Studies On The Mechanism Of Interaction Between Baculoviruses And Mammalian Cells

Recently, there is a new observation that baculoviruses as heterogenous viruses can be transduced into a variety of mammalian cell lines. Although some groups have done a lot of excellent works in this field, the mechanism that baculovirus interacts with mammalian cells is not well clarified. In addition, baculoviruses belong to a large family Baculoviridae, which contains two genera: Nucleopolyhedrovirus (NPV) and Granuleovirus (GV). Nucleopolyhedroviruses are sub-divided into Group I and Group II NPV. One of the major differences between Group I and Group II NPV is that they have different envelope pretions. This difference may function different in the interaction of baculovirus and the mammalian cell types. The aims of this thesis are to analyze the efficiency of baculoviruses transducing into mammalian cells, to illuminate the response of mammalian cells stimulated by baculoviruses, to compare the function of different baculoviral envelop proteins in transduction. These studies provided a good groundwork in the application of baculoviruses as vectors of gene therapy. In Chapter one, an overview outlines the character of baculovirus, major on the mechanism of baculoviruses invading into insect cells as well as the structure and function of envelope proteins. We also introduce the basic concept of gene therapy and the viral vectors including baculoviral vector for gene therapy. Baculovirus AcMNPV has shown the ability of high efficiently transducing into a variety of mammalian cell types and exhibited the preference to hepatic cells. We demonstrated that AcMNPV mediated high efficiency gene transfer into different mammalian kidney cells.(Chapter 2). AcMNPV can transduce into some mammalian kidney cells and achived a high-level expression of reporter gene encoding for the green fluorescent protein (GFP) under heterogonous promoter was observed in primary mouse kidney cells and continuous kidney cell lines of mouse, hamster, monkey, pig, and human in vitro. The level of transgene expression exhibited viral dose dependent,which was enhanced by the addition of butyrate. Butyrate is a chemical agent that suppresses potential inhibition of expression. It inhibits histone deacetylase, which induces a hyperacetylation of chromatin and leads to the induction of the expression of repressed genes. In addition, Baculovirus transduction could also provided long-term target gene expression in kidney cell lines without cytotoxic effects. This is suggested that baculovirus could be used as vectors for gene transfer to kidney and other tissues such as liver. . AcMNPV was capable of stimulating antiviral activity in SMMC-7721 cells manifested by inhibition of Vesicular Stomatitis virus (VSV) replication. Recombinant AcMNPV was shown to induce interferons alpha/beta even in the absence of transgene expression in human SMMC-7721 cells, while it mediated transgene expression in BHK and L929 mammalian cells without an ensuing antiviral activity. So, there is no relationship between baculovirus inducing antiviral activity and mediating expression of transgene (Chapter 3). Above results were acquired by using AcMNPV, a group I NPV, as vector, which contains GP64 fusion protein. However, the group II NPV such as HaSNPV and SeMNPV encoding a different envelope protein F shows no detectable infectivity to all tested mammalian cell types (Chapter 3 and 4). Moreover, HaSNPV and SeMNPV of group ?? NPVs cannot stimulate antiviral activity in mammalian cells (chapter 3). When GP64 of AcMNPV was inserted into genome of HaSNPV, the recombinant HaSNPV containing dual envelope proteins –GP64 and F protein can transduce mammalian cells. The efficiency of transduction was also enhanced by the addition of butyrate. However, the efficiency of the recombinant HaSNPV transducing into mammalian with or without butyrate is much lower than AcMNPV. Even with lower transduction ratios overall, the range of transduced mammalian cell types with the recombinant HaSNPV is consistent with AcMNPV. These findings indicate that theGP64 and F proteins function differentially. The F protein functions only in insect cells, while the GP64 protein works both in insect and mammalian cells. Although group II NPVs do not transduce into mammalian cells, GP64 helps them to transfer into these cells. This suggests that group II NPVs may be developed to target vectors of gene therapy by rebuiltig envelope protein. So, this is a new way for study about baculoviruses as vectors of gene therapy.