Interactions between papillomavirus L1 and L2 capsid proteins

The human papillomavirus (HPV) capsid consists of 360 copies of the major capsid protein, L1, arranged as 72 pentamers on a T=7 icosahedral lattice, with substoichiometric amounts of the minor capsid protein, L2. In order to understand the arrangement of L2 within the HPV virion, we have defined and biochemically characterized a domain of L2 that interacts with L1 pentamers. We utilized an in vivo binding assay involving the coexpression of recombinant HPV type 11 (HPV11) L1 and HPV11 glutathione S-transferase (GST) L2 fusion proteins in Escherichia coli. In this system, L1 forms pentamers, GST=L2 associates with these pentamers, and L1+L2 complexes are subsequently isolated by using the GST tag on L2. The stoichiometry of L1:L2 in purified L1+L2 complexes was 5:1, indicating that a single molecule of L2 interacts with an L1 pentamer. Coexpression of HPV11 L1 with deletion mutants of HPV11 L2 defined an L1-binding domain contained within amino acids 396 to 439 near the carboxy terminus of L2. L2 proteins from eight different human and animal papillomavirus serotypes were tested for their ability to interact with HPV11 L1. This analysis targeted a hydrophobic region within the L1-binding domain of L2 as critical for L1 binding. Introduction of negative charges into this hydrophobic region by site-directed mutagenesis disrupted L1 binding. L1-L2 interactions were not significantly disrupted by treatment with high salt concentrations (2 M NaCl), weak detergents, and urea concentrations of up to 2 M, further indicating that L1 binding by this domain is mediated by strong hydrophobic interactions. L1+L2 protein complexes were able to form virus-like particles in vitro at pH 5.2 and also at pH 6.8, a pH that is nonpermissive for assembly of L1 protein alone. Thus, L1/L2 interactions are primarily hydrophobic, encompass a relatively short stretch of amino acids, and have significant effects upon in vitro assembly.     

Structure and evolution of the L11, L1, L10, and L12 equivalent ribosomal proteins in eubacteria, archaebacteria, and eucaryotes

The genes corresponding to the L11, L1, L10, and L12 equivalent ribosomal proteins (L11e, L1e, L10e, and L12e) of Escherichia coli have been cloned and sequenced from two widely divergent species of archaebacteria, Halobacterium cutirubrum and Sulfolobus solfataricus, and the L10 and four different L12 genes have been cloned and sequenced from the eucaryote Saccharomyces cerevisiae. Alignments between the deduced amino acid sequences of these proteins and to other available homologous proteins of eubacteria and eucaryotes have been made. The data suggest that the archaebacteria are a distinct coherent phylogenetic group. Alignment of the proline-rich L11e proteins reveals that the N-terminal region, believed to be responsible for interaction with release factor 1, is the most highly conserved region and that there is specific conservation of most of the proline residues, which may be important in maintaining the highly elongated structure of the molecule. Although L11 is the most highly methylated protein in the E. coli ribosome, the sites of methylation are not conserved in the archaebacterial L11e proteins. The L1e proteins of eubacteria and archaebacteria show two regions of very high similarity near the center and the carboxy termini of the proteins. The L10e proteins of all kingdoms are colinear and contain approximately three fourths of an L12e protein fused to their carboxy terminus, although much of this fusion has been lost in the truncated eubacterial protein. The archaebacterial and eucaryotic L12e proteins are colinear, whereas the eubacterial protein has suffered a rearrangement through what appear to be gene fusion events. Within the L12e derived region of the L10e proteins there exists a repeated module of 26 amino acids, present in two copies in eucaryotes, three in archaebacteria, and one in eubacteria. This modular sequence is apparently also present in the L12e proteins of all kingdoms and may play a role in L12e dimerization, L10e-L12e complex formation, and the function of the L10e-L12e complex in translation.     

Identification and characterization of NIF3L1 BP1, a novel cytoplasmic interaction partner of the NIF3L1 protein

The NIF3L1 protein is strongly conserved during evolution from bacteria to mammals and recently its function in neuronal differentiation has been demonstrated. In the present study we identified novel binding partners of human NIF3L1 by screening a HeLa cDNA-library using the yeast two-hybrid system. We could show that the NIF3L1 protein is interacting with itself and with the NIF3L1 binding protein 1 (NIF3L1 BP1), a novel protein of 23.67kDa bearing a putative leucine zipper domain. Furthermore, both interactions were confirmed using the mammalian two-hybrid system. Deletion analyses clearly demonstrated that a C-terminal region of 100 amino acids of the NIF3L1 BP1 is sufficient for the interaction with NIF3L1. The NIF3L1 BP1 is ubiquitously expressed and cotransfection experiments revealed that NIF3L1 and NIF3L1 BP1 interact in the cytoplasm of human LNCaP cells. This study provides novel insights into the cellular function of the NIF3L1 protein.     

Expression of the L11-L1 operon in mutants of Escherichia coli lacking the ribosomal proteins L1 or L11

Summary Using a variety of immunological techniques, the supernatant levels of ribosomal proteins were measured in mutants lacking the ribosomal proteins L1 or L11, and in wild-type strains. There was a 2.5–5-fold elevation of protein L11 level in the supernatant of strains lacking protein L1, compared to wild-type. In contrast, there was no elevation, but rather a diminution, in the corresponding L1 level in strains lacking protein L11, compared to wild-type. These results are consistent with a model for the control of expression of the L11-L1 operon in which protein L1 is an inhibitor of expression of that operon, but protein L11 is not. The supernatant concentrations of other proteins were indistinguishable in all strains.     

人乳头瘤病毒16型L1和L2基因表达产物的鉴定

目的:构建人乳头瘤病毒(human papillomavirus,HPV)16型晚期基因L1及L2的原核表达质粒,并验证目的蛋白的表达.方法:用限制性酶切及连接的方法构建原核表达质粒pET3a-16 L1和pET3a-16 L2,通过SDS-PAGE及Westen blot检测目的融合蛋白的表达.结果:在大肠菌中诱导表达的L1蛋白分子量约为57 KD,L2蛋白分子量约为90 KD.结论:该实验结果为HPV16型预防性基因工程亚单位疫苗的研制和诊断试剂的研究开发奠定了基础.     

布鲁氏菌L7/L12蛋白的基因克隆与原核表达

本研究对羊布鲁氏菌L7/L12蛋白进行了表达和纯化。首先从布鲁氏菌M5基因组中克隆L7/L12目的基因片段,连接至pMD-19T载体,转化入E.coli DH5α感受态细胞,PCR鉴定及测序鉴定正确后对其进行双酶切,构建重组质粒pGEX-6P-1-L7/L12并利用E.coli BL21(DE3)进行诱导表达。羊布鲁氏菌L7/L12基因片段大小为375 bp。SDS-PAGE检测蛋白大小为13 kD,与预测值相符。Western blotting方法检测其免疫学特性。实验结果表明,成功构建了pGEX-6P-1-L7/L12原核表达载体,并在大肠杆菌中成功表达了L7/L12重组蛋白,Western blotting法检测其具有免疫反应。本实验为下一步研究蛋白功能及布鲁氏菌新型疫苗的研制提供了实验基础。     

The complete primary structure of ribosomal proteins L1, L14, L15, L23, L24 and L29 from Bacillus stearothermophilus

The amino acid sequences of ribosomal proteins L1, L14, L15, L23, L24 and L29 from Bacillus stearothermophilus have been completely determined. This has been achieved by sequence analyses of peptides derived from enzymatic digestions of the proteins with trypsin, chymotrypsin, pepsin, Staphylococcus aureus protease, and Armillaria mellea protease as well as by chemical cleavage with hydroxylamine and cyanogen bromide. Based on the primary structures of the six proteins, their secondary structures were predicted using four different computer prediction programs. A comparison of the amino acid sequences of the studied proteins from B. stearothermophilus with the homologous proteins from Escherichia coli revealed that in four proteins (L1, L15, L24 and L29) between 40-50% of the residue in the sequences are identical, whereas this value is significantly higher (69%) for L14 and lower (28%) for L23. The distribution of those amino acid residues which are identical in the corresponding proteins from the two bacteria is not random along the protein chain: some regions are highly conserved whereas others are not. This finding indicates that the regions which are conserved during evolution are important for the spatial structure and/or function of the protein.     

Interactions of ribosomal protein L1 with ribosomal and messenger RNAs

Crystal structures of unbound protein L1 and of its complexes with ribosomal an messenger RNAs are analyzed. It is shown that the values of the apparent association rate constant for L1-RNA depend on conformation of unbound protein L1. It is suggested that L1 binds to rRNA with higher affinity than to mRNA because of additional interactions between domain II of L1 and the loop rRNA region, which is absent in mRNA.     

Roles of the human Rad51 L1 and L2 loops in DNA binding

The human Rad51 protein, a eukaryotic ortholog of the bacterial RecA protein, is a key enzyme that functions in homologous recombination and recombinational repair of double strand breaks. The Rad51 protein contains two flexible loops, L1 and L2, which are proposed to be sites for DNA binding, based on a structural comparison with RecA. In the present study, we performed mutational and fluorescent spectroscopic analyses on the L1 and L2 loops to examine their role in DNA binding. Gel retardation and DNA-dependent ATP hydrolysis measurements revealed that the substitution of the tyrosine residue at position 232 (Tyr232) within the L1 loop with alanine, a short side chain amino acid, significantly decreased the DNA-binding ability of human Rad51, without affecting the protein folding or the salt-induced, DNA-independent ATP hydrolysis. Even the conservative replacement with tryptophan affected the DNA binding, indicating that Tyr232 is involved in DNA binding. The importance of the L1 loop was confirmed by the fluorescence change of a tryptophan residue, replacing the Asp231, Ser233, or Gly236 residue, upon DNA binding. The alanine replacement of phenylalanine at position 279 (Phe279) within the L2 loop did not affect the DNA-binding ability of human Rad51, unlike the Phe203 mutation of the RecA L2 loop. The Phe279 side chain may not be directly involved in the interaction with DNA. However, the fluorescence intensity of the tryptophan replacing the Rad51-Phe279 residue was strongly reduced upon DNA binding, indicating that the L2 loop is also close to the DNA-binding site.     

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.     

Bacterial expression and purification of human papillomavirus type 18 L1

The human papillomavirus (HPV) 18 L1 gene, which encodes the L1 major capsid protein, was isolated from a female patient in Pusan, Korea Republic and was cloned into pGEX-4T-1 vector. The HPV-18L1 gene was expressed in Escherichia coli as a fusion protein with a glutathione-S-transferase (GST) tag. The soluble recombinant fusion protein, GST-18 L1 fusion, was isolated to high purity. HPV-18 L1 was purified from the GST-18 L1 fusant after biotinylated thrombin cleavage, and then the treated thrombin was removed serially using streptavidin conjugated resin. The purified HPV-18 L1 was confirmed by western blotting using a rabbit anti-denatured papillomavirus polyclonal antibody. The virus-like particles (VLP) from the purified full-length 18 L1 protein without any extra amino acid sequences was observed through the analysis of the electron microscope. This is the first study to report the expression and purification of HPV-18 L1 in E. coli. This expression and purification system offers a simple method of expressing and purifying HPV L1 protein, and could potentially be an effective route for the development and manufacturing of highly purified HPV-18 L1-based cervical cancer vaccines.     

The N-terminal sequence protein of L7/L 12 is responsible for its dimerization.

Ultracentrifuge studies of intact protein L7/L12, of its fragments 27--120, 1--74 and 74--120 and of protein L7/L12 with oxidized methionine residues, indicate that the N-terminal sequence of the protein L7/L12 is responsible for its dimerization. The symmetry model of the dimer is discussed.     

Neutralization of bovine papillomavirus by antibodies to L1 and L2 capsid proteins.

We have generated four mouse monoclonal antibodies (MAbs) to bovine papillomavirus virions that bound type-specific, adjacent, and conformationally dependent epitopes on the L1 major capsid protein. All four MAbs were neutralizing at ratios of 1 MAb molecule per 5 to 25 L1 molecules, but only three effectively blocked binding of the virus to the cell surface. Therefore, antibodies can prevent papillomavirus infection by at least two mechanisms: inhibition of cell surface receptor binding and a subsequent step in the infectious pathway. The neutralizing epitopes of the bovine papillomavirus L2 minor capsid protein were mapped to the N-terminal half of L2 by blocking the neutralizing activity of full-length L2 antiserum with bacterially expressed peptides of L2. In addition, rabbit antiserum raised against amino acids 45 to 173 of L2 had a neutralizing titer of 1,000, confirming that at least part of the N terminus of L2 is exposed on the virion surface.     

The acidic ribosomal phosphoprotein of eukaryotes and its relationship to ribosomal proteins L7 and L12 of Escherichia coli.

The acidic ribosomal phosphoprotein, Lgamma, of Krebs II ascites cells was further characterized and compared with proteins L7 and L12 of Escherichia coli. Ribosomal protein Lgamma was selectively removed from 60S sibosomal subunits by 50% ethanol and 1M-NH4Cl, and antibodies raised against protein Lgamma cross-reacted with E. coli protein L12 in immunodiffusion experiments. These and other, previously reported, data support the proposal that the uekaryotic counterpart of E. coli proteins L7 and L12 is phosphorylated.     

Dimer state of protein L7/L12 and EF-G-dependent reactions of ribosomes.

A number of different monomer and dimer derivatives of protein L7/L12 has been studied in EF-G-dependent reactions on the ribosome. It has been shown that only dimer derivatives of protein L7/L12 are able to interact with the ribosome. This means that it is the dimer forms of protein L7/L12 that are present in the functionally active ribosome. It is likely that the N-terminal sequence of protein L7/L12 is responsible for dimerization of the protein in solution and at the same time contributes mainly to the interaction of the protein L7/L12 dimer with the ribosome. The results obtained suggest that there are four copies of protein L7/L12 in the translating ribosome.     

Attachment sites of primary binding proteins L1, L2 and L23 on 23 S ribosomal RNA of Escherichia coli

The attachment sites of the primary binding proteins L1, L2 and L23 on 23 S ribosomal RNA of Escherichia coli were examined by a chemical and ribonuclease footprinting method using several probes with different specificities. The results show that the sites are confined to localized RNA regions within the large ribonuclease-protected ribonucleoprotein fragments that were characterized earlier. They are as follows: (1) L1 recognizes a tertiary structural motif in domain V centred on two interacting internal loops; the main protein interaction sites occur at the internal loop/helix junctions. (2) The L2 site constitutes a single irregular stem/loop structure in the centre of domain IV where non-Watson-Crick pairing is likely to occur. (3) L23 recognizes a tertiary structural motif involving a single terminal loop structure and part of an adjacent internal loop at the centre of domain III. Each of the three primary binding proteins, whose presence is essential for ribosomal assembly, has been associated with important ribosomal functions: L1 lies in the E-site for deacylated tRNA binding while L2 and L23 have been implicated in the P and A substrate sites, respectively, of the peptidyl transferase centre. Moreover, each of the protein sites, but particularly those of L2 and L23, lies at the centre of RNA domains where they can maximally influence both the assembly of secondary binding proteins and the function of the RNA region.     

Codon optimization of the human papillomavirus type 58 L1 gene enhances the expression of soluble L1 protein in Saccharomyces cerevisiae

The effect of codon optimization of L1 gene on the production of the L1 protein of human papillomavirus (HPV) was investigated in a yeast expression system. Saccharomyces cerevisiae was transformed with a plasmid containing either the wild type (WS)-HPV type 58 L1 (HPV58 L1) gene or a codon-optimized (MO)-HPV58 L1 gene. The proportion of soluble L1 protein expressed from MO-HPV58 L1 was significantly higher than that expressed from WS-HPV58 L1. Moreover, the amount of purified MO-HPV58 L1 protein recovered was 2.5-fold higher than the amount of WS-HPV58 L1 protein. Codon optimization of HPV58 L1 gene thus increases the proportion of soluble L1 protein and the amount of purified product that can be used as antigen to generate vaccines.     

人乳头瘤病毒18型L1蛋白的包涵体和可溶性表达及纯化

目的:原核表达系统表达人乳头瘤病毒18型(HPV18)L1蛋白,建立包涵体和可溶性表达的L1蛋白的纯化方法。方法:构建重组表达质粒p GEX-4T-1-HPV18 L1,在大肠杆菌BL21中以包涵体和可溶性方式表达HPV18 L1蛋白。通过超声波破碎菌体、洗涤包涵体、碱变性、透析复性和谷胱甘肽(GST)琼脂糖凝胶4B亲和层析纯化包涵体蛋白;在菌体中加入三磷酸腺苷(ATP)和3.5 mol/L尿素孵育后,GST 4B亲和层析纯化可溶性蛋白,凝血酶酶切。SDS-PAGE和Western印迹鉴定表达和纯化产物。结果:SDS-PAGE结果表明,HPV18 L1蛋白以包涵体和可溶性方式在大肠杆菌BL21内高效表达,均产生相对分子质量约为86 000的HPV18 L1-GST融合蛋白。Western印迹结果显示,包涵体纯化后获得的融合蛋白降解条带较多;而可溶性蛋白纯化后获得的融合蛋白未降解,凝血酶酶切后得到HPV18 L1蛋白,可与HPV18 L1蛋白单克隆抗体结合。结论:采用原核系统表达了HPV18 L1-GST融合蛋白,分别建立了包涵体和可溶性蛋白的纯化方法,获得HPV18 L1蛋白,为其进一步应用奠定了基础。     

Localization of proteins L4, L5, L20 and L25 on the ribosomal surface by immuno-electron microscopy

Summary Ribosomal proteins L4, L5, L20 and L25 have been localized on the surface of the 50S ribosomal subunit of Escherichia coli by immuno-electron microscopy. The two 5S RNA binding proteins L5 and L25 were both located at the central protuberance extending towards its base, at the interface side of the 50S particle. L5 was localized on the side of the central protuberance that faces the L1 protuberance, whereas L25 was localized on the side that faces the L7/L12 stalk. Proteins L4 and L20 were both located at the back of the 50S subunit; L4 was located in the vicinity of proteins L23 and L29, and protein L20 was localized between proteins L17 and L10 and is thus located below the origin of the L7/L12 stalk.