Spodoptera Is The Identification Of Su (var) 3-9 And Its Role In Baculovirus Infection And The Construction Of Sustained Enhanced Recombinant Baculovirus

Part Ⅰ Identification and characterization of the Spodoptera Su(var)3-9 histone H3K9 trimethyltransferase and its effect in AcMNPV infectionPost-translational modifications on N-terminus of core histones, including methylation, acetylation, phosphorylation, ubiquitination and glycosylation, etc, could affect the affinity between histones, DNA and a variety of protein factors and change the status of chromatin and gene expression. Because these modifications play a vital role in the development of the organism and gene expression regulation, etc, and have the similar effect as the genetic code, they are also known as the "histone code". Site and state specific methylations of histone Lysine or Arginine are catalyzed by a family of proteins containing SET domain. Among various histone methyltransferases (HMTs), the SUV39 family member Su(var)3-9 specifically triggers H3K9 tri-methylation. The resulted product, H3K9mer3, can be bound by heterochromatin protein 1 (HP1), thus participates in heterochromatin formation and gene silencing. Su(var)3-9 can also directly interact with the Chromo-Shadow domain of HP1 through the N-terminus dimerization region. Moreover, Su(var)3-9 interacts with various epigenetic factors such as DMNT and HDAC through bridge proteins HP1 and methyl-CpG-binding protein 2 (MeCP2), therefore is involved in the complex epigenetic regulation network.Virus infection usually affects multiple host biological processes and also triggers host antiviral responses. Epigenetic factors are often involved in these virus-host interactions as it has been demonstrated that the genomes of several DNA viruses could form a chromatin-like structure which was subject to epigenetic modifications. On the other hand, viruses might make the use of cellular epigenetic factors to ensure their own replication. In Sf9 cells, infection by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) also induces various changes of host cells, such as alternation of cellular cytoskeleton, cell cycle arrest in G2/M, a global decrease of cellular mRNA levels and protein synthesis shut-off, etc. It has been suggested that AcMNPV genome also has the nucleosome-like structure within infected cells and several epigenetic drugs could affect viral genome replication and gene transcription. But whether and how HMTs, especial Su(var)3-9, participate in AcMNPV-insect cell interaction is still an unsolved mystery.In this report, histone H3K9 trimethyltransferase gene Su(var)3-9 and its functionally unrelated splice variant eIF2y were identified and cloned in three Spodoptera insects, Spodoptera frugiperda, Spodoptera exigua and Spodoptera litura. Analysis of the encoded amino acid sequences of Spodoptera Su(var)3-9 revealed that they are highly conserved evolutionarily. Su(var)3-9 protein was found to be expressed and localized in the nucleus in Sf9 cells, using homemade Su(var)3-9 specific antiserum generated in mice. Su(var)3-9 were found to interact with histone H3, and HP1a, HP1b, the genes of which were also cloned and identified in this study. A dose-dependent enzymatic activity was found at both 27 ℃ and 37 ℃ in vitro, with higher activity found at 27 ℃. In vivo enzymatic activity analysis also confirmed its histone methylation ability and the subsequent effect on chromatin compaction and repression of cellular genes.Furthermore, the transcription of Su(var)3-9 was found to increase at late time of AcMNPV infection, although the levels of most cellular mRNA decreased. In addition, AcMNPV replication and viral genes transcription were positively or negatively altered, respectively, when host cells were either pretreated with specific inhibitor of Su(var)3-9, Chaetocin, or transfected with transient expression vector of Su(var)3-9.In summary, the current study shows, for the first time, that Su(var)3-9 might participates in host genes shut-down and repression on viral replication during AcMNPV infection. It provided a new insight for the understanding virus-host interaction, as well as the better exploitation of AcMNPV-insect protein expression system.Part Ⅱ Enhanced transgene expression in mammalian cells by recombinant baculovirus vector containing Bovine papillomavirus type 1 replication elementsThe baculovirus AcMNPV has been widely studied as a transgene expression vector. Compared with other commonly used viral vectors, baculovirus system possesses multiple advantages such as non-replicative genome and no viral gene transcription in mammalian cells; easily manipulated to produce high-titer virus; large capability of foreign genes, etc. Meanwhile, the transient nature and low level of transgene expression limit the use of baculovirus vectors in mammalian systems. Further improving the transgene expression level and extending the period of transgene expression will be important for its application.Bovine papillomavirus type 1 (BPV-1) is a small DNA virus that establishes persistent infection with multi-copies of extra chromosomal viral genome. Replication initiation and stable episomal maintenance of viral genome are mainly determined by the cis-element upstream regulatory region (URR) and two viral early stage proteins El and E2, all other replication factors are supplied by host cells. Furthermore, in the presence of E2, URR has been reported to be able to transactivate adjacent heterologous promoters.In the current study, Bovine papillomavirus type 1 (BPV-1) cis-element URR and trans-elements E1, E2, which are essential for BPV-1 genome replication and maintenance, were inserted into baculovirus genome. Transgene expression efficiency in several mammalian cells transduced by the recombinant baculoviruses and control virus was compared. The cytotoxicity of the recombinant viruses was also evaluated. The results showed that the recombinant baculovirus containing URR and E1, E2 had significantly enhanced expression of enhanced green fluorescence protein (EGFP) in HEK293, HeLa, BHK-21, CNE, CHO-K1 and MDCK cells.The period of transgene expression was also extended. No obvious cytotoxicity was observed, compared with the control virus.In summary, coexistence of BPV-1 URR and El, E2 were essential and sufficient to improve the performance of baculovirus in mediating gene expression in various mammalian cells, without extra negtive effect on cell growth, compared with the control virus. These results would facilitate the application of baculovirus as a more powerful transgene vehicle for protein production and gene therapy.