Identification of the human papillomavirus type 6b L1 open reading frame protein in condylomas and corresponding antibodies in human sera.

Genital warts (condylomata acuminata) are among the most frequent sexually transmitted infections. Human papillomavirus type 6 (HPV-6), which is etiologically related to a majority of these lesions, has not been propagated in tissue culture. We generated two forms of HPV-6 viral antigens: a chemically synthesized oligopeptide (referred to as the C-terminal synthetic peptide) corresponding to residues 482 to 495 of the 500-amino-acid-long L1 open reading frame (ORF), and a bacterially expressed 54-kilodalton (kDa) fusion protein containing the N-terminal 13 amino acids encoded by the lambda bacteriophage cII gene followed by one vector-insert junctional residue and 462 amino acids of the L1 ORF sequence (residues 39 to 500). The cII-L1 fusion protein was specifically recognized by an antipeptide serum directed against the N-terminal 13 amino acids derived from the cII gene, an antiserum raised against the C-terminal synthetic peptide, and a genus-specific serum prepared by immunization with disrupted viral capsids. The 54-kDa fusion protein was purified, and the sequence of its first 36 amino acids was determined and found to be as predicted by the DNA sequence. Both the genus-specific anticapsid serum and the antiserum raised against the fusion protein identified authentic L1 ORF proteins in HPV-1-induced (58 kDa) and HPV-6/11-induced (56 kDa) papillomas. The synthetic peptide antiserum recognized the 56- to 58-kDa protein in HPV-6-induced warts, but not in HPV-1- or HPV-11-infected specimens. Using the fusion protein as antigen in immunoassays, we were able to detect the corresponding antibodies in human sera.     

Identification of the E5 open reading frame of human papillomavirus type 16.

Sequencing of the E5 open reading frame (ORF) of human papillomavirus type 16 revealed an additional nucleotide, a thymidine residue, at position 3903 compared with the original sequence (Seedorf et al., Virology 145:181-185, 1985). The additional T had two effects; first, in reading frame 2, in which the original E5 ORF was predicted, the additional T changed the reading frame downstream of position 3903 to create an ORF, which we designated E5, that terminated at position 4018 and potentially encoded a 52-amino-acid polypeptide. Secondly, in reading frame 3, a new ORF was created (positions 3807 to 4097), which we propose is the authentic papillomavirus type 16 E5 ORF. It contained a methionine residue and encoded an additional 82 amino acids. Both ORFs have been cloned into bacterial expression vectors (pATH), and the fusion proteins have been used to generate polyclonal antibodies in rabbits.     

Human immunodeficiency virus type 1-neutralizing monoclonal antibodies which react with p17 core protein: characterization and epitope mapping.

Monoclonal antibodies (MAbs) to human immunodeficiency virus type 1 were produced. Two antibodies reacted with the 17-kilodalton core protein (p17) of the virus and with its polyprotein precursor. To various degrees, each MAb neutralized infection by the cell-free virus. With a series of sequential overlapping hexapeptides which represent the p17 gene product, the epitopes identified by the MAbs were defined. The epitopes localize to overlapping regions near the amino terminus of the protein. Soluble synthetic peptides which span the antibody-binding sites of interest were demonstrated to competitively inhibit the reactivity of p17 MAbs, thus confirming the location of virus-neutralizing sites within the core protein.     

The L2 open reading frame of human papillomavirus type 1a encodes a minor structural protein carrying type-specific antigens.

The proteins encoded by the open reading frames of papillomavirus genomes and the minor polypeptides detected in purified virions are still poorly defined. We show here by its expression in Escherichia coli that the open reading frame L2 of human papillomavirus type 1a codes for a minor structural protein of Mr 76,000. Antisera raised against a truncated L2-beta-galactosidase fusion protein in which the conserved N-terminal region of L2 is missing are type specific for human papillomavirus type 1 virions and are reactive at high dilutions. Expression of the L2-encoded type-specific antigens thus provides a powerful new tool for the identification of papillomaviruses.     

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.     

Translation of the human papillomavirus type 16 E7 oncoprotein from bicistronic mRNA is independent of splicing events within the E6 open reading frame.

In this study we investigated the translational capacities of bicistronic and spliced mRNAs originating from the E6 and E7 regions of the high-risk genital human papillomavirus type 16 (HPV-16) and the low-risk HPV-11. For HPV-16 it was found, unexpectedly, that E7 protein could be translated from full-length bicistronic E6-E7 mRNAs. E6*I and E6*II splicing events were not required for E7 synthesis, nor did splicing increase the efficiency of E7 translation significantly. In cells, E7 synthesis from all known naturally occurring mRNA structures was very inefficient compared with that from synthetic monocistronic controls, suggesting that HPV-16 employs translational mechanisms to restrict E7 protein levels. For HPV-11, only RNAs initiated at the P264 promoter, located within the E6 open reading frame, were capable of providing an efficient template for E7 synthesis. P264-initiated mRNAs were as efficient in vivo as monocistronic controls, suggesting that the low-risk HPV-11 does not limit E7 synthesis by translational mechanisms. A detailed analysis of HPV-16 templates by using site-directed mutagenesis showed that the majority of ribosomes which ultimately translate E7 have not reinitiated after translating some or all of the upstream open reading frames. The data support a model in which the failure of 40S ribosomal initiation complexes to recognize the E6 AUG renders them capable of proceeding efficiently to translate E7.     

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.     

Human papillomavirus type 16 open reading frame E7 encodes a transforming gene for rat 3Y1 cells.

Human papillomavirus type 16 (HPV 16) DNA is capable of morphologically transforming rat 3Y1 cells. The expression plasmids, constructed from the simian virus 40-based expression vector pSV2-0 and specific DNA fragments from the putative early region of the HPV 16 genome, were tested for their transforming capacity. Among the various pSV2 plasmids, only those containing the intact E7 coding region were found to produce foci of the transformed rat cells which could grow in a soft-agar medium. The data indicate that expression of the HPV 16 E7 open reading frame is sufficient to induce focal transformation of rat cells.     

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.     

Identification of B-epitopes in the human papillomavirus 18 E7 open reading frame protein.

A panel of murine mAb raised against a MS2 replicase/HPV 18 E7 fusion protein included 23 reactive by ELISA with HPV 18 E7 determinants. A total of 19 of the 23 recognized linear epitopes in the N-terminal region of the E7 molecule, while the other four were deduced by binding inhibition assays to recognize conformational determinants in this region. All tested antibodies precipitated a 14-kDa peptide doublet that corresponded with the predicted size of the E7 protein, from HeLa cells, but not from HPV 16 E7 containing CaSki cells. HPV 18 E7 protein was detected by immunolabeling with electron microscopy in both the nucleus and the cytoplasm of HeLa cells with the greater proportion occurring in the cytoplasm. No antibody reacted specifically by indirect immunofluorescence with HeLa cells. Weak cross-reactivity of some mAb with the E6 MS2-replicase fusion protein of HPV 16 was detected by ELISA, but no protein of the appropriate size was immunoprecipitated from CaSki cells. It is concluded that the B cell epitopes on the HPV 18 E7 transforming protein are located in the N-terminal region of the molecule and that some are weakly cross-reactive with HPV 16 E6 protein. E7 protein is either present in HeLa cells at a concentration too low to be detected by indirect immunofluorescence, or the N-terminal epitopes are masked by protein conformation or interaction with cellular or other viral components.     

Partite expression of the bovine papillomavirus E1 open reading frame in Escherichia coli.

Six recombinants were constructed which expressed portions of the bovine papillomavirus E1 open reading frame as OmpF/E1/beta-galactosidase tribrid fusion proteins in Escherichia coli. Rabbit sera containing E1-specific antibodies were generated against five of these six fusion proteins (which together constitute 74% of the full-length E1 open reading frame). The individual fusion proteins and their cognate antisera will be useful reagents for defining the structure and function of the BPV E1 protein(s).     

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.     

Evidence for translational repression of the SOCS-1 major open reading frame by an upstream open reading frame

The suppressor of cytokine signalling 1 protein (SOCS-1) belongs to a novel family of cytokine inducible factors which function as inhibitors of the JAK/STAT pathway. While SOCS-1 previously has been described as a single-exon gene, here we present evidence for an additional 5' exon, separated by a 509 bp intron from exon 2. Exon 1 and part of exon 2 contain an open reading frame of 115 nt, ending one nucleotide upstream of the major reading frame. Using SOCS-1-promoter/luciferase constructs, we investigated which sequences are involved in the regulation of SOCS-1 expression. Serial promoter deletion clones indicate the localization and functionality of SP1, interferon-stimulated responsive elements (ISRE), and interferon-gamma-activated sites (GAS) promoter elements in the SOCS-1 5' flanking region. We present evidence that the upstream open reading frame (uORF) represses the translation of the downstream major open reading frame (mORF). Mutating the start codon of the uORF relieves this repression. Our data indicate that expression of the SOCS-1 protein is repressed on translational level by a mechanism, which bears similarities to that postulated for genes like retinoic acid receptor beta2 (RARbeta2), S-adenosylmethionine-decarboxylase (AdoMetDC), Bcl-2, and others.     

Characterization of a major neutralizing epitope on human papillomavirus type 16 L1

Persistent infection with human papillomavirus type 16 (HPV-16) is strongly associated with the development of cervical cancer. Neutralizing epitopes present on the major coat protein, L1, have not been well characterized, although three neutralizing monoclonal antibodies (MAbs) had been identified by using HPV-16 pseudovirions (R. B. Roden et al., J. Virol. 71:6247-6252, 1997). Here, two of these MAbs (H16.V5 and H16.E70) were demonstrated to neutralize authentic HPV-16 in vitro, while the third (H16.U4) did not. Binding studies were conducted with the three MAbs and virus-like particles (VLPs) composed of the reference L1 sequence (114K) and three variant L1 sequences: Rochester-1k (derived from viral stock DNA), GU-1 (derived from cervical biopsy DNA), and GU-2 (derived from biopsy DNA, but containing some sequence changes likely to be artifactual). While all three MAbs bound to 114K and Rochester-1k VLPs, GU-1 VLPs were not recognized by H16.E70, and both H16.E70 and H16.V5 failed to bind to GU-2 VLPs. Site-directed mutagenesis was used to replace disparate amino acids in the GU-2 L1 with those found in the 114K L1. Alteration of the amino acid at position 50, from L to F, completely restored H16.V5 binding and partially restored H16.E70 binding, while complete restoration of H16.E70 binding occurred with GU-2 VLPs containing both L50F and T266A alterations. Immunization of mice with L1 variant VLPs revealed that GU-2 VLPs were poorly immunogenic. The L50F mutant of GU-2 L1, in which the H16.V5 epitope was restored, elicited HPV-16 antibody responses comparable to those obtained with 114K VLPs. These results demonstrate the importance of the H16.V5 epitope in the generation of potent HPV-16 neutralizing antibody responses.     

Specific inactivation of inhibitory sequences in the 5' end of the human papillomavirus type 16 L1 open reading frame results in production of high levels of L1 protein in human epithelial cells

The expression of human papillomavirus type 16 late genes encoding virus capsid proteins L1 and L2 is restricted to terminally differentiated epithelial cells in the superficial layers of the squamous epithelium. We wish to understand the molecular mechanisms that determine the levels of expression of the human papillomavirus type 16 late genes. We have previously shown that the L1 coding region contains inhibitory sequences. Here we extend previous findings to show that the 5' end of the L1 gene contains strong inhibitory sequences but that the 3' end does not. We show that the first 514 nucleotides of the L1 coding region contain multiple inhibitory elements that act independently of one another and that the major inhibitory element is located within the first 129 nucleotides of the L1 gene. Introduction of point mutations in the inhibitory elements in the 5' end of the L1 gene which altered the RNA sequence without affecting the protein sequence specifically inactivated the inhibitory elements and resulted in production of high levels of human papillomavirus type 16 L1 mRNA and protein in human epithelial cells. Furthermore, we show that inhibitory sequences are present in the L1 coding regions of multiple human papillomavirus types, demonstrating that these elements are conserved among the human papillomaviruses, and suggest that they have an important function in the viral life cycle.     

Varicella-zoster virus open reading frame 4 protein is functionally distinct from and does not complement its herpes simplex virus type 1 homolog, ICP27.

Varicella-zoster virus (VZV) open reading frame 4 (ORF4) encodes a putative immediate-early protein which is homologous to herpes simplex virus type 1 (HSV-1) ICP27 on the basis of gene location and similarity in amino acid sequence. In transient expression assays, however, ORF4 and ICP27 exhibit different properties. ICP27 alone has little activity on target plasmids, but it acts as a transactivator or a transrepressor in the presence of other HSV-1 transactivators. In contrast, ORF4 directly transactivates plasmids containing homologous or heterologous promoters and has no apparent transrepressing activity. To further illuminate the functional similarities and differences between ORF4 and ICP27, Vero cell lines which express ORF4 under the inducible metallothionein promoter were constructed. Cell lines expressing functionally active ORF4 protein upregulated the expression of transfected VZV target plasmids but were unable to efficiently complement HSV-1 ICP27 mutants. These results indicate that, despite structural similarities, VZV ORF4 and HSV-1 ICP27 behave differently in transient expression assays and may play different roles in virus replication.     

Phylogenetic position of an uncharacterized Brazilian strain of bovine papillomavirus in the genus Xipapillomavirus based on sequencing of the L1 open reading frame

The use of PCR assays with degenerate primers has suggested the existence of numerous as yet uncharacterized bovine papillomaviruses (BPV). Despite the endemic nature of BPV infections, the identification of BPV types in Brazilian cattle is still only sporadic. However, in a recent analysis of a partial segment of the L1 gene, we observed notable diversity among the BPV types detected. The aim of this study was to determine the phylogenetic position of the previously identified wild strain BPV/BR-UEL2 detected in the state of Paraná in Brazil. Since previous analysis of the partial L1 sequence had shown that this strain was most closely related to BPV type 4, genus-specific primers were designed. Phylogenetic analysis using complete L1 ORF sequences revealed that BPV/BR-UEL2 was related to BPV types classified in the genus Xipapillomavirus and shared the highest L1 nucleotide sequence similarity with BPV type 4 (78%). This finding suggests that BPV/BR-UEL2 should be classified as a potential new type of BPV in the genus Xipapillomavirus.