal papillomaviruses (1). Interestingly, the transforming activity varies greatly in vivo in the different types of HPV, ranging from high to very low acitivy; some of the molecular correlates of this phenomenon have been described in vitro (2, 13). A possible implication of these genes in cell cycle regulation was investigated in candHPV87. The E6 and E7 putative coding regions of candHPV87 were amplified and cloned in frame into the EcoRV site of an expression vector based on sequences from Human Foamy Virus (HFV) (29). This vector features very strong and pleiotropic expression from the internal promoter of HFV, enhanced by the positive feedback of the bel1 viral transactivator coexpressed by the vector. The cloned insert is expressed as a fusion autocatalytic protein, which processes itself proteolytically to yield the original polypeptide coded by the insert. As controls of high- and low-risk HPV types, the E6 protein from HPV6, E7 from HPV11, and E6 and E7 from HPV16 were cloned in the same vector. The vector alone, which expresses the same viral transactivator and a small unrelated polypeptide, was also included in the experiments as a control. All these constructs were used to transfect NIH 3T3 mouse fibroblasts and the HaCat human keratinocyte-derived cell line (3). Eight-well square plates (Costar, Cambridge, Mass.) were used for culturing NIH 3T3 mouse fibroblast cells (a kind gift of H. Suarez) in Dulbecco's modified minimal essential medium supplemented with 4% newborn calf serum (HyClone, Logan, Utah). The HaCat human keratinocyte-derived cell line (obtained from V. Manni) was grown in the same conditions. Cells were transfected with 400 ng (per well) of DNA by using cationic liposomes (Lipofectin; Bethesda Research Laboratories, Gaithersburg, Md.). Two parameters were monitored after transfection: replication kinetics and focus formation. To study the growth of HaCat- and NIH 3T3-transfected cells, the cells were seeded at 200,000 and 100,000 cells per well, respectively, before transfection and were harvested and counted 4 days thereafter. Figure 3a and b, showing a set of three independent experiments, document the increase in cell growth compared to that of controls (vector alone) and indicate that HPV constructs were all able to enhance cell division (at Student's t-test analysis, this was statistically significant for all HPVs tested except for HPV11 E6 in HaCat cells and for low-risk E6 and E7 in 3T3 cells). This effect was more evident in the case of HPV16, while candHPV87 proteins produced more moderate growth enhancement, although the growth was still greater than that of low-risk HPV proteins (P < 0.05 for HaCat cells for E6). This effect, reproducible in the three independent experiments, was similar in the two cell lines. The activity of the candHPV87 proteins was enhanced when the E6 and E7 coding sequences were cotransfected (each at half concentration) in the same well (P < 0.05 compared to that for E7 alone), suggesting cooperation of these two proteins in their growth-enhancing potential. No foci formation was observed in any experiment with HaCat cells, whereas NIH 3T3 cells showed foci formation after 20 days of unpassaged culture (Fig. 3c).

View larger version (54K): [in this window] [in a new window]   FIG. 3.   Growth enhancement properties of the E6 and E7 proteins of HPVs in transient transfection experiments and foci formation. (a) Growth of HaCat keratinocytic cells 4 days after transfection by using HFV-based vectors (values from three independent experiments, labeled 1, 2, and 3). (b) Growth of NIH 3T3 mouse fibroblasts 4 days after transfection by using HFV-based vectors (values from three independent experiments, labeled 1, 2, and 3). (c) Focus formation in NIH 3T3 fibroblasts (in foci per well). lipof., negative control in which no plasmid was transfected but cells were treated with Lipofectin.

Concluding remarks. Since the introduction and wide application of PCR procedures for the amplification of papillomaviruses from clinical samples, the number of new HPV types identified by these methods has grown steadily. It has also been demonstrated that HIV-1-infected individuals as well as transplant recipients, who nearly always (in contrast to the general population) are persistently and overtly infected by HPVs in the genital epithelia or in the skin, harbor HPV types seldom observed in the general population (16, 31, 38, 39). In this study, we identified 6 putative novel HPV genotypes, from both HIV-1-positive and -negative subjects, and characterized the complete genome of one of them, designated candHPV87 by the Human Papillomavirus Reference Laboratory. The phylogenetic analysis performed in this study showed that the candHPV87 genome clusters within the A3 group of papillomaviruses together with HPV61, HPV72, HPV83, HPV84, and candHPV86, which have been completely sequenced and characterized, and with a number of other putative HPV types (two of which are described in this work) identified as partial L1 sequences. HPV62, HPV72, HPV83, HPV84, and candHPV86 have been described as low- or intermediate-risk papillomaviruses (4, 22, 36, 37), whereas HPV61 has been found in association with vulvar intraepithelial neoplasia (23). candHPV87 was isolated from 4 subjects, 3 of whom were infected with HIV-1. In these 4 cases, HPV detection followed atypical cell findings (koilocytic atypia) at the Pap test, with no sign of progression to high-grade dysplasia. We also observed a putative new type (HAN2500) associated with condylomas in two HIV-1-positive subjects.