OBJECTIVETo study the potential transcriptional depression activities of HPV2 E2 proteins with mutations in different functional domains.

METHODSThe primers for constructing various E2 mutants were synthesized based on a HPV2 isolate containing several point mutations within E2 open reading frame. Different E2 mutations were generated by the method of extending PCR and inserted into plasmid pcDNA3. 1. Various recombinant mammalian expression plasmids pcDNA3. 1-E2 were co-transfected into HeLa cells together with a CAT-reporter plasmid pBLCAT-LCR containing HPV-2 prototype LCR, respectively. The transcriptional repression activities of the E2 mutants were evaluated by detection of CAT expression values.

RESULTSCompared with the full-length prototype E2, removals of both N- and C-terminal domains abolished E2 transcriptional repressive activities. The point mutations in the transactivation domain (nt 3037), the internal hinge region (nt 3387) and DNA binding domain (nt 3697) showed remarkable inhibition on its transcriptional depression function.

CONCLUSIONThe transcriptional regulation activity of HPV2 E2 is related with its DNA binding and transactivation domains. The exchanges of the single amino acid within E2, derived from a HPV2 isolate, abolish significantly the repressive effect on viral promoter in the context of full-length E2.

HeLa Cells ; Humans ; Oncogene Proteins, Viral ; genetics ; Papillomaviridae ; genetics ; Promoter Regions, Genetic ; genetics ; Transcriptional Activation ; genetics

OBJECTIVE: To investigate the molecular mechanism of Wnt5a and Epstein-Barr virus latent membrane protein 1 (LMP1) aberrant expression in the nasopharyngeal carcinogenesis and to estimate if it can act as a molecular marker for nasopharyngeal cancer (NPC). METHODS: Immunohistochemistry combined with previously made tissue microarrays were used to study the expression of Wnt5a and LMP1 in the nasopharyngeal carcinogenesis tissues. We investigated the role of over expression of Wnt5a and LMP1 in the development and progression of NPC and their relation with the clinicopathological features of NPC and whether they could act as molecular markers in benign and malignant NPC. RESULTS: The positive percentage of Wnt5a and LMP1 protein expression in the NPC was significantly increased as compared with that in atypically hyperplastic nasopharyngeal epithelium, hyperplastic nasopharyngeal epithelium and histologically normal nasopharyngeal epithelium (P<0.05, P<0.01, and P<0.01). Wnt5a and LMP1 proteins were significantly higher in atypically hyperplastic nasopharyngeal epithelium than those in the hyperplastic nasopharyngeal epithelium and normal nasopharyngeal epithelium (P<0.05 and P<0.01). The positive expression of Wnt5a and LMP1 proteins in clinical T3 and T4 staged NPC was higher than that in clinical T1 and T2 staged NPC (P<0.01 and P<0.05). The positive expression of Wnt5a protein in the NPC with lymph node metastasis was higher than that in the NPC without lymph node metastasis (P<0.01). The positive percentage of LMP1 protein was significantly increased in non-keratinizing carcinoma compared with undifferentiated carcinoma and keratinizing carcinoma (P<0.05 and P<0.05). The expression of Wnt5a protein in the NPC had significant positive correlation with LMP1 (r=0.354, P<0.001). Combined molecular phenotype of both Wnt5a and LMP1 expression was a good marker to distinguish NPC from non-cancerous nasopharyngeal epithelium. CONCLUSION The expression of Wnt5a and LMP1 protein in the NPC is positively correlated, and both wnt5a and LMP1 protein play important roles in the nasopharyngeal carcinogenesis either together or successively promoting the malignant transformation of nasopharyngeal epithelium and the development and progression of NPC. Both Wnt5a and LMP1 positive expression may act as good markers for NPC differential diagnosis.
Biomarkers, Tumor ; genetics ; metabolism ; Carcinogenesis ; Humans ; Nasopharyngeal Neoplasms ; genetics ; metabolism ; pathology ; Oncogene Proteins, Viral ; genetics ; metabolism ; Proto-Oncogene Proteins ; genetics ; metabolism ; Tissue Array Analysis ; Viral Matrix Proteins ; genetics ; metabolism ; Wnt Proteins ; genetics ; metabolism ; Wnt-5a Protein

OBJECTIVETo research the application of the single time detection of HPV E6/E7 mRNA and HC2-HPV-DNA in cervical screening project.

METHODSWe detected both HPV E6/E7 mRNA and HC2-HPV-DNA of each sample which collected from 130 cervical disease patients' cervix during Jan. 2008 and July. 2009. TCT results were taken as standard to evaluate the diagnostic accuracy of the above two test methods in detecting high-grade cervical disease.

RESULTS82.3% (107/130)women were confirmed to infect HPV by HC2-HPV-DNA detection, and 40.0% (52/130) women were confirmed to infect HPV by HPV E6/E7 mRNA detection, there was no significant difference between the two results (chi2 = 24.5, P < 0.05). The sensitivity, specificity, positive predictive value, negative predictive value of HC2-HPV-DNA detection were 90.1%, 22.1%, 37.4% and 82.6%, respectively. The sensitivity, specificity, positive predictive value, negative predictive value of HPV E6/E7 mRNA detection were 65.9%, 73.3%, 55.8% and 80.8%, respectively.

CONCLUSIONIn clinical cervical screening project of single time, the combination of HC2-HPV-DNA detection and HPV E6/E7 mRNA detection wick take on more potential value than applying each of them alone. RNA;

Adult ; Alphapapillomavirus ; genetics ; isolation & purification ; DNA, Viral ; genetics ; Female ; Genetic Techniques ; Humans ; Middle Aged ; Oncogene Proteins, Viral ; genetics ; Papillomavirus Infections ; virology ; RNA, Viral ; genetics ; Vaginal Smears ; Young Adult

The heterologously expressed L1 protein of human papilomavirus 16 can assembly into virus-like particles (VLPs), which has been used as prophylactic vaccine for cervical carcinoma. To express L1 protein in Hansenula polymorpha, we analyzed the codon usage of the native gene of L1 protein and redesigned the encoding sequence according to the codon bias of H. polymorpha. We used assembly PCR to synthesize the native gene HPV16L1-N and the codon optimized gene HPV16L1. The synthesized genes were cloned into pMOXZa-A vector to generate plasmids pMOXZ-HPV16N and pMOXZ-HPV16. The expression cassettes MOXp-HPV16L1(N)-AOXTT were cloned into YEp352 vector and transferred into H. polymorpha. After methanol inducement, the expression of L1 protein in H. polymorpha was detected from the codon optimized gene HPV16L1 rather than the native gene HPVI6L1-N. The parameters for induced cultivation for strain HP-U-16L with HPV16L1 were investigated in shaking flask cultures. After induced cultivation in YPM (pH 7.0) medium supplemented with methanol to a final concentration of 1.0% every 12 h at 37 degrees C for 72 h, the recombinant produced 78.6 mg/L of L1 protein. This work offers the possibility for the production of prophylactic vaccine for cervical carcinoma by H. polymorpha.
Capsid Proteins ; biosynthesis ; genetics ; Cloning, Molecular ; Codon ; genetics ; Genetic Vectors ; genetics ; Human papillomavirus 16 ; genetics ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

BACKGROUNDTo select the mutants of HPV type 16 E6 and E7 genes suitable for construction of vaccine for treatment of cervical cancer.

METHODSE6 and E7 genes were modified by site-directed mutagenesis. Several recombinant vaccina viruses were constructed by inserting the E6 or E7 mutants into the genome of vaccina virus Tiantan strain and employed to study their antigenicity.

RESULTSWestern blot assay showed that the E6 ?mutant? with substitution of Gly for Leu at amino acid site 50 and E7 mutant with substitution of Gly for Cys-24 and Glu-26 had no effect on their stability and antigenicity, but change of the Cys at position 91 of E7 dramatically reduced its stability and antigencity. Conclusion The results confirmed that the Zinc-finger structure at the E7 C-terminal? plays an important role in the integrity and stability of E7 protein.

Animals ; Antibodies, Viral ; biosynthesis ; Female ; Mice ; Mice, Inbred C57BL ; Mutagenesis, Insertional ; Oncogene Proteins, Viral ; genetics ; immunology ; Papillomaviridae ; genetics ; Papillomavirus E7 Proteins ; Repressor Proteins ; Vaccinia virus ; immunology ; Zinc Fingers

This study was aimed to identify pET21b-HPV16E2/BL21(DE3) strain and to optimize the expression of human papillomavirus type 16 (HPV16) E2 protein by orthogonal analysis. Four influence factors on two levels were selected to increase the target protein quantity. The four factors were induction time, induction temperature, inductor concentration and cell density. The quantity of HPV16 E2 protein was used as the evaluation parameter. Induced by IPTG, HPV16 E2 protein was analyzed by SDS-PAGE and Western Blot. Target protein was analyzed by GIS imaging system to quantify the protein level. SPSS13. 0 software was applied to analyze the result. Data showed that the expression strain pET211rHPV16 E2/BL21(DE3) was identified correctly. HPV16 E2 protein expressed mainly at insoluble form. The 42KD protein band was identified by SDS-PAGE and Western blot. Orthogonal test was applied on influence factor analysis and expression optimization successfully. Main influence factors were inductor concentration and induction temperature. The optimimum condition of maximum expression quantity was 37 degrees C, 7h, 1.0 mmol/L IPTG and OD600 1.0. In this experiment, orthogonal test could not only be used to analyze the influential factors and promote the target protein expression, but also be used to provide a better experiment method for molecular biological study.
DNA-Binding Proteins ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; Human papillomavirus 16 ; metabolism ; Humans ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; Papillomavirus Infections ; virology ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVETo establish an immortalized oral epithelial cell line.

METHODSNormal human oral epithelial cells were transfected with HPV16E6/E7 open reading frames using recombinant retroviral system pLXSN. Expression of HPV16E6 and E7 protein were tested by Western blot in three kinds of cells. To define cellular biological characterization of HPV16E6/E7 transfected cells, a series analysis were performed, including protraction of growth curve, HE staining, immunocytochemical staining and scanning electron microscope observation. The tumorigenicity was assessed by colony formation and transplanting the cells into nude mice.

RESULTSHuman oral epithelial cells transfected with HPVE6/E7 has been in culture for over 18 months. The cell line was named HIO615. Western blot analysis showed HIO615 expressed HPV16 E6 and E7 protein. HIOC were positive for cytokeratin, tonofibril and desmosome as observed by scanning electron microscope. The number of large colonies of dense multilayer cells was low (0.77%). No tumor developed in nude mice injected subcutaneously with HIOEC.

CONCLUSIONA human immortalized oral epithelial cell line induced by HPV16E6 and E7 has been successfully established.

Animals ; Cell Line, Transformed ; Cell Transformation, Viral ; Humans ; Mice ; Mice, Inbred BALB C ; Mouth Mucosa ; cytology ; ultrastructure ; virology ; Oncogene Proteins, Viral ; genetics ; Papillomavirus E7 Proteins ; Repressor Proteins

OBJECTIVETo evaluate the immune potency of recombinant adenovirus combined with rAAV1 vector expressing HPV16L1 protein in mice.

METHODSThe rAdV and rAAV1 vector containing codon-modified HPV16L1 gene was constructed using Admax and AAVmax packaging system respectively. C57 BL/6 mice were immunized with purified rAdV and rAAV1 vector through intramuscular and intranasal inoculation routes, and the titer of neutralizing antibody was determined by neutralization assay based HPV16 pseudovirus.

RESULTSIntramuscular immunization by rAAV1-mod. HPV16L1 or combined with rAd-mod. HPV16L1 can induce higher titer of neutralizing antibody in serum than that of other groups. The titer of neutralizing antibody of intranasal groups is significantly lower than that of intramuscular group, although the prime-boost strategy using in intranasal group was effective to enhance the specific humoral immunity.

CONCLUSIONThe rAAV1-mod. HPV16L1 combined with rAd-mod. HPV16L1 can induce higher titer of neutralizing antibody in serum through intramuscular route than that of other groups at the 16th week after the first immunization.

Adenoviridae ; genetics ; immunology ; Adenoviridae Infections ; immunology ; Animals ; Antibodies, Viral ; immunology ; Dependovirus ; genetics ; immunology ; Immune System Phenomena ; Immunization ; Mice ; Mice, Inbred C57BL ; Oncogene Proteins, Fusion ; immunology ; Oncogene Proteins, Viral ; immunology ; Recombinant Proteins ; genetics ; immunology

BACKGROUNDHeat shock protein 70 (HSP70) is expressed highly in epithelial tumours associated closely with human papillomavirus 16 (HPV16) infections. However, evidence about the direct relationship between HSP70 expression and HPVs infections are still lacking. In the present study, we examined the expression of HSP70 in keratinocytes introduced with HPV16 E6/E7 oncogenes.

METHODSStable transfected cells were established by transfection of the plasmids pLXSN16E6/E7 into cultured primary keratinocytes and subsequently selected by plasmid specific selection antibiotic (G418) at the required concentration. The expression of HSP70 in pLXSN16E6/E7 transfected keratinocytes was determined by Western blot. The correlation of HSP70 expression and E6/E7 transfection was further confirmed by doubly labelled immunofluorescent staining.

RESULTSCompared to non-transfected keratinocytes, there was a significant trend for higher levels of HSP70 in pLXSN16E6/E7 transfected keratinocytes. Doubly labelled immunofluorescent staining experiment showed that the co-localization of HPV16 E6/E7 and HSP70 in transfected keratinocytes was observed and increased expression of HSP70 was strongly associated with the transfection of HPV16 E6/E7.

CONCLUSIONSOur studies demonstrated increased levels of HSP70 proteins in keratinocytes stably transfected by HPV16 E6/E7 oncogenes. It suggests that the expression of HSP70 is modulated by HPV16 E6/E7 proteins, which may be involved in HPV16 E6/E7 induced immortalization.

Cells, Cultured ; HSP72 Heat-Shock Proteins ; biosynthesis ; Humans ; Keratinocytes ; metabolism ; Oncogene Proteins, Viral ; genetics ; Papillomavirus E7 Proteins ; Repressor Proteins ; genetics ; Transfection

Common warts are close associated with HPVs infection. In this study, we amplified and sequenced the LCR fragment and E2 gene of HPV-2 that infected the patient of extensive common wart with cutaneous horns, and we constructed the recombinant CAT-reporter plasmids pBLCAT-LCR containing HPV-2 prototype or variant LCR and mammalian expression plasmids pcDNA3. 1-E2 containing prototype or variant E2 ORF individually. The promoter activities of HPV-2 variant and the transcriptional repression activities of the mutated E2 protein were evaluated by transient transfection into HeLa cells. The results showed that there were several mutations in LCR and E2 gene of HPV-2 variant. Compared with the prototype, the viral early promoter activity of variant was significantly increased uder the control of LCR. Compared with the wild type E2 protein, the transcriptional repression activities of the mutated E2 protein was abolished partially. We speculate herein that increased promoter activities and decreased repression effect of the mutated E2 protein are linked, at least partially, with the clinical phenotypes of the uncommon huge common wart.
DNA-Binding Proteins ; genetics ; physiology ; Humans ; Mutation ; Oncogene Proteins, Viral ; genetics ; physiology ; Papillomaviridae ; genetics ; Promoter Regions, Genetic ; Repressor Proteins ; physiology ; Warts ; virology