Human Papillomavirus Type 6a Research Articles

Refined physical map of the human PAX2/HOX11/NFKB2 cancer gene region at 10q24 and relocalization of the HPV6AI1 viral integration site to 14q13.3-q21.1.

BackgroundChromosome band 10q24 is a gene-rich domain and host to a number of cancer, developmental, and neurological genes. Recurring translocations, deletions and mutations involving this chromosome band have been observed in different human cancers and other disease conditions, but the precise identification of breakpoint sites, and detailed characterization of the genetic basis and mechanisms which underlie many of these rearrangements has yet to be resolved. Towards this end it is vital to establish a definitive genetic map of this region, which to date has shown considerable volatility through time in published works of scientific journals, within different builds of the same international genomic database, and across the differently constructed databases.ResultsUsing a combination of chromosome and interphase fluorescent in situ hybridization (FISH), BAC end-sequencing and genomic database analysis we present a physical map showing that the order and chromosomal orientation of selected genes within 10q24 is CEN-CYP2C9-PAX2-HOX11-NFKB2-TEL. Our analysis has resolved the orientation of an otherwise dynamically evolving assembly of larger contigs upstream of this region, and in so doing verifies the order and orientation of a further 9 cancer-related genes and GOT1. This study further shows that the previously reported human papillomavirus type 6a DNA integration site HPV6AI1 does not map to 10q24, but that it maps at the interface of chromosome bands 14q13.3-q21.1.ConclusionsThis revised map will allow more precise localization of chromosome rearrangements involving chromosome band 10q24, and will serve as a useful baseline to better understand the molecular aetiology of chromosomal instability in this region. In particular, the relocation of HPV6AI1 is important to report because this HPV6a integration site, originally isolated from a tonsillar carcinoma, was shown to be rearranged in other HPV6a-related malignancies, including 2 of 25 genital condylomas, and 2 of 7 head and neck tumors tested. Our finding shifts the focus of this genomic interest from 10q24 to the chromosome 14 site.

Cell-type-specific separate regulation of the E6 and E7 promoters of human papillomavirus type 6a by the viral transcription factor E2.

Gene expression of human papillomaviruses (HPV) is tightly controlled by cellular factors and by the virally encoded E2 protein through binding to distinct sites within the regulatory noncoding region. While for the high-risk genital papillomaviruses a single promoter drives the expression of all early genes, a second promoter present in the E6 open reading frame of the low-risk HPV type 6 (HPV6) would allow an independent regulation of E6 and E7 oncogene expression. In this report, we provide the first evidence that E2 regulates both early promoters of HPV6 separately and we show that promoter usage as well as E2 regulation is cell type dependent. Among the different epithelial cell lines tested, only RTS3b cells allowed an expression pattern similar to that observed in naturally infected benign condylomas. While the E6 promoter was repressed by E2 to 50% of its basal activity, the E7 promoter was simultaneously stimulated up to fivefold. Activation of the E7 promoter was mediated predominantly by the binding of E2 to the most promoter-distal E2 binding site. Repression of the E6 promoter depended on the presence of two intact promoter-proximal binding sites. Mutation of both of these repressor binding sites reversed the effect of E2 on the E6 promoter from repression to activation. In contrast, in HT3 cells we observed an E2-mediated activation of the E6 promoter in the context of the wild-type noncoding region. This indicated that repression of the E6 promoter by binding of E2 to both promoter-proximal binding sites did not function in the cellular environment provided by HT3 cells. These data suggest that the separate regulation of the E6 and E7 promoters of HPV6 is mediated through successive occupation of binding sites with different affinities for E2 depending on the intracellular concentration of E2 and on the cellular environment provided by the infected cell.

Expression of the major capsid protein of human papillomavirus type 11 in Saccharomyces cerevisae

The major capsid protein L1 of papillomaviruses expressed recombinantly or in infected cells has the intrinsic ability to form virus-like particles (VLPs) which display conformational epitopes necessary to elicit high-titered, virus-neutralizing antibodies. We have shown previously that the L1 gene of human papillomavirus type 6a (HPV6) can be expressed efficiently in Saccharomyces cerevisae (Sc) as VLPs. However, when we attempted to express the L1 gene cloned from the closely related HPV11 in yeast, few VLPs were observed in crude lysates. The lower expression level of HPV11 L1 protein was found to result from a truncation of the HPV11 L1 mRNA. Since sequence requirements for transcriptional termination in yeast are still unclear, the HPV6 L1 gene was used as the basis for the complete synthetic reconstruction of the entire 1506-bp HPV11 L1 gene. Expression of this HPV6/11 hybrid L1 gene in yeast resulted in predominantly full-length L1 mRNA and a > 7-fold increased level of production of HPV11 VLPs compared to that expressed by the wild-type HPV11 L1 gene. The VLPs were shown to display the conformational epitopes important to elicit virus-neutralizing antibodies

Stimulation of Human Papillomavirus Type 1a DNA Replication by a Multimerized AT-Rich Palindromic Sequence

Replication of most papillomaviruses requires the viral E1 and E2 proteins and an origin of replication containing the E1 and E2 binding sites. In the case of human papillomavirus type 1a (HPV-1a), the E1 protein alone is sufficient for DNA replication although the E2 protein significantly stimulates replication. We have further analyzed the role ofcis-acting sequences and the E1 and E2 proteins in HPV-1a replication. Previous studies have shown that a 60-bp region lacking the E2 binding sites but containing an imperfect 16-bp AT-rich palindrome corresponding to the putative E1 binding site contains the minimal origin of replication (ori). Using a transient replication assay, we demonstrate that duplication of this 60-bp region causes a severalfold increase in replication. Synthetic oligonucleotides containing a 39-bp region centered around the above palindromic sequence supported only low-level replication in the presence of either E1 alone or both E1 and E2, but replication was significantly increased in the presence of multiple copies of this sequence. Plasmids containing a 19-bp sequence which includes the AT-rich palindrome failed to replicate, but multiple copies of this region supported replication in the presence of both E1 and E2 to significant levels. The results presented indicate that the HPV-1a E1 protein is capable of recruiting all the cellular factors required for replication. Our results also suggest that multimerization of the AT-rich palindromic sequence may result in a significant increase in the recruitment of the E1 protein to the origin, thereby stimulating replication. This increased targeting of the E1 protein to the origin containing multiple copies of the putative E1 binding site may be functionally similar to the targeting of E1 to the origin by E2.

Deletion in the L1 open reading frame of human papillomavirus type 6a genomes associated with recurrent laryngeal papilloma

Human papillomavirus (HPV) type 6a genomes with a large deletion in their L1 open reading frames (ORF) were found in two of five recurrent cases of laryngeal papilloma. One of these mutant HPVs had a 186 base pair (bp) deletion near the N-terminus end of the L1 ORF, which encodes a major capsid protein. The other had a 454 bp deletion at the C-terminus end of L1 at which is located a nuclear localising signal (NLS). No other large deletion or insertion was found in the remaining regions of all five HPV6a genomes. The laryngeal papillomas which harboured the mutant viruses showed typical hyperplasia and pathological changes as observed in tumours induced by the wild-type virus. The biological significance of the two large deletions in the late region of HPV6a associated with laryngeal papilloma is discussed.

Sequence Determination of Human Papillomavirus Type 6a and Assembly of Virus-like Particles in Saccharomyces cerevisiae

Human papillomavirus 6a (HPV6a), the most abundant HPV6 subtype, was detected in a vulvar condyloma acuminatum. The complete genome of HPV6a was cloned, and its DNA sequence was shown to be over 97% identical to the HPV6b sequence. Of the eight open reading frames (ORFs) of HPV6a, only the imputed amino acid sequence of the major capsid protein L1 was identical to the corresponding HPV6b sequence; all other HPV6a ORFs showed amino acid changes compared to the HPV6b ORFs. The HPV6a L1 or the L1 + L2 ORFs were expressed in the yeast Saccharomyces cerevisiae. Self-assembly of the L1 capsid protein into virus-like particles (VLPs) was demonstrated both in the L1 as well as L1 + L2 coexpressing yeast strains. Copurification of the L1 and L2 proteins showed complex formation of the L1 and L2 proteins in the yeast-derived VLPs of coexpressing strains.

E1 protein of human papillomavirus type 1a is sufficient for initiation of viral DNA replication.

Previous studies on transient replication of papillomaviruses have shown an absolute requirement for the viral E1 and E2 proteins in DNA replication. Here we demonstrate that for human papillomavirus type 1a (HPV-1a) DNA, the E1 protein alone is sufficient for in vivo replication of plasmids containing the viral origin of replication. Replication was origin-specific and required the presence of a DNA sequence containing a putative E1 binding site, but the E2 binding sites were dispensable. In the presence of the E1 protein, E2 stimulated replication of plasmids containing the E1 and E2 binding sites, but no stimulation was observed when the origin plasmids lacked E2 binding sites. Conversely, in the presence of E1 alone, the E2 binding sites did not affect replication. Plasmids containing the replication origins of HPV-6b, HPV-18, and bovine papillomavirus type 1 (BPV-1) also replicated efficiently in the presence of the HPV-1a E1 and E2 proteins. However, plasmids containing the origins of HPV-6b and HPV-18 failed to replicate in the presence of HPV-1a E1 alone, whereas a plasmid containing the BPV-1 origin replicated to lower levels than the HPV-1a origin-containing plasmid. These results suggest that replication from papillomaviral origins in the presence of E1 alone is presumably dependent on the strength of E1-origin interactions. Additionally, E1-dependent replication is stimulated by the E2 protein in the presence of E2 binding sites.

Analysis of human papillomavirus types in exophytic condylomata acuminata by hybrid capture and Southern blot techniques.

Exophytic condylomata acuminata of the external genitalia of 40 patients were analyzed for human papillomavirus (HPV) DNA by the Southern blot and hybrid capture methods. All lesions were initially analyzed by the Southern blot method by using a mixture of HPV type 6, 11, 16, and 18 whole genomic probes. Southern blots demonstrated characteristic PstI restriction patterns of HPV type 6, 11, or 16 in all but one lesion. HPV 6 subtypes accounted for 28 of 39 HPV-positive lesions. Twenty-seven of these 28 lesions contained HPV type 6a, and 1 lesion contained HPV type 6c. Eight lesions contained HPV type 11 and three contained HPV type 16. Two of the three condylomata acuminata containing HPV type 16 were obtained from solid-organ transplant recipients receiving immunosuppressive medications. The third lesion containing HPV type 16 was a typical exophytic condyloma acuminatum from a woman with previously resected vulvar carcinoma. The hybrid capture assay detected HPV DNAs in all lesions except the Southern blot-negative lesion. Twenty-five lesions were positive for the A probe only (HPV types 6 and 11 and related types). All of these lesions were found to contain HPV type 6 or 11 sequences in the Southern blot assay. The remaining 14 lesions were positive for both the A probe and the B probe (HPV types 16 and 18 and related types). The strongest signal in these 14 lesions by the hybrid capture assay was consistent with the result of the Southern blot assay in all but one case. We conclude that (i) HPV type 6a is the most common type found in these lesions, (ii) HPV type 16 may be present more often in exophytic condylomata acuminata from immunosuppressed individuals, (iii) hybrid capture is a useful tool for documenting the presence of HPV sequences in DNAs from exophytic condylomata acuminata, and (iv) in samples containing multiple HPV types, hybrid capture allows detection of minority HPV types.

Transfection of cos cell with human papillomavirus type-1a and -2a DNA

Transfection of cos cell with human papillomavirus type-1a and -2a DNA

Transfection of human keratinocytes with human papillomavirus type-1a, -2a and -4 DNA

Transfection of human keratinocytes with human papillomavirus type-1a, -2a and -4 DNA

A common function for polyoma virus large-T and papillomavirus E1 proteins?

Nucleotide sequencing has revealed a common genetic organization for three papillomaviruses: BPV-1 (bovine papillomavirus type 1), HPV-1 (human papillomavirus type 1a) and HPV-6 (human papillomavirus type 6b). Several open reading frames, corresponding to as yet uncharacterized proteins, were observed in these genomes in the region that is required for oncogenic transformation by BPV-1 and for plasmidial maintenance of its genome. The longest of these frames, E1, is also the most conserved between the three viruses; we have compared the amino acid sequence of its putative product ('E1 protein') with those of the large-T proteins of three polyoma viruses and report here significant homologies in their carboxy-terminal halves, extending for over 200 amino acids. Moreover, similar secondary structures were predicted in this region, especially in two blocks of homologous residues, which correspond in the large-T proteins of polyoma and simian virus 40 (SV40) viruses to sites involved in the ATPase and nucleotide-binding activities. These observations suggest that the papillomavirus E1 proteins might have a function in common with the polyoma virus large-T proteins (which are required for the initiation of viral DNA replication). As it was suggested recently that the E1 gene product is involved in maintaining the BPV-1 genome as a plasmid in transformed cells, we speculate that the structural features conserved in these otherwise very different viruses are general characteristics of eukaryotic proteins involved in the control of DNA replication.

Uneven distribution of methylation sites within the human papillomavirus la genome: possible relevance to viral gene expression

A homogeneous preparation of human papillomavirus type 1a (HPV-1a) DNA resisted complete cleavage by the methylation-sensitive restriction endonuclease HhaI. Ten fragments additional to those predicted from the known HPV-1a DNA sequence were resolved by agarose gel electrophoresis of the HhaI-cleaved viral DNA. By determining the composite structures of the additional HhaI viral fragments, evidence was found for part-methylation of six of the thirteen HhaI sites. Two of the modified HhaI sites were localized to the 3'-end of the putative early gene region. The other four modified Hha-I sites were situated within the L1 open reading frame of the putative late gene region. Ten successive restriction endonuclease sites occurring close to and within an area of high CG density which surrounds the 5' end of the putative early gene region, were not modified detectably. The possible relevance of DNA methylation to the control of HPV-1a gene expression in epidermal cells is discussed.

DNA sequence and genome organization of genital human papillomavirus type 6b.

The complete nucleotide sequence of the circular double-stranded DNA of the genital human papillomavirus type 6b (HPV6b) comprising 7902 bp was determined and compared with the DNA sequences of human papillomavirus type 1a (HPV1a) and bovine papillomavirus type 1 (BPV1). All major open reading frames are located on one DNA strand only. Their arrangement reveals that the genomic organization of HPV6b is similar to that of HPV1a and BPV1. The putative early region includes two large open reading frames E1 and E2 with marked amino acid sequence homologies to HPV1a and BPV1 which are flanked by several smaller frames. The internal part of E2 completely overlaps with another open reading frame E4. The putative late region contains two large open reading frames L1 and L2. The L1 amino acid sequences are highly conserved among analyzed papillomavirus types. By sequence comparison, potential promoter, splicing and polyadenylation signals can be localized in HPV6b DNA suggesting possible mechanisms of genital papillomavirus gene expression.