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 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.     

Accumulation of RNA homologous to human papillomavirus type 16 open reading frames in genital precancers.

The accumulation of human papillomavirus type 16 (HPV-16)-specific RNAs in tissue sections from biopsies of patients with genital precancers was studied by in situ hybridization with single-stranded 35S-labeled RNA. These analyses revealed that the most abundant early-region RNAs were derived from the E4 and E5 open reading frames (ORFs). RNAs homologous to the E6/E7 ORFs were also detected, whereas RNAs homologous to the intervening E1 ORF were not. This suggests that the E4 and E5 mRNAs are derived by splicing to the upstream E6/E7 ORFs, consistent with studies of HPV-11 in condylomata (L. T. Chow et al., Cancer Cells (Cold Spring Harbor) 5:55-72, 1987). Abundant RNAs homologous to the 5' portion of L1 were also detected. These RNAs were localized to the apical strata of the epithelium. HPV-16 RNAs accumulated in discrete regions of these lesions, and when present were most abundant in the upper cell layers of the precancerous epithelium. RNAs homologous to early ORFs were also detected in some germinal cells within the basal layer of the epithelium.     

Cloning and sequencing of a putative Escherichia coli [NiFe] hydrogenase-1 operon containing six open reading frames.

DNA encompassing the structural genes of an Escherichia coli [NiFe] hydrogenase has been cloned and sequenced. The genes were identified as those encoding the large and small subunits of hydrogenase isozyme 1 based on NH2-terminal sequences of purified subunits (kindly provided by K. Francis and K. T. Shanmugam). The structural genes formed part of a putative operon that contained four additional open reading frames. We have designated the operon hya and the six open reading frames hyaA through F. hyaA and hyaB encode the small and large structural subunits, respectively. The nucleotide-derived amino acid sequence of hyaC has a calculated molecular mass of 27.6 kilodaltons, contains 20% aromatic residues, and has four potential membrane-spanning regions. Open reading frames hyaD through F could encode polypeptides of 21.5, 14.9, and 31.5 kilodaltons, respectively. These putative peptides have no homology to other reported protein sequences, and their functions are unknown.     

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.     

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).     

Expression of the E2 open reading frame of papilloma viruses BPV1 and HPV6b in Escherichia coli

A new expression vector, pRA10, has been constructed for the expression of the open reading frames (ORF) of bovine (B) and human (H) papilloma viruses (PV). This vector is a derivative of pAJ pi and contains 15 restriction sites proximal to the lambda PL promoter, offering considerable versatility for insertion of different ORFs. This vector was used specifically to express the E2 ORF gene products from BPV1 and HPV6b at high level in Escherichia coli. The genuine nature of these proteins was demonstrated by restriction map analysis of expression vector plasmids to insure proper orientation, nucleotide sequence analysis to demonstrate in-frame insertion, E2 ORF protein production by expression-vector plasmids, and not by appropriate controls and, immunoprecipitation of E2 proteins by antibody specific for a common N-terminal sequence derived from the expression vector.     

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.     

The production of human papillomavirus type 16 L1 vaccine product from Escherichia coli inclusion bodies

The major capsid protein L1 of the human papillomavirus type 16 (HPV16) has been previously expressed recombinantly in Escherichia coli cells as inclusion bodies (IBs). The HPV16 L1 protein offers potential as a vaccine candidate against cervical cancer, but the reported E. coli process is limited in its ability to economically produce significant quantities of material. In this study, a scaleable laboratory process for the purification of recombinant His-tagged L1 protein and its processing to give an immunogenic product is developed. The performances of ion-exchange chromatography (IEX) and immobilised metal affinity chromatography (IMAC) for the purification of L1 protein in the presence of concentrated denaturant are compared. IEX was found to be superior to IMAC when taking into account the complexity of operation, cost of adsorbent, selectivity and purity of the final product. Following purification, reduction of denaturant concentration was performed by dilution to yield a product suitable for formulation. The simplicity and ease of scale-up of dilution makes it an attractive option for process scale production and superior to the existing approach employing dialysis. It was found that direct dilution of denaturant into suitable buffer can give rise to products which have neutralising conformational epitopes identified by strong antibody-binding properties, as assessed by ELISA with a conformational monoclonal antibody. Analysis of the results showed negative main effects of protein concentration and PEG addition on antibody-binding yields, but positive main effects of the addition of detergent and L-arginine to the buffer. The diluted product had antigenic properties as assessed by ELISA and may be formulated easily for use by diafiltration and the addition of adjuvant. This work demonstrates the feasibility of producing viral vaccines using E. coli and scaleable unit operations.     

Identification of immunoreactive antigens of human papillomavirus type 6b by using Escherichia coli-expressed fusion proteins.

Human papillomavirus (HPVs) infect the genital epithelium and are found in proliferative lesions ranging from benign condylomata to invasive carcinomas. The immunological response to these infections is poorly understood because of the lack of purified viral antigens. In this study, bacterially derived fusion proteins expressing segments of all the major open reading frames (ORFs) of HPV type 6b (HPV-6b) have been used in Western blot (immunoblot) assays to detect antibodies directed against HPV-encoded proteins. The most striking reactivities present in sera from patients with genital warts were to the HPV-6b L1 ORF protein and, to a lesser extent, to the HPV-6b L2 ORF protein. Two cases of reactivity to HPV-6b E2 ORF were observed, but no reactivities were seen with other HPV-6b constructs. Two sera reacted with the HPV-16 L2 fusion protein, and two sera reacted with the HPV-16 E4 protein. The antibodies directed against the HPV-6b fusion proteins showed no cross-reactivity with comparable regions of the HPV-16 ORFs. This assay provides a useful approach for further studies of HPV serology.     

Human antibodies react with an epitope of the human papillomavirus type 6b L1 open reading frame which is distinct from the type-common epitope.

Recombinant proteins encoded by the human papillomavirus type 6b (HPV6b) L1 open reading frame react with sera from patients with condylomata acuminata and also react with rabbit antiserum raised against sodium dodecyl sulfate-disrupted bovine papillomavirus type 1 (BPV1) virions. To map the immunoreactive epitopes, a series of procaryotic expression plasmids was made which contained a nested set of 3' to 5' deletions in the HPV6b L1 open reading frame. The deleted plasmids expressed a set of carboxy to amino terminus truncated fusion proteins. Regions containing the immunoreactive epitopes were mapped by determining which of the deleted fusion proteins retained reactivity with sera in Western immunoblot assays. The coding sequence for a human antibody-reactive linear epitope mapped between HPV6b nucleotide coordinates 7045 and 7087, and the rabbit anti-BPV1-reactive epitope coding sequence mapped between coordinates 6377 and 6454. Synthetic peptides derived from the epitope mapping were reacted with sera in enzyme-linked immunosorbent assay. Human sera reacted with synthetic peptide QSQAITCQKPTPEKEKPDPYK (HPV6b L1 amino acids 417 through 437). Rabbit anti-BPV1 and rabbit antisera raised against HPV16 L1 recombinant proteins reacted with the synthetic peptide DGDMVDTGFGAMNFADLQTNKSDVPIDI (HPV6b L1 amino acids 193 through 220). Human sera which reacted with HPV6b L1 fusion proteins cross-reacted with an HPV11 L1 fusion protein but did not react with fusion proteins encoded by HPV1a, HPV16, or HPV18. Rabbit anti-BPV1 reacted with L1 fusion proteins encoded by all of these HPV types. In contrast to the type-common (rabbit anti-BPV1-reactive) epitope, the human antibody-reactive epitope appears to be relatively HPV type specific.     

Chimeric human papillomavirus type 16 (HPV-16) L1 particles presenting the common neutralizing epitope for the L2 minor capsid protein of HPV-6 and HPV-16

Both the Human papillomavirus (HPV) major (L1) and minor (L2) capsid proteins have been well investigated as potential vaccine candidates. The L1 protein first oligomerizes into pentamers, and these capsomers assemble into virus-like particles (VLPs) that are highly immunogenic. Here we examine the potential of using HPV type 16 (HPV-16) L1 subunits to display a well-characterized HPV-16 L2 epitope (LVEETSFIDAGAP), which is a common-neutralizing epitope for HPV types 6 and 16, in various regions of the L1 structure. The L2 sequence was introduced by PCR (by replacing 13 codons) into sequences coding for L1 surface loops D-E (chideltaC-L2), E-F (chideltaA-L2), and an internal loop C-D (chideltaH-L2); into the h4 helix (chideltaF-L2); and between h4 and beta-J structural regions (chideltaE-L2). The chimeric protein product was characterized using a panel of monoclonal antibodies (MAbs) that bind to conformational and linear epitopes, as well as a polyclonal antiserum raised to the L2 epitope. All five chimeras reacted with the L2 serum. ChideltaA-L2, chideltaE-L2, and chideltaF-L2 reacted with all the L1 antibodies, chideltaC-L2 did not bind H16:V5 and H16:E70, and chideltaH-L2 did not bind any conformation-dependent MAb. The chimeric particles elicited high-titer anti-L1 immune responses in BALB/c mice. Of the five chimeras tested only chideltaH-L2 did not elicit an L2 response, while chideltaF-L2 elicited the highest L2 response. This study provides support for the use of PV particles as vectors to deliver various epitopes in a number of locations internal to the L1 protein and for the potential of using chimeric PV particles as multivalent vaccines. Moreover, it contributes to knowledge of the structure of HPV-16 L1 VLPs and their derivatives.     

Immortalization and altered differentiation of human keratinocytes in vitro by the E6 and E7 open reading frames of human papillomavirus type 18.

The E6-E7 region of human papillomavirus types 16 and 18 is selectively retained and expressed in cervical carcinoma cells. In cultured human keratinocytes, expression of the E6 and E7 open reading frames of human papillomavirus type 18, under the control of its homologous promoter, resulted in high-frequency immortalization. Furthermore, by using a system that allows for stratification of keratinocytes in vitro (raft system), we observed that the morphological differentiation of these E6-E7 immortalized cells was altered such that parabasal cells extended throughout most of the epithelium, with abnormal nuclei present in the upper regions. Examination of E6-E7-expressing cell lines in the raft system at a later passage revealed that complete loss of morphological differentiation had occurred. E7 alone was a much less effective immortalizing agent than E6 and E7 together and acted only minimally to alter morphological differentiation in vitro. No such activities were found for E6 alone. High-frequency transformation of human epithelial cells thus appears to require expression of both E6 and E7 gene products.     

人乳头瘤病毒16型L1蛋白的克隆及表达

采用PCR技术从宫颈癌组织中扩增人乳头瘤病毒16型(Human papillomavirus type16,HPV16)L1全长基因片段,目的片段克隆到pMD18T载体后经酶切鉴定及测序确认。构建重组原核表达质粒pGEX4T1-L1,转化大肠杆菌E.coliBL21,IPTG诱导表达出以非可溶性蛋白形式存在的表达蛋白,该重组蛋白的表达量占菌体总蛋白的17%,免疫印迹检测表明,表达蛋白与宫颈癌病人血清出现特异性反应。成功构建了重组原核表达质粒pGEX4T1-L1,并且在原核细胞中得到表达,为进一步研究L1蛋白的免疫学活性及疫苗的研发奠定了基础。     

Construction and genetic analysis of dicistronic polioviruses containing open reading frames for epitopes of human immunodeficiency virus type 1 gp120.

On the basis of previous studies of dicistronic (dc) polioviruses that carried two internal ribosomal entry sites (L. Alexander, H.-H. Lu, and E. Wimmer, Proc. Natl. Acad. Sci. USA 91:1406-1410, 1994; A. Molla, S. K. Jang, A. V. Paul, Q. Reuer, and E. Wimmer, Nature [London] 356:255-257, 1992), we have constructed a variety of dc polioviruses which express foreign genetic elements that were inserted either between two internal ribosomal entry site elements upstream of the poliovirus open reading frame (pPNENPO derivatives) or upstream of the open reading frame for the poliovirus proteinase 2Apro (pDI-E2A derivatives). Surprisingly, the addition of an N-terminal secretory pathway signal sequence to the open reading frame of the inserted foreign sequences (specifying either truncated versions of human immunodeficiency virus type 1 [HIV-1] gp120 or chloramphenicol acetyltransferase) resulted in a null phenotype, whereas removal of the signal sequence led to the production of viable viruses. Constructs that carried a foreign gene with a signal sequence were negative in RNA synthesis, an observation that suggested a very early block in viral replication. The insertion of transmembrane sequences downstream of the leader sequence did not reverse the replication block. Studies of dc polioviruses that encoded the truncated versions of HIV-1 gp120 showed an increase in genetic stability that correlated with a decrease in the size of the insert. A dc construct that contained a minigene encoding the principal neutralization determinant of HIV-1 produced a stable virus that retained the foreign sequence through multiple passages in cultured cells. These data indicate that dc polioviruses have potential as vaccines for the expression of small foreign epitopes.     

Expression of Escherichia coli heat-labile enterotoxin b subunit (LTB) in carrot (Daucus carota L.)

We expressed the B subunit of enterotoxigenic Escherichia coli heat-labile enterotoxin (LTB) encoded by a synthetic codon-optimized gene in carrot. An Agrobacterium-mediated transformation method was used. Thirty independent transgenic lines were regenerated via somatic embryogenesis after 6 months in culture and were transferred to a greenhouse. GM1-ELISA assay was used to assess LTB protein content in mature taproots. Some transgenic lines expressed LTB up to 0.3% of the total soluble protein, which is tenfold higher than the expression levels reported earlier using the native bacterial gene in plants. Immunological assay confirmed proper assembly of the pentameric complex and in vitro activity of the recombinant LTB protein, suggesting that it can be functional in prevention of diarrhea.     

The nucleotide sequence of an Escherichia coli operon containing genes for the tRNA(m1G)methyltransferase, the ribosomal proteins S16 and L19 and a 21-K polypeptide.

The nucleotide sequence of a 4.6-kb SalI-EcoRI DNA fragment including the trmD operon, located at min 56 on the Escherichia coli K-12 chromosome, has been determined. The trmD operon encodes four polypeptides: ribosomal protein S16 (rpsP), 21-K polypeptide (unknown function), tRNA-(m1G)methyltransferase (trmD) and ribosomal protein L19 (rplS), in that order. In addition, the 4.6-kb DNA fragment encodes a 48-K and a 16-K polypeptide of unknown functions which are not part of the trmD operon. The mol. wt. of tRNA(m1G)methyltransferase determined from the DNA sequence is 28 424. The probable locations of promoter and terminator of the trmD operon are suggested. The translational start of the trmD gene was deduced from the known NH2-terminal amino acid sequence of the purified enzyme. The intercistronic regions in the operon vary from 9 to 40 nucleotides, supporting the earlier conclusion that the four genes are co-transcribed, starting at the major promoter in front of the rpsP gene. Since it is known that ribosomal proteins are present at 8000 molecules/genome and the tRNA-(m1G)methyltransferase at only approximately 80 molecules/genome in a glucose minimal culture, some powerful regulatory device must exist in this operon to maintain this non-coordinate expression. The codon usage of the two ribosomal protein genes is similar to that of other ribosomal protein genes, i.e., high preference for the most abundant tRNA isoaccepting species. The trmD gene has a codon usage typical for a protein made in low amount in accordance with the low number of tRNA-(m1G)methyltransferase molecules found in the cell.