Biomedical subjects

K Yoshiike

Publications and source records attributed to K Yoshiike.

At least 19 recordsLinked to original sources

PubMed2001

Nasal immunization of mice with peptide having a cross-neutralization epitope on minor capsid protein L2 of human papillomavirus type 16 elicit systemic and mucosal antibodies.

A common cross-neutralization epitope for human papillomavirus types 6 and 16 (HPV 6 and 16) is present in the region of amino acids (aa) 108-120 of HPV-16 minor capsid protein, L2. We nasally immunized Balb/c mice with a synthetic peptide with the 13 aa HPV 16 L2 sequence, and examined the antibodies elicited. ELISA showed that the immunization induced predominantly IgG and IgA antibodies cross-binding to L1/L2-capsids of HPVs 6, 16, and 18 in sera and in vaginal secretions, respectively. The serum containing the IgG antibody and the vaginal wash containing the IgA antibody neutralized HPV 16 pseudovirions and HPV 11 authentic virions, as shown by surrogate infectivity assays. From their cross-binding activity for HPV 16 and 18, the peptide-induced antibodies can probably cross-neutralize most of the genital HPVs. The peptide-induced neutralizing activity in vaginal wash was comparable to that induced by nasally immunization with HPV 16 L1-capsids. Unlike Balb/c, C57BL/10, which has different MHC class II, did not respond to the peptide immunization, but aa substitutions in the peptide to fulfill the requirement for the C57BL/10 agretope rendered the modified peptides immunogenic. The results provide a basis for development of a peptide vaccine against broad-spectrum of genital HPVs for humans.

Administration, Intranasal↗

PubMed2001

Human papillomavirus type 16 minor capsid protein l2 N-terminal region containing a common neutralization epitope binds to the cell surface and enters the cytoplasm.

The first step of papillomavirus infection is believed to be binding of major capsid protein L1 to the cell surface without involvement of minor capsid protein L2, but the viral infectivity can be neutralized either by anti-L1 or anti-L2 antibody. To understand the role of L2 in human papillomavirus (HPV) infection, we examined a segment of HPV type 16 (HPV16) L2, which contains a neutralization epitope common to HPV6, for its involvement in adsorption and penetration of the capsids. Preincubation of monkey COS-1 cells with a synthetic peptide having amino acids (aa) 108 to 120 of HPV16 L2 reduced the susceptibility of COS-1 cells to infection with HPV16 pseudovirions. Confocal microscopy showed that the green fluorescence protein (GFP) fused with the L2 peptide was found to bind to the surface of a HeLa cell, an HPV18-positive human cancer cell line, at 4 degrees C and to enter the cytoplasm after subsequent incubation at 37 degrees C. Flow cytometry showed that fused GFP did not bind to HeLa cells that had been treated with trypsin. Besides COS-1 and HeLa cells, some human and rodent cell lines were detected by flow cytometry to be susceptible to binding with fused GFP, showing a tendency of epithelial cells toward higher susceptibility. Substitutions at aa 108 to 111 inhibited fused GFP from binding to HeLa cells and reduced the infectivity in COS-1 cells of the in vitro-constructed pseudovirions. The results suggest that L2 plays an important role in enhancing HPV infection through interaction between the N-terminal region and a cellular surface protein, facilitating penetration of the virions and determining part of the tropism of HPVs.

Amino Acid Sequence↗

PubMed2000

Adeno-associated virus type 2 nonstructural protein Rep78 suppresses translation in vitro.

Adeno-associated virus type 2 nonstructural protein Rep78 [621 amino acids (aa) long] affects the expression of various cellular and viral genes. In this study we examined the effects of Rep78 on expression of the luciferase gene from the human cytomegalovirus immediate-early promoter in HeLa cells and on translation of RNA encoding luciferase in rabbit reticulocyte lysate. When Rep78 and luciferase were coexpressed, the luciferase activity decreased despite increased levels of luciferase mRNA in the cells. Purified Rep78 or Rep68 fused with Escherichia coli maltose binding protein suppressed translation of luciferase RNA in vitro, but Rep52/40 fusion proteins did not. A mutated Rep78, which is 520 aa long and truncated at its C-terminus, did suppress the in vitro translation, whereas a similarly truncated Rep78 of 420 aa did not. The results indicate that Rep78/68 function to suppress gene expression through translation inhibition, which requires the N-terminal region contained within aa 1-520.

Animals↗

PubMed2000

DNA vaccination of mice with plasmid expressing human papillomavirus 6 major capsid protein L1 elicits type-specific antibodies neutralizing pseudovirions constructed in vitro.

Human papillomavirus 6 (HPV 6) causes benign condylomata. As a model for HPV vaccine development, we tested a HPV 6 DNA vaccine candidate, constructed by subcloning the major capsid protein (L1) gene into an expression plasmid having the cytomegalovirus promoter, for its immunogenicity in BALB/c mice. Three intracutaneous inoculations of the plasmid with a gene gun at 2-week intervals elicited anti-L1 serum antibodies. The antibodies were found to recognize highly type-specific, conformation-dependent epitopes, including those to neutralize pseudovirions capable of inducing beta-galactosidase in infected monkey COS-1 cells. The data support the idea that immunization with DNA capable of expressing HPV L1 can be used as an HPV vaccine strategy for humans.

Animals↗

PubMed2000

Enhancer-promoter activity of human papillomavirus type 16 long control regions isolated from cell lines SiHa and CaSki and cervical cancer biopsies.

Expression of human papillomavirus 16 (HPV-16) oncogenes is markedly higher in cervical cancer cells than in precancerous cells, and the elevated expression is believed to be required for the malignant phenotypes. We compared cancer cell lines CaSki (with 200 to 400 copies of HPV-16 DNA per cell) and SiHa (with one to two copies of HPV-16 DNA per cell) for the E7 expression in cells and the enhancer-promoter activity of the isolated viral long control region (LCR). Although these parameters per cell were 10-fold higher in CaSki than in SiHa, the levels of the E7 mRNA and protein per HPV DNA copy were 10- to 20-fold higher in SiHa than in CaSki. Characterization of the isolated LCRs showed that, whereas the LCR from CaSki resembled the prototype in structure and activity, the LCR from SiHa, with a deletion of 38 base pairs, enhanced transcription from P97 as assayed by using a plasmid capable of expressing luciferase. The upregulation appeared to be due to removal of one of the silencer YY1-binding sites. Furthermore, we isolated and characterized LCRs from 51 cervical cancer patients' biopsies. Among them, one with a deletion including YY1-binding sites and the other with a substitution in a YY1-motif were found to enhance the transcription. These findings suggest that mutation affecting YY1-motifs in the LCR is one of the mechanisms enhancing the viral oncogene expression in the course of progression of cancer cells.

DNA, Viral↗

PubMed1999

Inhibition of replication of HIV-1 at both early and late stages of the viral life cycle by single-chain antibody against viral integrase.

Retroviruses including HIV-1 integrates a DNA copy of their RNA genome into cellular DNA of the infected cell. This reaction, essential and unique to replication of retroviruses, is mediated by the viral enzyme, integrase (IN). We constructed a recombinant gene encoding a single-chain, antigen-binding peptide (scAb2-19), which interacted with a carboxyl terminal part of HIV-1 IN. HeLa CD4 cells expressing scAb2-19 localized in either cytoplasmic or nuclear compartment were resistant to HIV-1 infection at an multiplicity of infection (MOI) of 0.25 or 0.063, but the resistance was overcome when MOI was increased to 1. To determine whether this resistance was due to inhibition of the early events, transduction experiments were performed with a replication-incompetent HIV-1 vector carrying bacterial lacZ driven by an internal Tat-independent cytomegalovirus immediate early promoter. Both cytoplasmic and nuclear expressions of scAb2-19 resulted in decrease in the transduction efficiency on HeLa CD4 cells. This implies that an early step of replication--before or during integration--was affected by the scAb2-19. Furthermore, cytoplasmic expression of scAb2-19 did not affect the viral amount released from the cells transfected with HIV-1 infectious clone DNA (pLAI). However, infectivity relative to reverse transcriptase activity was lower for virions released from the 293T cells cotransfected with pLAI and the cytoplasmic scAb2-19 expression plasmid than for those released from the 293T cells transfected with pLAI alone. This implies that scAb2-19 reduced infectivity of released virions by interfering a late step of the viral replication. The single-chain, antigen-binding peptide molecule may prove useful not only for studies of the functions of IN and its role in the viral life cycle but also for developing a gene therapy strategy against AIDS.

Amino Acid Sequence↗

PubMed1999

Common neutralization epitope in minor capsid protein L2 of human papillomavirus types 16 and 6.

Studies of virus neutralization by antibody are a prerequisite for development of a prophylactic vaccine strategy against human papillomaviruses (HPVs). Using HPV16 and -6 pseudovirions capable of inducing beta-galactosidase in infected monkey COS-1 cells, we examined the neutralizing activity of mouse monoclonal antibodies (MAbs) that recognize surface epitopes in HPV16 minor capsid protein L2. Two MAbs binding to a synthetic peptide with the HPV16 L2 sequence of amino acids (aa) 108 to 120 were found to inhibit pseudoinfections with HPV16 as well as HPV6. Antisera raised by immunizing BALB/c mice with the synthetic peptide had a cross-neutralizing activity similar to that of the MAb. The data indicate that HPV16 and -6 have a common cross-neutralization epitope (located within aa 108 to 120 of L2 in HPV16), suggesting that this epitope may be shared by other genital HPVs.

Animals↗

PubMed1998

Human papillomavirus oncoprotein E6 binds to the C-terminal region of human minichromosome maintenance 7 protein.

Oncoprotein E6 of the human papillomavirus (HPV) associated with cervical cancer (HPV-16 and -18) degrades tumor suppressor protein p53, but seems to have p53-independent transforming functions. We searched for other cellular targets for the N-terminal region of HPV-16 E6 using a yeast two-hybrid system. The E6 was found to bind to the C-terminal region of a human minichromosome maintenance 7 (hMCM7) protein, which is a component of replication licensing factors. The full-length hMCM7 translated in vitro was capable of binding to bacterially expressed E6. In yeast cells the E6s of the cancer-associated HPVs (HPV-16, -18, and -58) bound to hMCM7 more strongly than those of the HPVs associated with a benign tumor (HPV-6 and -11). Binding of E6 with hMCM7 may cause chromosomal abnormalities found in the human cells expressing E6s of oncogenic HPVs.

Binding Sites↗

PubMed1998

A surface immunodeterminant of human papillomavirus type 16 minor capsid protein L2.

We used human papillomavirus type 16 (HPV-16) particles composed of capsid proteins L1 and L2 (L1/L2 capsids) as an antigen to produce mouse monoclonal antibodies (MAbs). Of 18 MAbs recognizing surface epitopes of L1/L2 capsids, 1 was an anti-L1 MAb and 17 were anti-L2 MAbs. Seven of 11 anti-L2 MAbs recognizing linear epitopes wer found to bind to a synthetic peptide with an HPV-16 L2 sequence of amino acids (aa) 69-81, which is within a highly conserved region among different HPVs. The synthetic peptide reacted with the human sera that had been shown to be positive for an antibody against HPV-16, -18, -58, or -6b capsids composed of L1 alone. The data suggest that the HPV-16 L2 region of aa 69-81 contains a type-common immunodeterminant exposed on the surface of HPV virions.

Animals↗

PubMed1998

Retroviral vector targeting human cells via c-Kit-stem cell factor interaction.

Targeted gene transfer into hematopoietic stem cells by retroviral vectors would greatly facilitate the development of in vivo strategies for stem cell gene therapy. We engineered a recombinant retroviral vector that can target human cells expressing a c-Kit receptor via a ligand-receptor interaction. The ecotropic (Moloney murine leukemia virus) envelope protein was modified by insertion of a sequence encoding the N-terminal 161 amino acids of murine stem cell factor (mSCF), the ligand for murine c-Kit. The chimeric envelope protein was correctly processed and incorporated into viral particles as efficiently as the wild-type envelope protein. Virions pseudotyped with the chimeric envelope proteins bound to 293 cells expressing murine c-Kit (293KIT) preferentially; however, they could not transduce any c-Kit-positive cells under conventional conditions. They could transduce 293KIT cells in the presence of chloroquine, and HEL cells expressing human c-Kit on a fibronectin fragment (CH296)-coated dish. The fact that recombinant mSCF in the medium at the time of transduction greatly reduced the efficiency of both gene deliveries implies that the vector utilized the mSCF-c-Kit interaction for the initial step of transduction in either case. The vector may prove useful for targeting cells expressing c-Kit on their surface.

Cells, Cultured↗

PubMed1998

In vitro construction of pseudovirions of human papillomavirus type 16: incorporation of plasmid DNA into reassembled L1/L2 capsids.

Lack of permissive and productive cell cultures for the human papillomaviruses (HPVs) has hindered the study of virus-neutralizing antibodies and infection. We developed a cell-free system generating infectious HPV16 pseudovirions. HPV16 L1/L2 capsids, which had been self-assembled in insect cells (Sf9) expressing virion proteins L1 and L2, were disassembled with 2-mercaptoethanol (2-ME), a reducing agent, and reassembled by removal of 2-ME in the presence of a beta-galactosidase expression plasmid. Plasmid DNA purified together with the reassembled capsids was resistant to DNase I digestion. The reassembled pseudovirions mediated DNA transfer to COS-1 cells, as monitored by induced beta-galactosidase activity. Transfer was inhibited by anti-HPV16 L1 antiserum but not by antisera against L1s of HPV6 and HPV18. Construction in vitro of HPV pseudovirions containing marker plasmids would be potentially useful in developing methods to assay virus-neutralizing antibodies and to transfer exogenous genes to HPV-susceptible cells.

Animals↗

PubMed1997

Characterization of the cloned promoter of the human initiation factor 4AI gene.

Protein synthesis initiation factor 4AI (eIF-4AI) is ubiquitously expressed in eucaryotic cells. We characterized the eIF-4AI gene promoter cloned from human fibroblasts. The minimal promoter, localized to a region in the 5'-noncoding region adjacent to the first exon, consisted of approximately 300 base pairs (bp), 80% of which were identical with those in the corresponding mouse promoter. The minimal promoter contained TATA and CAAT motifs and consensus sequences binding to SP1 (three sites) and AP2 (one site). Deletion analyses of the promoter revealed that a 24-bp region near 5'-end of the minimal promoter was essential for the efficient transcription, although the AP2 site in the region was dispensable. A fluorescence polarization assay suggested that the plus strand of the 24-bp region, despite the lack of known consensus sequences binding to transcription factors except for AP2, bound to unknown nuclear protein(s) in a sequence specific manner.

Animals↗

PubMed1997

Antibodies to human papillomavirus 16, 18, 58, and 6b major capsid proteins among Japanese females.

Among genital human papillomaviruses (HPVs), the so-called high-risk (HPV 16, 18, etc.) and intermediate-risk (HPV 58, etc.) viruses are believed to be etiologically associated with cervical cancer. To estimate the extent of infection with common HPVs among Japanese females, we examined 328 sera from healthy donors (201) and patients with cervical intraepithelial neoplasia (CIN) (22), cervical cancer (67), and condyloma acuminatum (CA) (38) for IgG antibodies against L1 capsid protein by enzyme-linked immunosorbent assay using virus-like particles of HPVs 16, 18, 58 and 6b (low-risk) as antigens. Antibodies recognizing conformational epitopes were found in the sera from both the patients and the healthy donors. The prevalences of anti-HPV 16, 18, and 58 antibodies in the sera from the patients with CIN (45%) and cervical cancer (49%), and that of anti-HPV 6b in the sera from the patients with CA (55%), were significantly higher than those in the sera from the age-matched healthy donors (12%, 14%, and 23%, respectively). Anti-HPV 16 was not found in some of the sera from patients with HPV 16-DNA positive CIN or cervical cancer, suggesting that HPV infection may not always induce production of anti-capsid antibodies or that the level of antibodies may not always be maintained until development of CIN or cancer. Some of the sera contained antibodies against more than one type of HPV, suggesting that the donors had been infected with different HPVs. The type-specific antibodies against capsid L1 protein of one type of HPV may not be able to prevent infections with other types of HPVs.

Adult↗

PubMed1996

Human endogenous retrovirus K10 encodes a functional integrase.

We cloned a human endogenous retrovirus K1O DNA fragment encoding integrase and expressed it as a fusion protein with Escherichia coli maltose-binding protein. Integrase activities were measured in vitro by using a double-stranded oligonucleotide as a substrate mimicking viral long terminal repeats (LTR). The fusion protein was highly active for both terminal cleavage and strand transfer in the presence of Mn2+ on the K1O LTR substrate. It was also active on both Rous sarcoma virus and human immunodeficiency virus type 1 LTR substrates, whereas Rous sarcoma virus and human immunodeficiency virus type 1 integrases were active only on their corresponding LTR substrates. The results strongly suggest that K1O encodes a functional integrase with relaxed substrate specificity.

Amino Acid Sequence↗

PubMed1995

Mutational analysis of human papillomavirus type 16 E6 protein: transforming function for human cells and degradation of p53 in vitro.

The E6 oncoprotein of human papillomavirus type 16 (HPV 16) [151amino acids (AA) long] contains four metal-binding motifs, C-X-X-C, and is postulated to form two 29-AA finger-like structures in the N-terminal and C-terminal halves, which mediate degradation of p53 and binding to p53, respectively. We constructed a series of E6 mutants with single-AA substitutions in these finger regions (AAs 34-62 and 107-135) and examined their transforming function for human embryonic kidney (HEK) cells in conjunction with HPV 16 E7 and their interaction with human p53 in vitro. The mutants with substitution of L for F-37, G for L-50, S for Y-54, and P for L-110, which did not transform HEK cells, showed markedly lowered activity to direct degradation of p53. The mutants with substitutions of G for R-39, G for V-42, G for Y-43, L for F-47, and G for V-53 lost the transforming function, but they could mediate degradation of p53 at levels comparable to the activities of the wild-type and transforming mutants. Like the wild type, all of the E6 mutants were localized by immunofluorescence to the nuclei of human TS21B cells or monkey COS-1 cells, except for the E6 mutant with substitution of G for Y-43 whose expression was undetectable. The levels of E6 mutants metabolically labeled in COS-1 cells were comparable to those of the transforming E6s. The data indicate that E6-directed degradation of p53 is necessary but not sufficient for HPV 16-mediated transformation of of HEK cells.

Amino Acid Sequence↗

PubMed1995

Occurrence of the antibody against human papillomavirus type 16 virion protein L2 in patients with cervical cancer and dysplasia.

Infection with HPV 16 is believed to be a major risk factor for cervical cancer. To correlate HPV 16 infection and carcinogenesis in the cervix, we examined by ELISA 326 sera from healthy females and patients with cervical cancer, cervical intraepithelial neoplasia, or dysplasia, for the presence of IgG antibodies against HPV 16 virion protein L2 expressed in Escherichia coli. Whereas 2 of 208 were positive in the healthy females, 4 of 23 and 6 of 90 were positive in the patients with cervical cancer and dysplasia, respectively. The findings indicate that infection with HPV 16 is related to cancer and dysplasia of the cervix. The anti-L2 antibody did not occur coincidentally with the antibodies against the HPV 16 early proteins E4 and E7, which are specifically but independently associated with patients with cervical cancer.

Adolescent↗

PubMed1993

Association of the antibodies against human papillomavirus 16 E4 and E7 proteins with cervical cancer positive for human papillomavirus DNA.

Occurrence of the antibodies against human papillomavirus (HPV) 16 proteins E4 and E7 is specifically but independently associated with cervical cancer. To correlate HPV DNA and antibody data, we examined the biopsy specimens and sera, by polymerase chain reaction (PCR) and by ELISA, respectively, from 51 patients with cervical cancer (including 3 recurrent cases) and 22 with cervical intra-epithelial neoplasia. Consensus primers for the L1 region were used for PCR and bacterially expressed, purified fusion protein HPV-16 E4 and non-fusion protein HPV-16 E7 were used for ELISA. HPV-16 DNA and other HPV types were detected in 17 and 25, respectively, out of 51 cases of cervical cancer. Ten out of the 17 HPV-16-DNA-positives were positive either for anti-E4 or for anti-E7: positivities for anti-E4, for anti-E7, and for both were 6/17, 5/17 and 1/17 respectively. Three anti-E7-positives consisted of those for HPV-33, -52 and -58 DNA, suggesting that limited cross-reaction occurred between the HPV types. Among the HPV-16-DNA-positive cases of cancer, lymph-node or distant metastasis was recorded more frequently in the seropositives than in the seronegatives. Our results show that the HPV-16 anti-E4 or anti-E7 occurs in some, but not in all, of the HPV-16-DNA-positive cases, and support the hypothesis that the presence of the HPV-16 antibodies can be used as a marker for possible metastasis.

Adult↗

PubMed1993

Growth dependence of human papillomavirus 16 DNA-positive cervical cancer cell lines and human papillomavirus 16-transformed human and rat cells on the viral oncoproteins.

The dependence on human papillomavirus (HPV) oncoproteins of the growth of cervical cancer cell lines [C4-1, HeLa (both containing HPV 18 DNA), CaSki and SiHa (both containing HPV 16 DNA)], HPV 16-transformed human embryonic kidney cells, and HPV 16-transformed rat brain and 3Y1 cells was examined by using antisense RNA approaches. The cells were transfected with plasmids expressing RNA antisense to the HPV 16 or 18 open reading frames E6E7, together with plasmids expressing the hygromycin B resistance gene, and drug-resistant colonies were scored three weeks later. In all the human cell lines, the efficiency of colony formation was lowered by RNA antisense to the resident HPV type. Some of the rat cell lines responded to the antisense plasmids, but some did not. From a nonresponding rat tumor line (3Y1HP-1T), cell clones with various levels of E7 protein were isolated after transfection with the antisense plasmid, and were examined for anchorage-independent growth in soft agar. The colonies formed by the clones with lower E7 levels tended to be smaller and fewer than those formed by the clones with higher E7 levels. These findings strongly suggest that some of the transformed or cancer phenotypes of cells in vitro are dependent, even after extensive passages and malignant changes, on expression of the oncoproteins of the resident HPV.

Animals↗