BRD4S interacts with viral E2 protein to limit human papillomavirus late transcription.

The E2 protein encoded by human papillomaviruses (HPV) is a sequence-specific DNA-binding protein that recruits viral and cellular proteins. Bromodomain-containing protein 4 (BRD4) is a highly conserved interactor for E2 proteins that has been linked to E2's functions as transcription modulator, activator of viral replication and segregation factor for viral genomes. In addition to BRD4, a short form of BRD4 (BRD4S) is expressed from the BRD4 gene which lacks the C-terminal domain of BRD4. E2 proteins interact with the C-terminal motif (CTM) of BRD4, but a recent study suggested that the phospho-dependent interaction domain (PDID) and the basic interaction domain (BID) in BRD4 also bind to E2. These domains are also present in BRD4S. We now find that HPV31 E2 interacts with the isolated PDID domain in living cells and also with BRD4S which is present in detectable amounts in HPV-positive cell lines and is recruited into HPV31 E1 and E2 induced replication foci. Overexpression and knockdown experiments surprisingly indicate that BRD4S inhibits activities of E2. In line with that, the specific knockdown of BRD4S in the HPV31-positive CIN612-9E cell line induces mainly late viral transcripts. This occurs only in undifferentiated but not differentiated cells in which the productive viral replication cycle is induced. These data suggest that the BRD4S-E2 interaction is important to prevent HPV late gene expression in undifferentiated keratinocytes which may contribute to immune evasion and HPV persistence.ImportanceHuman papillomaviruses (HPV) have coevolved with their host by using cellular factors like bromodomain-containing protein 4 (BRD4) to control viral processes such as genome maintenance, gene expression and replication. We here show that, in addition to the C-terminal motif in BRD4, the phospho-dependent interaction domain in BRD4 interacts with E2 proteins which enable the recruitment of BRD4S, the short isoform of BRD4, to E2. Knock-down and overexpression of BRD4S reveals that BRD4S is a negative regulator of E2 activities. Importantly, the knockdown of BRD4S induces mainly L1 transcripts in undifferentiated CIN612-9E cells, which maintain replicating HPV31 genomes. Our study reveals an inhibitory role of BRD4S on HPV transcription, which may serve as an immune escape mechanism by the suppression of L1 transcripts and thus contribute to the establishment of persistent HPV infections.

Orf Virus-Based Therapeutic Vaccine for Treatment of Papillomavirus-Induced Tumors.

Orf virus (ORFV) represents a suitable vector for the generation of efficient, prophylactic antiviral vaccines against different pathogens. The present study investigated for the first time the therapeutic application of ORFV vector-based vaccines against tumors induced by cottontail rabbit papillomavirus (CRPV). ORFV-CRPV recombinants were constructed expressing the early CRPV gene E1, E2, E7, or LE6. In two independent experiments we used in total 23 rabbits which were immunized with a mixture of the four ORFV-CRPV recombinants or empty ORFV vector as a control 5 weeks after the appearance of skin tumors. For the determination of the therapeutic efficacy, the subsequent growth of the tumors was recorded. In the first experiment, we could demonstrate that three immunizations of rabbits with high tumor burden with the combined four ORFV-CRPV recombinants resulted in significant growth retardation of the tumors compared to the control. A second experiment was performed to test the therapeutic effect of 5 doses of the combined vaccine in rabbits with a lower tumor burden than in nonimmunized rabbits. Tumor growth was significantly reduced after immunization, and one vaccinated rabbit even displayed complete tumor regression until the end of the observation period at 26 weeks. Results of delayed-type hypersensitivity (DTH) skin tests suggest the induction of a cellular immune response mediated by the ORFV-CRPV vaccine. The data presented show for the first time a therapeutic potential of the ORFV vector platform and encourage further studies for the development of a therapeutic vaccine against virus-induced tumors.IMPORTANCE Viral vectors are widely used for the development of therapeutic vaccines for the treatment of tumors. In our study we have used Orf virus (ORFV) strain D1701-V for the generation of recombinant vaccines expressing cottontail rabbit papillomavirus (CRPV) early proteins E1, E2, LE6, and E7. The therapeutic efficacy of the ORFV-CRPV vaccines was evaluated in two independent experiments using the outbred CRPV rabbit model. In both experiments the immunization achieved significant suppression of tumor growth. In total, 84.6% of all outbred animals benefited from the ORFV-CRPV vaccination, showing reduction in tumor size and significant tumor growth inhibition, including one animal with complete tumor regression without recurrence.

The contribution of SP100 to cottontail rabbit papillomavirus transcription and replication.

SP100 proteins are components of nuclear domain 10 structures and have been implicated as inhibitors of human papillomavirus (HPV) replication. In this study, we have addressed the role of SP100 in tumour formation by the cottontail rabbit (Sylvilagus floridanus) papillomavirus (CRPV or SfPV1) in a rabbit model. Tissue culture studies using rabbit keratinocyte lines indicated that rabbit SP100 is an interferon-beta-inducible gene similar to its human counterpart. Stable knockdown of SP100 by shRNA in a cell line harbouring CRPV genomes resulted in a decrease of viral early transcripts. In contrast, infection of domestic rabbits with recombinant CRPV genomes expressing short hairpin (sh)RNAs directed against SP100 did not reveal changes in tumour formation rate, tumour size or early viral transcript levels. However, late viral transcript levels and viral genome copies were consistently lower in CRPV/shSP100-induced tumours than in the control, but these differences did not reach statistical significance. In summary, this study suggests that rabbit SP100 is not an inhibitor but an activator of CRPV replication and transcription.

Controlling the dynamics of an open many-body quantum system with localized dissipation.

We experimentally investigate the action of a localized dissipative potential on a macroscopic matter wave, which we implement by shining an electron beam on an atomic Bose-Einstein condensate (BEC). We measure the losses induced by the dissipative potential as a function of the dissipation strength observing a paradoxical behavior when the strength of the dissipation exceeds a critical limit: for an increase of the dissipation rate the number of atoms lost from the BEC becomes lower. We repeat the experiment for different parameters of the electron beam and we compare our results with a simple theoretical model, finding excellent agreement. By monitoring the dynamics induced by the dissipative defect we identify the mechanisms which are responsible for the observed paradoxical behavior. We finally demonstrate the link between our dissipative dynamics and the measurement of the density distribution of the BEC allowing for a generalized definition of the Zeno effect. Because of the high degree of control on every parameter, our system is a promising candidate for the engineering of fully governable open quantum systems.

The E8 domain confers a novel long-distance transcriptional repression activity on the E8E2C protein of high-risk human papillomavirus type 31.

Infections with high-risk human papillomaviruses (HPVs) are the major risk factor for the development of anogenital cancers. Viral E2 proteins are involved in viral DNA replication and regulation of transcription. Repression of the viral P97 promoter by E2 proteins has been implicated in the modulation of the immortalization capacity and DNA replication properties of high-risk HPVs. Analysis of the cis and trans requirements for repression of the HPV type 31 (HPV31) P97 promoter, however, revealed striking differences between the full-length E2 and the E8E2C fusion protein which were due to conserved residues W6 and K7 of the E8 domain. In contrast to E2, E8E2C completely inhibited the P97 promoter from a single promoter-distal E2 binding site. This novel long-distance repression activity of the E8 domain also enabled E8E2C to inhibit the HPV6a P2 promoter and minimal-promoter constructs containing E2 binding sites. Thus, E8E2C may represent the master repressor of viral gene expression during a high-risk HPV infection, and changes in the activity of E8E2C might contribute to the progression of high-risk HPV-induced lesions.

The E8E2C protein, a negative regulator of viral transcription and replication, is required for extrachromosomal maintenance of human papillomavirus type 31 in keratinocytes.

The viral E2 protein is a major regulator of papillomavirus DNA replication. An important way to influence viral replication is through modulation of the activity of the E2 protein. This could occur through the action of truncated E2 proteins, called E2 repressors, whose role in the replication cycle of human papillomaviruses (HPVs) has not been determined. In this study, using cell lines that contain episomal copies of the "high-risk" HPV type 31 (HPV31), we have identified viral transcripts with a splice from nucleotide (nt) 1296 to 3295. These transcripts are similar to RNAs from other animal and human papillomaviruses and have the potential to fuse a small open reading frame (E8) to the C terminus of E2, resulting in an E8E2C fusion protein. E8E2C transcripts were present throughout the complete replication cycle of HPV31. A genetic analysis of E8E2C in the context of the HPV31 genome revealed that mutation of the single ATG of the E8 gene, introduction of a stop codon downstream of the ATG, or disruption of the splice donor site at nt 1296 led to a dramatic 30- to 40-fold increase in the transient DNA replication levels in both normal and immortalized human keratinocytes. High-level expression of E8E2C from heterologous vectors was found to inhibit E1-E2-dependent DNA replication of an HPV31 origin of replication construct as well as to interfere with E2's ability to transactivate reporter gene constructs. In addition, HPV31 E8E2C strongly repressed the basal activity of the major viral early promoter P97 independent of E2. E8E2C may therefore exert its negative effect on viral DNA replication through modulating E2's ability to enhance E1-dependent DNA replication as well as by regulating viral gene expression. Surprisingly, HPV31 genomes that were unable to express E8E2C could not be maintained extrachromosomally in human keratinocytes in long-term assays despite high transient DNA replication levels. This suggests that the E8E2C protein may play a role in copy number control as well as in the stable maintenance of HPV episomes.

Human papillomavirus life cycle: active and latent phases.

Productive infection by human papillomaviruses (HPV) is dependent upon the differentiation of the host cell. Following entry into basal epithelial cells, HPV genomes are established as autonomous replicating extrachromosomal elements and a low level of HPV expression occurs. Upon differentiation of infected cells, productive replication and expression of capsid genes is induced resulting in the synthesis of progeny virions. Evidence from immunosuppressed patients as well as individuals with recurring laryngeal papillomatosis suggest that certain HPV types can exist in a latent state. In latently infected cells, HPV DNA may be present but no differentiation-dependent synthesis of virions occurs. The presence of a latent state for HPVs can be a determining factor in the effectiveness of therapeutic methods for treatment of infections.

Transactivation by the E2 protein of oncogenic human papillomavirus type 31 is not essential for early and late viral functions.

The activation of transcription and of DNA replication are, in some cases, mediated by the same proteins. A prime example is the E2 protein of human papillomaviruses (HPVs), which binds ACCN6GGT sequences and activates heterologous promoters from multimerized binding sites. The E2 protein also has functions in replication, where it complexes with the virally encoded origin recognition protein, E1. Much of the information on these activities is based on transient-transfection assays as well as biochemical analyses; however, their importance in the productive life cycle of oncogenic HPVs remains unclear. To determine the contributions of these E2 functions to the HPV life cycle, a genetic analysis was performed by using an organotypic tissue culture model. HPV type 31 (HPV31) genomes that contained mutations in the N terminus of E2 (amino acid 73) were constructed; these mutants retained replication activities but were transactivation defective. Following transfection of normal human keratinocytes, these mutant genomes were established as stable episomes and expressed early viral transcripts at levels similar to those of wild-type HPV31. Upon differentiation in organotypic raft cultures, the induction of late gene expression and amplification of viral DNA were detected in cell lines harboring mutant genomes. Interestingly, only a modest reduction in late gene expression was observed in the mutant lines. We conclude that the transactivation function of E2 is not essential for the viral life cycle of oncogenic HPVs, although it may act to moderately augment late expression. Our studies suggest that the primary positive role of E2 in the viral life cycle is as a replication factor.

Activation of papillomavirus late gene transcription and genome amplification upon differentiation in semisolid medium is coincident with expression of involucrin and transglutaminase but not keratin-10.

The life cycle of the papillomaviruses is closely linked to host cell differentiation, as demonstrated by the fact that amplification of viral DNA and transcription of late genes occur only in the suprabasal cells of a differentiated epithelium. Previous studies examining the pathogenesis of papillomavirus infections have relied on the use of organotypic raft cultures or lesions from patients to examine these differentiation-dependent viral activities. In this study, we used a simple system for epithelial differentiation to study human papillomavirus (HPV) late functions. We demonstrate that the suspension of HPV-infected keratinocytes in semisolid medium containing 1.6% methylcellulose for 24 h was sufficient for the activation of the late promoter, transcription of late genes, and amplification of viral DNA. These activities were shown to be linked to and coincide with cellular differentiation. Expression of the late protein E1(wedge)E4 and amplification of viral DNA were detected in the identical set of cells after suspension in methylcellulose. This technique was also used to analyze the differentiation properties of the cells which expressed the late protein E1(wedge)E4. While induction of the spinous layer markers involucrin and transglutaminase was compatible with late promoter induction, expression of the differentiation-specific keratin-10 was shown not to be required for HPV late functions. Interestingly, while the majority of normal human keratinocytes induced filaggrin expression by 24 h, this marker of the granular layer was induced in a smaller subset of HPV type 31 (HPV-31)-positive cells at this time point. The HPV-31-positive cells which expressed filaggrin did not induce the late protein E1(wedge)E4. Use of the methylcellulose system to induce epithelial differentiation coupled with the ability to perform a genetic analysis of HPV functions by using transfection of cloned viral DNA will facilitate the study of the regulation of the papillomavirus life cycle.

Differential requirements for conserved E2 binding sites in the life cycle of oncogenic human papillomavirus type 31.

Human papillomavirus (HPV) E2 proteins regulate viral replication by binding to sites in the upstream regulatory region (URR) and by complex formation with the E1 origin recognition protein. In the genital HPV types, the distribution and location of four E2 binding sites (BS1 to BS4) which flank a single E1 binding site are highly conserved. We have examined the roles of these four E2 sites in the viral life cycle of HPV type 31 (HPV31) by using recently developed methods for the biosynthesis of papillomaviruses from transfected DNA templates (M. G. Frattini et al., Proc. Natl. Acad. Sci. USA 93:3062-3067, 1996). In transient assays, no single site was found to be necessary for replication, and mutation of the early promoter-proximal site (BS4) led to a fourfold increase in replication. Cotransfection of the HPV31 wild-type (HPV-wt) and mutant genomes with expression vectors revealed that E1 stimulated replication of HPV31-wt as well as the HPV31-BS1, -BS2, and -BS3 mutants. In contrast, increased expression of E2 decreased replication of these genomes. Replication of the HPV31-BS4 mutant genome was not further increased by cotransfection of E1 expression vectors but was stimulated by E2 coexpression. In stably transfected normal human keratinocytes, mutation of either BS1, BS3, or BS4 resulted in integration of viral genomes into host chromosomes. In contrast, mutation of BS2 had no effect on stable maintenance of episomes or copy number. Following growth of stably transfected lines in organotypic raft cultures, the differentiation-dependent induction of late gene expression and amplification of viral DNA of the BS2 mutant was found to be similar to that of HPV31-wt. We were unable to find a role for BS2 in our assays for viral functions. We conclude that at least three of the four E2 binding sites in the URRs of HPVs are essential for the productive viral life cycle. The specific arrangement of E2 binding sites within the URR appears to be more important for viral replication than merely the number of sites.

Differential effects of the splice acceptor at nucleotide 3295 of human papillomavirus type 31 on stable and transient viral replication.

In human papillomavirus type 31 (HPV-31), the E1--E4 and E5 open reading frames are expressed from polycistronic mRNAs. The major polycistronic mRNAs which encode E1--E4 and E5 are spliced messages which utilize a splice acceptor at nucleotide (nt) 3295 (SPA3295). Our laboratory recently developed a recombinant system for the synthesis of HPVs following immortalization of primary keratinocytes with cloned HPV-31 genomes (M. G. Frattini et al., Proc. Natl. Acad. Sci. USA 93:3062-3067, 1996). These immortalized cell lines are capable of maintaining HPV-31 DNA as episomes and induce the synthesis of virions in organotypic raft culture. In this study, we used these methods to begin an analysis of the roles of E1--E4 and E5 in HPV pathogenesis by mutating the major splice at nt 3295. Mutation of SPA3295 did not significantly alter the ability of HPV-31 genomes to replicate transiently in keratinocytes, nor did the mutation affect the immortalization potential of HPV-31. However, genomes carrying the SPA3295 mutation were not stably maintained as viral episomes, and the resulting immortalized keratinocyte cell line contained multiple, integrated copies of the mutated HPV-31 DNA. Northern analysis indicated that cell lines immortalized with the mutant HPV-31 expressed transcripts which were similar in size and abundance to wild-type messages, including those transcripts which rely on utilization of SPA3295. RNase protection and reverse transcription-PCR revealed that mutation of SPA3295 resulted in the utilization of a cryptic splice acceptor at nt 3298. These data suggest that the requirements for stable maintenance of HPV genomes are more stringent than those for transient replication and that factors which define these requirement rely on the major splice acceptor at nt 3295.

E2 represses the late gene promoter of human papillomavirus type 8 at high concentrations by interfering with cellular factors.

The late gene promoter P7535 of the epidermodysplasia verruciformis-associated human papillomavirus type 8 (HPV8) is regulated by the viral E2 protein. Transfection experiments performed with the human skin keratinocyte cell line RTS3b and P7535 reporter plasmids revealed transactivation at low amounts and a repression of basal promoter activity at high amounts of E2 expression vector. This repression was promoter specific and correlated with the amount of transiently expressed E2 protein. Mutational analyses revealed that the negative regulation of P7535 activity is mediated by the low-affinity E2 binding site P2, which is separated by one nucleotide from the P7535 TATA box. Biochemical and genetic analyses suggested that repression is due to a displacement of the TATA-box binding protein by E2 and an interference of E2 with promoter-activating cellular factors that specifically recognize the P2 sequence. The high conservation of the P2 sequence among several papillomaviruses (epidermodysplasia verruciformis-associated HPVs, HPV1, cottontail rabbit papillomavirus, and bovine papillomavirus type 1) in the vicinity of the late gene promoter cap site suggests that an interplay of E2 and cellular factors at this sequence element is important for the expression of structural proteins.

Differentiation-dependent transcription of the epidermodysplasia verruciformis-associated human papillomavirus type 5 in benign lesions.

Human papillomavirus type 5 (HPV 5) induces cutaneous lesions and persists in skin carcinomas of patients with epidermodysplasia verruciformis (EV). We investigated the expression pattern of HPV 5 in biopsies from benign skin lesions of EV patients by cDNA analysis and in situ hybridization. Nine different cDNAs could be generated from total RNA of one of these lesions by reverse transcription and PCR amplification with HPV 5-specific primers. We could identify two major splice donors: one was found in the E6-proximal part of the noncoding region (NCR), and the other just downstream of the first ATG codon of ORF E1. Each of the characterized transcripts was processed at one or the other donor site and the two corresponding leader exons were found in combination with both 3'-early and late exons. Two transcripts appear to be specific for EV-associated papillomaviruses: one species might encode an E1--E2C fusion protein, and the other mRNA (NCR/E2) is probably encoding for the full-length E2 protein. According to the results of the cDNA analysis, riboprobes were designed for in situ hybridization experiments to study the cell differentiation-dependent expression of the different exons. Only the E7/E1 and E4 probes led to strong signals almost throughout the epithelium. The signals generated by the 5'-E2 and E1 probe increased with cell differentiation and were mainly confined to the nucleus. The NCR, E6, E7, L2, and L1 probes yielded more or less strong signals in the terminally differentiated epidermal layers. The difference in the cell differentiation-dependent expression of the 5'-early region exon (probe E7/1) and L2/L1 exons may point to a differentiation-dependent processing of transcripts.

Low-affinity E2-binding site mediates downmodulation of E2 transactivation of the human papillomavirus type 8 late promoter.

The constitutively active promoter P7535 of the epidermodysplasia verruciformis-associated human papillomavirus type 8 (HPV8) is transactivated by the viral E2 protein. The distribution of potential E2-binding sites (ACCN6GGT) in the viral transcription control region is highly conserved among epidermodysplasia verruciformis-associated human papillomaviruses and differs completely from that of other papillomaviruses. To investigate the role of E2-binding sites P0 to P4 in P7535 regulation, we analyzed their binding affinities in gel retardation experiments using a full-length HPV8 E2 protein expressed from a recombinant baculovirus. Binding site P1 within a transcriptional silencer showed the highest affinity, followed by P0 within the L1 gene and P3 downstream of P7535. P2, 33 nucleotides upstream of the mRNA cap site, and P4 were very weak binders. There is some indication that the number of A/T pairs in the nonconserved core of the recognition sequence is critical for the binding of HPV8 E2. Transient transfection experiments were carried out with an HPV8 E2 expression vector and reporter plasmids containing mutated E2-binding sites in the context of the HPV8 regulatory region. The knockout of the strongest binding site P1 sufficed to clearly diminish transactivation. P0, P3, and P4 mutations had little effect on their own, whereas double mutations P01 and P34 strongly reduced E2 inducibility. Both mutations in P2 severely affected constitutive promoter activity but had opposite effects on transactivation. They revealed an inverse correlation between E2-binding strength and the extent of E2 transactivation. This finding suggests that P2 mediates a negative control of P7535 by E2, counteracting E2 transactivation exerted via the four distal E2 target sequences.

Prevalence of deletions of YY1-binding sites in episomal HPV 16 DNA from cervical cancers.

Expression of the oncogenes E6 and E7 of human papillomavirus 16 (HPV 16) appears enhanced in pre-malignant and malignant genital tumors. We recently identified a transcriptional silencer upstream of the oncogene promoter P97, comprising 4 binding sites for the cellular YY1 protein. The analysis of the long transcriptional control regions (LCR) of episomal HPV 16 DNAs from primary tumors and lymph-node metastases of 6 patients with cervical cancer revealed deletions and point mutations of YY1 binding sites in 4 cases. To test for the activity of the P97 promoter, the mutated LCRs were cloned in a luciferase reporter gene vector. A point mutation in YY1-recognition site 4, which prevents DNA-protein interaction, did not affect promoter activity, probably due to compensation by the overlapping YY1-binding site 3. However, 5.5- to 6.5-fold increased luciferase expression was obtained under the control of 3 shortened LCRs lacking 2 to 4 YY1-binding sites. A point mutation in YY1-recognition site 2, which was previously shown to stimulate P97 3.5-fold, could be detected in the HPV 16 LCRs from both primary tumor and metastasis, indicating that the mutation is a stable characteristic of HPV 16 DNA associated with the individual cancer. These findings suggest that deletions or mutations of YY1-binding sites play a significant role in over-expression of viral oncogenes and tumor progression.

The E6/E7 promoter of extrachromosomal HPV16 DNA in cervical cancers escapes from cellular repression by mutation of target sequences for YY1.

Human papillomavirus type 16 (HPV16) induces squamous intraepithelial lesions of the cervical mucosa which may develop into invasive cancer. The expression of viral oncogenes in advanced neoplasias appears increased relative to the proliferating cell layers of low grade lesions raising questions about molecular mechanisms of deregulation of transcription. In a lymph node metastasis of a cervical cancer, we observed full-length HPV16 plasmids and molecules with a small deletion, which was mapped to the long control region (LCR). Both wild type and shortened LCR were amplified by PCR, cloned into the promoter test plasmid pBLCAT6 and sequenced to identify a 107 bp deletion from position 7794 to 7901 in the short LCR. CAT expression in cervical cancer-derived HT3, SiHa and CaSki cells appeared 5- to 6-fold increased under the control of the short LCR. This could be traced back to elevated levels of mRNA initiated at the viral oncogene promoter. A slight further increase in CAT expression was noted in the presence of the HPV16 E2 protein which is probably due to the deletion of one E2 binding site and consequent relief from E2 repression. Computer-assisted sequence analysis and band-shift experiments with purified YY1 protein and wild type or mutated oligonucleotides identified four binding sites for this cellular transcriptional repressor within the promoter-proximal segment of the HPV16 LCR, three of which were removed by the deletion. A LCR fragment comprising these YY1 binding sites was cloned in front of the heterologous thymidine kinase gene promoter and suppressed CAT expression 3- to 4-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Late promoter of human papillomavirus type 8 and its regulation.

Human papillomavirus type 8 (HPV8) belongs to the HPV types associated with skin carcinomas of patients with epidermodysplasia verruciformis (EV). Its noncoding regulatory sequences (NCR) were shown to drive the expression of the reporter gene chloramphenicol acetyltransferase (cat) in transient assays with human epithelial cells (HT3 cells). This constitutive activity could be enhanced by coexpression of the HPV8 transactivator protein E2. The analysis of 5' deletions of the NCR showed that the EV-specific sequence motif M33 and the neighboring AP1 site are essential for the promoter activity, whereas 44 nucleotides located immediately upstream of M33 are strongly inhibitory. The same effects were observed in simian virus 40-immortalized fetal keratinocytes (SV61 cells) and spontaneously immortalized skin keratinocytes (HaCaT cells). By using primer extension and RNase protection analyses two promoters could be identified within the HPV8 NCR. A nested set of weak signals, corresponding to start sites between positions 175 to 179, represented the previously described E6 promoter. The vast majority of transcripts was initiated at position 7535 and shown to undergo processing at an NCR-internal splice donor (positions 1 to 8). The promoter P7535 is similar to late promoters of other skin-associated papillomaviruses as far as localization, transcript structure, and sequence characteristics are concerned. To confirm that P7535-initiated transcripts proceed indeed to the L1 gene for the major capsid protein, viral mRNAs from an HPV8-induced lesion of a patient with EV were characterized by RNase protection and sequence analysis of polymerase chain reaction-amplified cDNAs. The NCR leader (positions 7535 to 4) appeared in two messages with three exons each. The third exon started with the second ATG codon of L1 in both cases; the short central exons from the 3' part of the early coding region were defined by a common splice acceptor site (position 3303) and different splice donor sites (positions 3443 and 3704).