Mutational analysis of open reading frame E4 of bovine papillomavirus type 1.

Open reading frame (ORF) E4 is a 353-base-pair ORF of bovine papillomavirus type 1. To determine the biological activities of this ORF in mouse C127 cells, we analyzed the effects of two constructed mutations which are predicted to prevent synthesis of ORF E4 proteins while leaving the amino acid sequence encoded by the overlapping ORF E2 unchanged. Neither mutation interfered with the abilities of the mutants to efficiently induce focus formation, induce growth in soft agarose, or transactivate an inducible bovine papillomavirus type 1 enhancer. Also, neither mutation prevented establishment of the viral DNA as an extrachromosomal plasmid in transformed cells. These results suggest that ORF E4 proteins are not required for these biological activities, and they are consistent with the observation of others (J. Doorbar, D. Campbell, R. J. A. Grand, and P. H. Gallimore, EMBO J. 5:355-362, 1986) that the ORF E4 protein of a human papillomavirus is associated with late gene expression during papilloma formation.     

Expression of human papillomavirus type 6 E1, E2, L1 and L2 open reading frames in Escherichia coli

Open reading frame (ORF) fragments (putative gene fragments) from human papillomavirus type 6b (HPV-6b) were inserted into the bacterial expression vector pHK413 to provide viral antigenic determinants. Approximately 86% of the entire L1 ORF, 82% of the E2 ORF, and 52% of the L2 ORF were expressed in Escherichia coli. The E1 ORF was cloned as two fragments. The constructions containing E1n (coding for the N-terminal region) and E1c (coding for the C-terminal region) expressed 27% and 16% of the E1 ORF, respectively. Protein encoded by the L1 ORF, but not that encoded by the L2 ORF, reacted with antibodies elicited by disrupted bovine papillomavirus. These reagents will be extremely useful in unravelling the HPV-6b replication cycle.     

The genomes of the animal papillomaviruses European elk papillomavirus, deer papillomavirus, and reindeer papillomavirus contain a novel transforming gene (E9) near the early polyadenylation site.

We report that the genomes of reindeer papillomavirus (RPV), European elk papillomavirus (EEPV), and deer papillomavirus (DPV) contain a short conserved translational open reading frame (ORF), E9, which is located between the E5 ORF and the early polyadenylation site. In RPV, DPV, and EEPV, E9 ORFs have the potential to encode extremely hydrophobic polypeptides of approximately 40 amino acids. In mouse C127 cells transformed by EEPV and RPV, there exists a unique, abundant mRNA species of approximately 700 nucleotides which has the capacity to encode an E9 polypeptide. This mRNA is transcribed from a previously unrecognized promoter at position 4030 in the EEPV genome. The EEPV E9 ORF exhibits weak transforming activity in C127 cells and primary rat embryo fibroblasts. We also show that EEPV E5 is the major oncogene in the EEPV genome when assayed in C127 cells, although it is less efficient in transformation than the E5 genes of bovine papillomavirus type 1, DPV, and RPV.     

E5 open reading frame of bovine papillomavirus type 1 encodes a transforming gene.

We have previously shown that the early region of the bovine papillomavirus type 1 genome contains two nonoverlapping segments that can independently induce the morphological transformation of cultured cells. The transforming gene from the 5' end of the early region is encoded by the E6 open reading frame. The second transforming segment was previously localized to a 2.3-kilobase fragment (2.3T) from the 3' end of the early region. To determine which of the four open reading frames (E2, E3, E4, and E5) located within 2.3T encodes a transforming gene, we have now introduced a series of insertion and deletion mutations into a clone (pHLB1) in which 2.3T is activated by the Harvey viral long terminal repeat, and we tested the mutants for their ability to induce focal transformation. Our results indicate that the E5 open reading frame, which could encode a low-molecular-weight hydrophobic peptide, is required for pHLB1-induced transformation of NIH 3T3 cells, but that the E2, E3, and E4 open reading frames are not.     

Identification of a 68-kilodalton nuclear ATP-binding phosphoprotein encoded by bovine papillomavirus type 1.

E1 is the largest open reading frame (ORF) of bovine papillomavirus type 1 (BPV-1) and is highly conserved among all papillomaviruses, maintaining its size, amino acid composition, and location in the viral genome with respect to other early genes. Multiple viral replication functions have been mapped to the E1 ORF of BPV-1, and evidence suggested that more than one protein was encoded by this ORF. We previously identified a small protein (M) whose gene consists of two exons, one encoded by the 5' end of the E1 ORF. We show here that a 68-kilodalton (kDa) phosphoprotein made from the E1 ORF can be detected in BPV-1-transformed cells, and we present evidence that this protein is encoded by sequences colinear with the entire E1 ORF. The full-length E1 protein immunoprecipitated from virally transformed cells and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis comigrates with a protein expressed from a recombinant DNA construct capable of producing only the complete E1 protein. In addition, two different antisera directed against polypeptides encoded from either the 3' or the 5' end of the E1 ORF both recognize the full-length E1 product. A mutation converting the first methionine codon in the ORF to an isoleucine codon abolishes BPV-1 plasmid replication and E1 protein production. Consistent with the notion that this methionine codon is the start site for E1, a mutant with a termination codon placed after the splice donor at nucleotide 1235 in E1 produces a truncated protein with the molecular mass predicted from the primary sequence as well as the previously identified M protein. When visualized by immunostaining, the E1 protein expressed in COS cells is localized to the cell nucleus. A high degree of similarity exists between the BPV-1 E1 protein and polyomavirus and simian virus 40 large-T antigens in regions of the T antigens that bind ATP. We show by ATP affinity labeling that the E1 protein produced in COS cells binds ATP and that this activity is abolished by a point mutation which converts the codon for proline 434 to serine. Furthermore, this mutation renders the viral genome defective for DNA replication, suggesting that the ATP-binding activity of E1 is necessary for its putative role in viral DNA replication.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transforming activity of E5a protein of human papillomavirus type 6 in NIH 3T3 and C127 cells.

Human papillomavirus type 6 (HPV-6) is the etiologic agent of genital warts and recurrent respiratory papillomatosis. We are investigating the mechanism by which this virus stimulates cell proliferation during infection. In this paper, we report that the E5a gene of HPV-6c, an independent isolate of HPV-11, is capable of transforming NIH 3T3 cells. The E5a open reading frame (ORF) was expressed under the control of the mouse metallothionein promoter in the expression vector pMt.neo.1, which also contains the gene for G418 resistance. Transfected cells were selected for G418 resistance and analyzed for a transformed phenotype. The transformed NIH 3T3 cells overgrew a confluent monolayer, had an accelerated generation time, and were anchorage independent. In contrast, E5a did not induce foci in C127 cells, but C127 cells expressing E5a did form small colonies in suspension. The presence of the 12-kilodalton E5a gene product in the transformed NIH 3T3 cells was shown by immunoprecipitation and was localized predominantly to nuclei by an immunoperoxidase assay. A mutation in the E5a ORF was engineered to terminate translation. This mutant was defective for transformation, demonstrating that translation of the E5a ORF is required for biological activity. This is the first demonstration of a transforming oncogene in HPV-6, and the differential activity of E5a in these two cell lines should facilitate future investigations on the mechanism of transformation.     

The product of the bovine papillomavirus type 1 modulator gene (M) is a phosphoprotein.

The M gene of bovine papillomavirus type 1 has been genetically defined as encoding a trans-acting product which negatively regulates bovine papillomavirus type 1 replication and is important for establishment of stable plasmids in transformed cells. The gene for this regulatory protein has been mapped in part to the 5' portion of the largest open reading frame (E1) in the virus. We constructed a trpE-E1 fusion gene and expressed this gene in Escherichia coli. Rabbits were immunized with purified fusion protein, and antisera directed against the product were used to identify the M gene product in virus-transformed cells. In this way a polypeptide with an apparent molecular mass of 23 kilodaltons was detected. The virus-encoded product is phosphorylated and can be readily detected by immunoprecipitation assays from cells transformed by the virus. Cells that harbor viral DNA without M as integrated copies do not produce this protein, whereas cells that harbor integrated viral genomes which are defective for another E1 viral gene important for plasmid replication, R, do produce this protein. The protein has an anomalously low electrophoretic mobility. An in vitro translation product of an SP6 RNA product of a sequenced cDNA predicts a molecular mass of 16 kilodaltons for the protein, and this in vitro translation product has an electrophoretic mobility identical to that of the in vivo immunoprecipitated protein. The results of these studies confirm our previous genetic studies which indicated that part of the E1 open reading frame defined a discrete gene product distinct from other putative products which may be encoded by this open reading frame.     

Codon Optimization of the Human Papillomavirus E7 Oncogene Induces a CD8+ T Cell Response to a Cryptic Epitope Not Harbored by Wild-Type E7

Codon optimization of nucleotide sequences is a widely used method to achieve high levels of transgene expression for basic and clinical research. Until now, immunological side effects have not been described. To trigger T cell responses against human papillomavirus, we incubated T cells with dendritic cells that were pulsed with RNA encoding the codon-optimized E7 oncogene. All T cell receptors isolated from responding T cell clones recognized target cells expressing the codon-optimized E7 gene but not the wild type E7 sequence. Epitope mapping revealed recognition of a cryptic epitope from the +3 alternative reading frame of codon-optimized E7, which is not encoded by the wild type E7 sequence. The introduction of a stop codon into the +3 alternative reading frame protected the transgene product from recognition by T cell receptor gene-modified T cells. This is the first experimental study demonstrating that codon optimization can render a transgene artificially immunogenic through generation of a dominant cryptic epitope. This finding may be of great importance for the clinical field of gene therapy to avoid rejection of gene-corrected cells and for the design of DNA- and RNA-based vaccines, where codon optimization may artificially add a strong immunogenic component to the vaccine.     

Identification of a transforming gene of human papillomavirus type 16.

Previously, we observed sequential two-step alteration, growth stimulation, and progression to a more malignant state in NIH 3T3 cells transfected by human papillomavirus type 16 (HPV-16) DNA. In this study, we prepared a cDNA library from RNA extracted from cells transfected with the HPV-16 DNA and isolated cDNA clones which had growth-stimulating activity. Analysis of these cDNA clones indicated that the E7 open reading frame alone is responsible for inducing both steps of this cell transformation.     

Identification of proteins encoded by the L1 and L2 open reading frames of human papillomavirus 1a.

The human papillomavirus 1 (HPV-1) virion is composed of two virally encoded proteins: a 57,000-molecular-weight polypeptide (57K polypeptide), which is the product of the L1 open reading frame (ORF), and a 78K polypeptide, which is derived from the L2 ORF. The 57K (L1) product, which represents the major structural component, appears to be disulfide cross-linked in virus particles. The 78K (L2) protein is a minor component of the virion and does not appear to be disulfide linked either to the L1 gene product or to itself. Analysis of virus particles banding at different buoyant densities revealed differences in the L2 content of heavy-full and light-full virions. Antiserum prepared against a bacterially expressed fragment of the L1 ORF was found by immunofluorescence to cross-react with HPV-2 and bovine papillomavirus 1 virions in wart sections. No cross-reactivity was observed with antisera prepared against either the N- or C-terminal halves of the L2-encoded protein. Similarly, antisera prepared against purified virus particles (disrupted and nondisrupted) reacted only with an expressed fragment of the L1 ORF and not with either L2-encoded polypeptides or proteins derived from the E1, E2, E4, E6, or E7 ORFs. This indicates that the L1 protein contains the papillomavirus common antigens.     

Cloning of monomeric human papillomavirus type 16 DNA integrated within cell DNA from a cervical carcinoma.

We have molecularly cloned and characterized monomeric human papillomavirus type 16 DNA with flanking cell DNA sequences from a cervical carcinoma. Determination of nucleotide sequence around the junctions of human papillomavirus and cell DNAs revealed that at the site of integration within cell DNA the cloned viral DNA had a deletion between nucleotides 1,284 and 4,471 (numbering system from K. Seedorf, G. Kr?mmer, M. Dürst, S. Suhai, and W. G. R?wekamp, Virology 145:181-185, 1985), which includes the greater part of E1 gene and the entire E2 gene. In the remaining part of the E1 gene, three guanines were found at the location where two guanines at nucleotides 1,137 and 1,138 have been recorded. This additional guanine shifted the reading frame and erased an interruption in the E1 gene described by Seedorf et al. The data strongly suggest that, like other papillomaviruses, human papillomavirus type 16 has an uninterrupted E1 gene.     

Human adenovirus early region 4 open reading frame 1 genes encode growth-transforming proteins that may be distantly related to dUTP pyrophosphatase enzymes.

An essential oncogenic determinant of subgroup D human adenovirus type 9 (Ad9), which uniquely elicits estrogen-dependent mammary tumors in rats, is encoded by early region 4 open reading frame 1 (E4 ORF1). Whereas Ad9 E4 ORF1 efficiently induces transformed foci on the established rat embryo fibroblast cell line CREF, the related subgroup A Ad12 and subgroup C Ad5 E4 ORF1s do not (R. T. Javier, J. Virol. 68:3917-3924, 1994). In this study, we found that the lack of transforming activity associated with non-subgroup D adenovirus E4 ORF1s in CREF cells correlated with significantly reduced protein levels compared to Ad9 E4 ORF1 in these cells. In the human cell line TE85, however, the non-subgroup D adenovirus E4 ORF1s produced protein levels higher than those seen in CREF cells as well as transforming activities similar to that of Ad9 E4 ORF1, suggesting that all adenovirus E4 ORF1 polypeptides possess comparable cellular growth-transforming activities. In addition, searches for known proteins related to these novel viral transforming proteins revealed that the E4 ORF1 proteins had weak sequence similarity, over the entire length of the E4 ORF1 polypeptides, with a variety of organismal and viral dUTP pyrophosphatase (dUTPase) enzymes. Even though adenovirus E4 ORF1 proteins lacked conserved protein motifs of dUTPase enzymes or detectable enzymatic activity, E4 ORF1 and dUTPase proteins were predicted to possess strikingly similar secondary structure arrangements. It was also established that an avian adenovirus protein, encoded within a genomic location analogous to that of the human adenovirus E4 ORF1s, was a genuine dUTPase enzyme. Although no functional similarity was found for the E4 ORF1 and dUTPase proteins, we propose that human adenovirus E4 ORF1 genes have evolved from an ancestral adenovirus dUTPase and, from this structural framework, developed novel transforming properties.     

Bovine papillomavirus type 1 3'' early region transformation and plasmid maintenance functions.

Human papillomavirus (HPV) 8 induces skin tumors which are at high risk for malignant conversion. The nucleotide sequence of HPV8 has been determined and compared to sequences of papillomaviruses with different oncogenic potential. The general organization of the HPV8 genome is similar to that of other types. Highly conserved, genus-specific sequences were found in open reading frames (ORFs) E1, E2, and L1. In ORFs E6, E7, and L2, HPV8 is more distantly related, but it was possible to differentiate subgenera in which HPV8 belonged to the HPV1-cottontail rabbit papillomavirus group. Sequences within ORF E4 and part of ORF L2 are rather type specific. HPV8 stands out by several unique features: the considerably reduced size of the noncoding region (397 base pairs), with a seemingly low potential for forming complex secondary structures; a cluster of putative promoter elements in the 3' half of ORF E1; an RNA polymerase III promoter-like sequence close to the C terminus of ORF E2; and of particular interest, the homology between the putative protein encoded by ORF E4 and the Epstein-Barr virus nuclear antigen 2 protein, which may reflect similar mechanisms in virus-mediated transformation.     

Varicella-Zoster Virus Open Reading Frame 48 Encodes an Active Nuclease

Based on a DNA sequence and relative genomic position similar to those other herpesviruses, varicella-zoster virus (VZV) open reading frame 48 (ORF48) is predicted to encode an alkaline nuclease. Here we report the cloning, expression, purification, and characterization of recombinant VZV ORF48 protein and a VZV ORF48 point mutation (T172P). Protein encoded by wild-type ORF48, but not mutant protein, displayed both endo- and exonuclease activity, identifying ORF48 as a potential therapeutic target in VZV disease since efficient viral replication requires viral nuclease activity.     

Reindeer papillomavirus transforming properties correlate with a highly conserved E5 region.

A papillomavirus was isolated from the epithelial layer of a cutaneous fibropapilloma on a Swedish reindeer (Rangifer tarandus). Reindeer papillomavirus (RPV) is morphologically indistinguishable from other papillomaviruses, but the restriction enzyme cleavage pattern of its genome is different. No sequence homology was detected between RPV DNA and the DNAs of bovine papillomavirus type 1 (BPV-1) and avian papillomavirus when hybridization was performed under stringent conditions. However, the RPV genome hybridized to the genome of the European elk papillomavirus and the deer papillomavirus under stringent conditions. A physical map of the RPV genome was constructed, and selected regions of the genome, covering the open translational reading frame (ORF) E5 and part of the E1 and L1 ORFs, were studied by nucleotide sequence analysis. The results made it possible to align the RPV genome with the genome of BPV-1. The E5 ORF of RPV has the potential to encode a 44-amino-acid, exceptionally hydrophobic polypeptide which is very similar to the E5 polypeptides of BPV-1 and deer and European elk papillomaviruses. RPV is oncogenic for hamsters and transforms C127 mouse cells in vitro. Several virus-specific mRNAs were detected in RPV-transformed C127 cells.     

Identification of early proteins of the human papilloma viruses type 16 (HPV 16) and type 18 (HPV 18) in cervical carcinoma cells.

We have sequenced 1730 bp of human papilloma virus type 18 (HPV 18) DNA containing the open reading frames (ORF) E6, E7, the N-terminal part of E1 and, additionally, 120 bp of the N-terminal part of L1. Based on these sequencing data, together with the human papilloma virus type 16 (HPV 16) DNA sequence published recently, we identified and cloned the ORF E6, E7, E1 and L1 of HPV 18 and the ORF E6, E7, E1, E4, E5, L2 and L1 of HPV 16 into prokaryotic expression vectors. The expression system used provides fusions to the N-terminal part of the MS2 polymerase gene controlled by the heat-inducible lambda PL promoter. Using the purified fusion proteins as immunogens we raised antisera against the proteins encoded by the ORF E6, E7 and E1 of HPV 18 as well as those encoded by the ORF E6, E7, E4 and L1 of HPV 16. By Western blot analysis we could show that the E7 gene product is the most abundant protein in cell lines containing HPV 16 or HPV 18 DNA. It is a cytoplasmic protein of 15 kd in the SiHa and the CaSki cell lines which contain HPV 16 DNA, and 12 kd in the HeLa, the C4-1 and the SW756 cell lines which contain HPV 18 DNA. These results were confirmed by in vitro translation of hybrid-selected HPV 16 and HPV 18 specific poly(A)+ RNA from SiHa, CaSki and HeLa cells. Additionally, these experiments led to the identification of an 11-kd E6 and a 10-kd E4 protein in the CaSki cell line as well as a 70-kd E1 protein in HeLa cells.     

Negative control of DNA replication in composite SV40-bovine papilloma virus plasmids

To identify DNA sequences that function in the control of DNA replication, we designed a hybrid replicon consisting of linked SV40 and BPV DNA sequences. In the composite SV40-BPV plasmid negative control encoded by BPV is dominant over the uncontrolled replication encoded by the positive factor, SV40 T antigen. Using a transient replication assay, we show that replication control requires three BPV elements. Two cis-acting sequences are closely linked to BPV replication origins. A third trans-acting element is encoded within the 5' part of the BPV E1 open reading frame (ORF) and is separable from the positive replication factor encoded within the 3' part of the same ORF. The controlled replication of SV-BPV composite replicons has enabled us to create permanent COS cell lines that stably maintain these plasmids as episomes.