The Direct Interaction Of Human Papillomavirus Type58E2Protein And E7Protein Inhibits E7's Oncogenic Activity

Objective Human Papillomavirus (HPV) are a family of non-enveloped and double-stranded small DNA virus that infect keratinocytes in differentiating epithelia and induce a series of benign and malignant hyperproliferative lesions. The virus is classified to two genotypes. One namely low-risk HPV, including HPV-1,2,6,11, et al., induces benign warts such as common warts and condyloma acuminate; The other namely high-risk HPV, including HPV-16,18,31,58et al., can lead to the development of malignant hyperproliferative lesions, such as cervical cancer.HPV-58is an important high-risk HPV type tightly associated with cervical cancer. The etiology studies indicated that the main prevalent region of HPV-58is in Asia, especially in Eastern Asia. HPV-58is also the third most prevalent HPV type found in cervical cancer patients in China following HPV-16and18. But it is much less studied than HPV-16and18on virology and oncogenesis. Although the HPV vaccine has already been used to protect human being from HPV-16or18infection, it do not play any protective role on inhibiting HPV-58infection. Therefore, HPV-58is necessary to be well studied.The length of HPV genome is around8kb, which can be divided into three regions. The long control region contains the elements for viral gene replication and transcription; the early gene region encode the early protein E1, E2, E4, E5, E6and E7; the late gene region encode two capsid protein L1and L2. E6and E7, the main oncoproteins, were well studied on the oncogenic mechanism. While the E2protein is a viral transcriptional factor, down-regulating the expression of E6and E7genes in cervical carcinoma cell lines. But E2gene is always broken when the viral genome integrates into the host genome. And the E2protein is lost in cervical cancer cells. So it is believed that E2deficiency and high expression of E6and E7is a critical step in process of cell transformation. Since E2is a muti-functional protein, it is still not clear how it works in carcinogenesis. The knowledge of E2function came from the studies of HPV-16or BPV, little work on HPV-58. So our study shed light on the tumor-suppressive function of HPV-58E2protein.Methods and Results First, the HPV-58E2truncations and58E7protein were expressed and purified from E. coli. for GST-pull down assay. We found the C-terminal domain plus hinge domain (225-358aa) of58E2, but not N-terminal domain (1-201aa), is able to bind to58E7. To confirm the direct interaction between58E2and58E7, co-immunoprecipitation assay was employed. Our result indicated the physical interaction between58E2and58E7in293T cell, and the binding domain of58E2with58E7was mapped to the hinge domain of58E2, especially244-254aa, which is essential for interacting with58E7.Interestingly, HPV E2-E7direct interaction exhibits type-specifity. We identified the interaction of58E2and58E7, as well as16E2and16E7, but failed to detect the cross-interaction of16E2and58E7, or58E2and16E7.Furthermore, The biological consequence of E2-E7interaction in HPV-58, especially in viral tumorigenesis was investigated. Firstly, we investigate if the58E2competes with pRb to bind58E7. The results showed that58E2did not competitively inhibit58E7binding to pRb. Next, the effects of58E2on the steady-state levels of pRb in58E7-expressing cells were assessed.58E2and its truncations were co-expressed with58E7in HaCat or NIH3T3cell. The results showed that only the truncations of58E2which interact with58E7can inhibit the pRb degradation and cell proliferation, while the.truncations of58E2which cannot bind to58E7, did not show any inhibition. Conclusion①HPV-58E2and E7protein directly interact with each other physically in293T cells. And the hinge domain of58E2is essential for binding to58E7.②The interaction between E2and E7is HPV type-specifity.③58E2inhibits58E7-induced pRb degradation and cell proliferation. Objective:Kaposi's sarcoma associated herpesvirus (KSHV) is one of seven currently known human cancer viruses. This virus causes Kaposi's sarcoma, a cancer commonly occurring in AIDS patients, as well as primary effusion lymphoma and some types of multicentric Castleman's disease. KSHV is a large double-stranded DNA virus with a protein covering that packages its nucleic acids, called the capsid, which is then surrounded by an amorphous protein layer called the tegument, and finally enclosed in a lipid envelope derived in part from the cell membrane.Most of virion proteins are located in the tegument layer, such as ORF11,21,23,33,45,52,63,64,75, etc. ORF45encodes an407amino acid (aa) immediately early protein, which only conserved in y herpesvirus. In Dr. Yuan lab, an virion-wide protein interaction was identified. It indicates that ORF45is able to widely interact with many capsid proteins, tegument proteins and envelop proteins. The previous study showed, wild type KSHV bacteria artificial chromosome (BAC36) stable transfection293T cell line induced by TPA and butyrate produced much more virion than ORF45mutant BAC36which was inserted a stop code at the beginning of ORF45(BAC-stop45). It suggests that ORF45might play an important role on KSHV assembly/egress.Lipid rafts (LRs) are component of dynamic membrane system in cell, which associate with many cell processes, including signal transduction, immune response, virus infection, assembly and egress. An increasing number of virion proteins were found in LRs, and participate in virion production. The LRs serve as a platform for virion assembly/egress. In this study, we attempt to shed light on the mechanism of ORF45in LR-mediated virion assembly/egress. Methods and results:Lipid rafts are more ordered and tightly packed than the surrounding bilayer, but float freely in the membrane bilayer. Researchers take advantage of lipid raft resistance to non-ionic detergents, such as Triton X-100, extracted lipid rafts from a plasma membrane under4℃. Because of their composition and detergent resistance, lipid rafts are also called Detergent Resistant Membranes (DRMs). Firstly, the DRMs were extracted from a KSHV infection cell line (BCBL-1) induced by TPA. The ORF45was detected in DRMs. To confirm the LR localization of ORF45is independent from other virion proteins, the DRMs were extracted from ORF45over-expression293T cell. ORF45still can be detected by western blot. A serial of ORF45deletions were constructed and employed in membrane flotation assay to identify the LR localization motif of ORF45. The residues297-300aa were confined as the key sites for LR localization of ORF45.Next, a serial of ORF45deletion mutants were transcomplemented into BAC-stop45stable transfection293T cell line, respectively. The lytic replication was induced by TPA and butyrate. The virions were collected and purified from cell culture medium. The viral genome copy numbers were analyzed by Real-time PCR. The results showed the ORF45-null mutant produced a noticeably lowered yield of progeny virions compared to the wild-type virus reconstituted cells. However, the yield of progeny virions of ORF45full length transcomplement set significantly increased, nearly caught up with BAC-36. Furthermore, the yield of progeny virions of ORF45mutants transcomplement sets were different. The yield of progeny virions of LR localization motif deletion set decreased closely to that of BAC-stop45set.In previous study, it is found that ORF45can activate ERK signal transduction pathway, through interacting with ASK. Additionally, LRs widely participate in assembly and transportation of cell signal molecular. So, we also investigated the association of ORF45LR localization with signal transduction. It suggested that only Δ19-77aa mutant which is disable to interact with ASK, cannot maintain phosphorylation of ERK. Whereas, other ORF45mutants including LR motif deletion still can maintain phosphorylation of ERK, collectively suggesting that LRs does not associate with ERK signal pathway.Conclusion:①The tegument protein ORF45of KSHV is a lipid raft (LR) associated protein, the LR localization motif of ORF45is located at297-300aa.②ORF45locates in LRs and plays an key role on virion assembly/egress.③There is no association of LR localization of ORF45with ERK signal transduction.