Studies On Roles Of E6 And E7 Proteins Of Low-Risk HPV Type 6 And Immunogenicity Of TB Hsp70-E7 Fusion Protein

Human Papillomavirus(HPV) is a circular double-straned DNA virus .Over 100 different genotypes of HPV have been identified. HPVs that infect the anogenital tract can be classified as either high risk or low risk on the basis of their clinical associations. High-risk HPVs causes cervical carcinoma and seems to be strongly associated with various malignant tumors. Condylomata acuminata, also known as Genital warts or venereal warts, is caused by the low risk HPVs, among them , the HPV 6 and 11 are the most prevalent viral types found in condyloma acuminate. Approximately 90% of condyloma acuminata are related to HPV types 6 and 11. HPVs infect keratinocytes in the basal layers of stratified epithelia at a variety of anatomical sites. The replicative phase of HPV infection is confined to more differentiated cells that have already exited the cell cycle and are non-permissive for DNA synthesis. Due to their limited coding capacity, HPVs have to use the cellular DNA synthesis machinery in order to replicate their genomes .Human papillomaviruses have evolved proteins to control the growth of the epithelial cells they infect. This was a necessity since these viruses require a metabolically active, dividing cell in order to complete their life cycle. In particular, the E6 and E7 proteins have the ability to abrogate growth and differentiation controls that would otherwise prevent epithelial cell growth and block viral propagation. In order tocell cycle regulatory proteins, thereby upregulating genes required for Gl/S transition and DNA synthesis. The ability of high-risk HPV E6 and E7 proteins to associate with tumor suppressors p53 and pRB respectively has been suggested as a major mechanism to overcome this obstacle. The early region of these HPV types encodes two oncoproteins, E6 and E7, which associate with and neutralize the cellular tumor suppressors p53 and retinoblastoma (pRb), respectively. The E7 protein targets the retinoblastoma protein, a critical component of cell cycle control. E7 binding of RB leads to release of sequestered E2F, enabling the cell cycle to progress. The E6 oncoprotein forms a complex with the cellular tumor suppressor protein p53, leading to degradation of p53 via ubiquitin-dependent proteolysis. Thus, E6 expression results in the loss of p53 function in cells. But the low-risk E7 or E6 proteins can not bind to Rb or p53 efficiently. In contrast, the physiological roles of the low-risk E6 and E7 proteins remain unclear. At present, little is known about their pathogenicity in the Genital warts.In order to study the biological activities of low risk papillomavirus E6 and E7O The retrovirus vector pLXSN-HPV6 -E6 and pBabe-HPV6 -E7 were constructed . After the retrovirus plasmid was packaged by PA317, the HaCaT cell was infected with these retroviruses and selected with G418 and puromycin. E6 and E7 gene expression was determined by PCR amplification of the cDNA after reverse transcription of cellular mRNA while the controls were their corresponding empty plasmid transduced cells. Cell proliferation of HPV6-E7 transduced HaCaT was increased as compared to the control cells by using MTT assay. Flow cytometry analysis showed that HaCaT cells expressing HPV 6E7 had a significant proportion of cells progressing into the S phage and the percentage of cells in Gl stage was decreased. Our studies indicate that expression of HPV6 E7 induces DNA synthesis and stimulates cells to enter S phase in HaCaT cells. These viruses can induce DNA replication in differentiating cells. In contrast , the growth of HPV6 E6 transduced HaCaT have no differencecompared with the control cells by using MTT assay and flow cytometry analysis. The results indicate that expression of HPV6 E6 may not affect the HaCaT cell life cycle. Because both proliferation and apotosis may play a role in a hyperplasia lesion, the effects of apoptosis were detected. In order to analyze the effect on apoptosis by HPV6 E6 or E7, flow cytometer detection was used. After the double stain of PI and annexin V FITC, we can see that less apoptosis cells could be seen in the HPV6 E6-expressing cells group, the role of low-risk E6 in blocking apoptosis may be in favour of proliferation and help virus-infected cells to escape immunological surveillance. While HPV6 E7-expressing cells show no significant difference to the empty plasmid control, which suggested HPV6-E7 may not affect apoptotic in HaCaT cell.In order to understand the mechanisms by which E6 and E7 modulate apoptosis and promote cell proliferation or pathways by which E6 or E7 could stimulate proliferation or inhibit apoptosis, we analyze the protein levels of p2U bcl-2 and c-Myc in transduced HaCaT cell. Western Blot showed that the HPV-6-E6 transduced HaCaT cells expressed higher levels of bcl-2whereas HPV-6-E7 transduced cells displayed the same levels of bcl-2 protein. HaCaT Cells expressing HPV-6 E6 which are resistant to apoptosis correlated with prolonged expression of Bcl-2. Thus, one possible mechanism of low-risk E6 blocking apoptosis is to upregulate bcl-2. Contrary to bcl-2, HPV-6-E7 transduced HaCaT cells expressed high levels of c-Myc. whereas HPV-6-E7 transduced cells displayed the same levels of c-Myc protein. As c-Myc plays a vital role in cell-cycle progression, deregulated expression of c-Myc can overcome cell-cycle arrest in order to promote cellular proliferation and c-Myc is able to induce cells to enter S phase through an Rb/E2F-independent pathway. The studies that c-Myc levels are increased in HPV6 E7-expressing cells indicate an important role for c-Myc in HPV6 E7-induced cell proliferation. p21 is an inhibitor of cyclin-dependent kinases (CDK), who has been shown to inhibit the activity of each member of the cyclin/Cdk family. Nosignificant differences were detected in the expression of p21 between the vector and either of the transduced cells. Our experiment indicates that the effects low risk HPV E6 or E7 act on p21 in a manner different to high risk HPV E6 and E7, theymay have no effect on the expression of p21.In brief, the major role of HPV6 E7 lies in dysregulating cell life cycle (promote cellular proliferation), whereas the functions of low risk E6 is associated with inhibiting apotosis. HaCaT cell infected with the E7 retrovirus exhibited increased levels of c-Myc protein and normal levels of bcl-2, whereas cells infected with E6 retroviruses displayed normal levels of c-Myc protein and increased levels of bcl-2. The results above suggests that HPV6 E6^ E7 play different roles in the development of hyperplasia lesion , and also supports the notion that they act cooperatively to create a favourable environment for viral replication, help the virus to escape immune recognition.C A is considered to be the most common type of sexually transmitted disease (STD). Because of genital warts is so common and prevalent and no effective virus-specific agent is available for the treatment of genital warts, the recurrence is relatively frequent. Treatment is not proven to reduce transmission to sexual partners nor to prevent progression to dysplasia or cancer. On the basis of this evidence, nowdays most of effort is under way to develop an HPV vaccine that targets these oncogenic HPV types because High risk types of HPV have been implicated as the primary etiologic agent of cervical cancer. Vaccines targeting the nononcogenic HPV types most commonly associated with genital warts are in urgent need . The HPV non-oncogenic proteins, E6 and E7,are important in the induction and maintenance of genital warts Vaccines or immunotherapies targeting E7 and/or E6 proteins may provide an opportunity to prevent and treat genital warts. One of the concerns about E7 and/or E6 protein vaccine is its limited potency. In our study, we have sought to enhance the immunogenicity of a therapeutic E7 protein vaccine for the treatment of genital warts with the use of the adjuvant Hsp70. Linkage of antigens to HSP represents apotential approach for increasing the potency of vaccines. Heat shock protein 70 is a conserved protein, which can aid the folding and transport of proteins. Hsp70 plays an important role in the antigen presentation pathway. Some findings suggested Hsp70 could be used as a molecular carrier to enhance the antigenicity of antigen linked to Hsp70. Many investigations have made HSP70 more attractive for use in immunotherapy. Using human papillomavirus type 6 E7 as a antigen, we evaluated the effect of linkage to Mycobacterium tuberculosis heat shock protein 70 (HSP70) on the potency of antigen-specific immunity generated by the fusion protein vaccines. The Hsp70 gene was cloned from Mycobacterium tuberculosis H37Rv by PCR, and the eukaryotic expression plasmids of Hsp70 was constructed. The HPV6-E7 DNA was amplified by PCR, and the eukaryotic expression plasmids of HPV6-E7 was constructed. The accuracy of these constructs was confirmed by DNA sequencing. The hsp70 and HPV6 E7were expressed in E coli., and the proteins were purified with GSTrap FF columns. The E7 and Hsp70 fusion gene prokaryotic expression plasmid (pGEX-E7-Hsp70) was constructed and expressed in the DH5a. The fusion protein was purified.The Balb/c mice were immunized with Hsp70, E7 and fusion proteins. Splenocytes were harvested 2 weeks after the last vaccination and cultured with E7 protein. The supernatants were harvested and assayed for the presence of IFN-7 >IL-2 and IL-4 using ELISA kits. Titres of IL-4, IL-2, and IFN-gamma were increased significantly in the mices immunized with E7-hsp70 fusion proteins as compared to other groups .The results of IFN-y, IL-2 and IL-4 ELISA demonstrated that the fusion protein vaccine could induce a higher number of HPV6—E7 specific T cells than other groups (p<0.05). Although cell-mediated immune responses to the virus are probably the most important factor in host resistance , humoral immunity, on the other hand, does appear to play a certain role in host responses or in treatment responses.The anti-HPV 6 E7 antibodies in the sera were determined by a direct ELISA. The fusion protein vaccine could induce...