Baculovirus Inhibition Of Experimental Studies Of The Epstein-barr Virus-associated Tumors

The baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) can efficiently transduce mammalian cells of various tissue and species origins.The large capacity for foreign genes,easiness to construct recombinant virus and obtain high viral titers,and no visible cytotoxicity are features that make baculovirus an attractive gene delivery vector for mammalian cells.In recent years, many efforts have been made to improve baculovirus mediated transgene expression, which laid good foundation for its application in gene therapy.Epstein-Barr virus(EBV) is aγ-herpesvirus closely associated with a growing number of malignancies,such as Burkitt's lymphoma,Hodgkin's disease, nasopharyngeal carcinoma,gastric carcinoma and so on.EBV has two distinct replication forms,the latent and lytic replication.Latent replication origin(OriP) and EBV nuclear antigen 1(EBNA-1) are two viral elements responsible for EBV latent maintenance.Other necessary factors are provided by the cells.Besides,OriP is also an EBNA-1-dependent enhancer,which significantly augments the expression of nearby genes,when bound by EBNA-1.The universal presence of EBV genome in tumor cells is a good target for EBV-related cancer therapy.One potential approach is to introduce heterologous expression of EBV immediate-early gene BRLF1(Rta), which may induce EBV lytic replication,and ultimately kill host cells.Efficient and specific gene delivery methods are needed to facilitate this strategy.In current research,a recombinant baculovirus containing Rta expression cassette (BV-R) was constructed.After transduced into EBV latently infected cell line D98/HR1,BV-R mediated Rta gene transcription and expression,which successfully reactivated EBV lytic replication in the presence of sodium butyrate.When infected at MOI of 200 pfu/cell,cell growth(48h p.i.) was inhibited by 17%or 25%in the absence or presence of sodium butyrate,respectively.A comparison of an adenovirus vector and BV-R,revealed that baculovirus was far less cytotoxic to EBV-negative HeLa cells.In order to improve the baculovirus to achieve better tumor cell killing effect,the EBV EBNA-1/OriP replication system was applied in baculovirus.Baculovirus vectors with Rta and OriP(BV-RO),or RTA,OriP and EBNA-1 gene (BV-ROE-CMV),were constructed.Compared with BV-R,both BV-RO and BV-ROE-CMV more efficiently reactivated EBV lytic replication in infected EBV-positive NPC cell lines Hone1-EBV,HK1-EBV and C666-1.EBV lytic protein RTA,EA-D and VCA were detected,and EBV genome copy numbers in infected Hone1-EBV cells increased by around 10 fold.Cell viability was reduced by 40%to 50%in BV-RO and BV-ROE-CMV infected EBV positive cells,while no remarkable effect was observed in EBV-negative Hone1 cells.To further examine the efficacy of baculovirus vectors for the inhibition of tumor growth,a nude mice model carrying Hone1-EBV derived tumor was established. Compared to the control groups,the treatment of either BV-RO or BV-ROE-CMV resulted in a significant reduction of tumor volume by about 70%.However,we could only detect the early lytic virus gene expression but not late gene,which suggested that EBV lytic replication was initiated but somehow not completed in treated NPC tumors.The massive cell death induced by BV-RO and BV-ROE-CMV was thought to be caused by RTA induced apoptosis,or by early events of EBV lytic replication, rather than the cytolysis.We for the first time successfully use baculovirus as a gene therapy vector for EBV related cancers.It is proved that the presence of OriP and EBNA-1 improved the performance of RTA-expressing baculovirus vectors.Since EBNA-1 is universally expressed in all kinds of EBV-positive cells but not in normal cells,BV-RO can specifically enhance RTA expression and strongly antagonize the growth of EBV-positive tumor cells and tissues.This improved baculovirus vector may become a good candidate for the therapy of EBV-related cancers in the clinical research and application.