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

Amino Acid Sequence ; Animals ; Birds ; Cell Transformation, Viral ; Herpesvirus 2, Gallid ; genetics ; physiology ; Marek Disease ; virology ; Models, Biological ; Molecular Sequence Data ; Oncogene Proteins, Viral ; genetics ; physiology

Active Transport, Cell Nucleus ; Capsid Proteins ; physiology ; Heparan Sulfate Proteoglycans ; physiology ; Humans ; Oncogene Proteins, Viral ; physiology ; Papillomaviridae ; genetics ; physiology ; Papillomavirus Infections ; etiology ; Viral Structural Proteins ; physiology ; Virion ; physiology ; Virus Assembly

Hydrogen peroxide is considered to be a dose- and time-dependent mediator in apoptotic and necrotic death. In this study, we examined the signaling of the E6 and E7 proteins with respect to apoptosis or necrosis after H2O2 injury using an in vitro model with overexpressed E6 or E7 genes. For this purpose, the E6 and E7 gene expressing astrocytes were exposed to 0.01 mM and 0.2 mM H2 O2 solutions. Twenty- four hours after treatment with the lower dosage(0.01 mM H2O2), control, E6-expressing cells suffered about 45% injury and LXSN-expressi ng cells decreased by 67% as assessed by LDH release. However, E7-expressing cells showed less injury, resulting in 20-30% of LDH release. Astrocytes expressing E6, E7, LXSN and mock-infected cells showed a typical apoptotic death patter n on the DNA gel after treatment with a low-dose of H2O2 (0.01 mM), however the y died from necrotic death after a high-dose (0.2 mM) H2O2. Overexpression of HPV-E7 genes protected the cells from apoptotic death after a low-dose of H2O2 and from necrotic death after a high-dose of H2O2, while the overexpression of E 6 genes from the necrotic death. E7 expressing astrocytes showed higher catalas e activity and the levels of E2F protein surged more than 100-folds compared with the control astrocytes. We believe that the activity of E7 protein to protect astrocytes from H2O2 injury was at least partly due to increased catalase, a scavenger protein.
Animal ; Apoptosis/*physiology ; Astrocytes/*drug effects/pathology/*physiology ; Cells, Cultured ; Hydrogen Peroxide/*pharmacology ; Mice ; Necrosis ; Oncogene Proteins, Viral/*genetics/*physiology ; Oxidants/*pharmacology ; Signal Transduction/physiology

To prepare carboxyl terminus truncated human papillomavirus type 58 L1 (HPV58L1) protein and evaluate its ability to form virus-like particles, the baculovirus and Sf-9 insect cells was used to express HPV58L1 protein, and pFastBac-Htb containing HPV58L1 gene sequence of carboxyl terminus truncation was generated. Then Sf-9 cells were infected with recombinant baculovirus. After being cultured, the post-infected cells expressing--HPV58L1 protein were harvested and analyzed by SDS-PAGE and Western blot. The ProBond purification system was used for protein purification. The bio-activity of purified protein was identified by mouse erythrocyte hemagglutination assay, and the VLP formation was examined with transmission electron microscope. Our results showed that the recombinant baculovirus was generated and the Sf-9 cells was infected with the recombinant baculovirus, and after collecting, total cellular proteins were extracted. Truncated HPV58L1 protein with MW 58KD was revealed by SDS-PAGE and confirmed by Western blot. The purified L1 proteins under native condition could cause mouse erythrocytes to agglutinate and form VLP. It is concluded that HPV58L1 protein with carboxyl terminus truncation could be efficiently expressed. In baculovirus Sf-9 cells expression system, the purified protein could self-assemble into virions in vitro, and induce agglutination of mouse erythrocytes, indicating that carboxyl terminus truncation does not interfere with the bioactivity of HPV58L1 protein.
Baculoviridae ; genetics ; metabolism ; Capsid Proteins ; Carbon Dioxide ; Humans ; Inclusion Bodies, Viral ; ultrastructure ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; immunology ; Papillomaviridae ; physiology ; Recombinant Proteins ; biosynthesis ; Virion ; growth & development ; physiology ; Virus Assembly

To identify the integration sites in the host genome for the hepatitis B virus (HBV)-encoded X protein (HBx) in hepatocellular carcinoma (HCC) biopsies that are positive for hepatitis B surface antigen (HBsAg). HCC biopsies were obtained from six patients that were HBV carriers, as demonstrated by the presence of HBsAg in their serum and sero-negativity for antibody to HBsAg. DNA was extracted from the tissue, fractionated, and circularized. Primers were designed according to the HBx sequence and used to amplify the circularized DNA templates by inverse polymerase chain reaction (IPCR). The amplified DNA fragments were checked by electrophoresis, cloned into the PMD18-T expression vector, and sequenced. Sequence alignment was performed by the Blast algorithms. Seven electrophoresis bands yielded 22 sequencing results, which represented a total of three HBx integration sites in the host genome: 19q12, 2q32.2, 22q12. The 19q12 integration site encompasses the CCNE1 gene, which encodes a G1/S-specific cyclin-E1. HBx-related integration sites exist in HBsAg-positive HCC biopsies. The CCNE1 gene may play a role in the development of HBx-related HCC.
Carcinoma, Hepatocellular ; blood ; genetics ; Cyclin E ; genetics ; DNA Primers ; DNA, Viral ; genetics ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B virus ; genetics ; physiology ; Humans ; Liver Neoplasms ; blood ; genetics ; Oncogene Proteins ; genetics ; Trans-Activators ; genetics ; Virus Integration

OBJECTIVETo elucidate the expression of tanscription factor Ets-1 mediated by EB virus encoded latent membrane protein 1 (LMP1) in nasopharyngeal carcinoma (NPC) cells.

METHODSLMP1-expressing NPC HNE2 cells regulated by Tet-on system (pTet-on-LMP1 HNE2) were used. Expression of LMP1 and Ets-1 was observed after induction with Doxycycline (Dox). Expression of Ets-1 mRNA and protein was detected by RT-PCR and Western blot, respectively. The phosphorylation level of Ets-1 protein was examined by co-immunoprecipitation. The DNA binding activity of Ets-1 was detected by electrophoretic-mobility shift assay (EMSA).

RESULTSAfter induction with Dox in pTet-on-LMP1 HNE2 cells, to some extent, the expression of Ets-1 mRNA and protein, its phosphorylation level and DNA binding activity were increased with enhancement of LMP1 expression.

CONCLUSIONLMP1 induces transcriptional activation and expression of Ets-1 which may contribute to the development of NPC.

Carcinoma, Squamous Cell ; metabolism ; pathology ; virology ; Cell Line, Tumor ; Doxycycline ; pharmacology ; Herpesvirus 4, Human ; Humans ; Nasopharyngeal Neoplasms ; metabolism ; pathology ; virology ; Phosphorylation ; Proto-Oncogene Protein c-ets-1 ; Proto-Oncogene Proteins ; biosynthesis ; genetics ; Proto-Oncogene Proteins c-ets ; RNA, Messenger ; biosynthesis ; genetics ; Transcription Factors ; biosynthesis ; genetics ; Viral Matrix Proteins ; biosynthesis ; genetics ; physiology

The inactivation of p53 and p105RB by viral proteins or by mutations plays a key role in the oncogenesis of cervical carcinoma. The E6 and E7 proteins of HPV type 16 can bind to p53 and p105RB tumor suppressor gene products, respectively. In the present study, we tested a simple in vivo model that could explain the interactions between HPV E6 oncoprotein and p53 tumor suppressor protein. Our results showed that the life span of normal cervical epithelial cells was increased up to 4.5 times when transfected with expression vector containing E6/E7 ORF of HPV type 16. However, these cells did not divide after second crisis. Therefore, we employed an established human epidermal keratinocytes, RHEK-1. When transfected with an expression vector containing E6 ORF of HPV type 16, RHEK-1 cells showed anchorage independent growth character. When RHEK-E6 cells were transfected with wild type p53 expression vector, the growth rate of the RHEK-E6 cells was diminished. After 48 hours of transfection, many cells showed apoptotic signal but no more apoptotic signal was observed thereafter. These results suggested that the overexpression of the wild type p53 could overcome the dysfunction of the p53 on the cell cycle regulation imposed by E6 protein although not being of physiological condition.
Animal ; Base Sequence ; Cells, Cultured ; Cervix Uteri/*cytology ; Female ; Genes, p53/*physiology ; Human ; Keratinocytes/*cytology ; Mice ; Molecular Sequence Data ; Oncogene Proteins, Viral/genetics/*physiology ; Papillomavirus, Human/*genetics ; Support, Non-U.S. Gov't ; Transfection

Human papillomavirus (HPV) is a common small DNA tumor virus that specifically infects squamous epithelial cells and causes benign or malignant epithelial lesions such as genital warts and cervical cancer. High-risk HPV is detected in specimens of more than 90% of cervical cancer. In the 7. 9 kb genome of HPV, E6 and E7 are the crucial viral oncoproteins that consistently maintained after viral integration into host cell genome. These two proteins interfere with cell proliferation and differentiation through interacting with important tumor suppressors including p53 and pRb. High-risk HPV E6/E7 also induces genomic instability, facilitating cell transformation.
Cell Differentiation ; Cell Proliferation ; Cell Transformation, Neoplastic ; metabolism ; pathology ; Female ; Genomic Instability ; Host-Pathogen Interactions ; Humans ; Oncogene Proteins, Viral ; genetics ; physiology ; Papillomaviridae ; genetics ; physiology ; Papillomavirus Infections ; metabolism ; pathology ; Uterine Cervical Neoplasms ; metabolism ; pathology ; virology

Adult ; Female ; Hepatitis C, Chronic ; virology ; Humans ; Liver Cirrhosis ; virology ; Male ; Middle Aged ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics ; Viral Core Proteins ; physiology