Juan Yin

An OriP/EBNA-1-based baculovirus vector with prolonged and enhanced transgene expression.

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has been explored as a gene delivery vehicle for a variety of mammalian cell lines. However, the transient expression nature due to its incapability to replicate in mammalian cells and insufficient transduction efficiency limit its application.

Functional and structural characterization of DNMT2 from Spodoptera frugiperda

Dear Editor, DNA methylation is one of the most important epigenetic marks and plays a central role in gene silencing in high eukaryotes. It is established by DNA methyltransferases (DNMTs), which are classified into three subfamilies. In contrast to DNMT1 and DNMT3, the biological function of DNMT2 remains elusive to date. DNMT2 was first identified in mouse and human and appears to be well conserved among eukaryotes (Okano et al., 1998; Yoder and Bestor, 1998). DNMT2 contains all 10 characteristic motifs of DNA cytosine MTases and a distinctive target recognition domain (TRD) between motifs VIII and IX, but lacks the N-terminal regulatory domains present in DNMT1 and DNMT3 (Dong et al., 2001; Goll and Bestor, 2005). Nevertheless, only weak residual DNA MTase activity could be detected in human DNMT2, and no DNA methylation changes could be observed in DNMT2 knockout mouse ES cells (Okano et al., 1998; Hermann et al., 2003). Intriguingly, human DNMT2 exhibits a notable MTase activity towards C38 of tRNA (Goll et al., 2006). To explore the biological function(s) of DNMT2 and the underlying molecular mechanism, we identified and cloned a DNMT2 homolog from the insect Spodoptera frugiperda (SfDNMT2). SfDNMT2 shares moderate to high sequence identities with other known DNMT2s and possesses all 10 characteristic motifs of DNA cytosine MTases (Supplementary Figure S1). We generated the SfDNMT2 specific antiserum in mouse for the immunostaining experiments. SfDNMT2 is found to exist in both the nucleus and the cytoplasm of the Sf9 cells (Figure 1A). An in vitro DNA methylation activity assay shows that SfDNMT2 can methylate DNA substrate (Figure 1B). The mutation of Cys231 [equivalent to Cys292 of the human DNMT2 which is required for the tRNA MTase activity (Jurkowski et al., 2008)] to Gln has no effect on its DNA MTase activity (Figure 1B); whereas mutation of the adjacent Cys230, which is conserved in some DNMT2s, significantly reduces its DNA MTase activity, and double mutation of Cys230 and Cys231 has a comparable effect (Figure 1B). The electrophoretic mobility shift assay results show that for both the wild-type and mutant SfDNMT2, DNA is not completely shifted but fades with increasing concentrations of SfDNMT2 (Figure 1C), indicating that SfDNMT2 has weak DNA-binding ability and the mutations have no significant effects on the substrate binding. We further tested the binding ability of SfDNMT2 for human tRNA using electrophoretic mobility shift assay, which shows that SfDNMT2 can also bind human tRNA (Figure 1D), suggesting that tRNA might also be a substrate for SfDNTM2. This is consistent with the observation that Drosophila DNMT2 has both low DNA MTase and tRNA MTase activities (Hermann et al., 2003; Goll et al., 2006). It was suggested that DNMT2 was originated from a DNA MTase which acquired an RNA MTase activity during evolution (Jurkowski and Jeltsch, 2011). It is possible that in some lower invertebrates, such as Drosophila, DNMT2 has an RNA MTase activity but retains a low DNA MTase activity, which results in a low genomic DNA methylation level; however, in higher mammals such as humans which contain more specific DNA MTases, DNMT2 becomes a more specific tRNA MTase. We further determined the crystal structure of SfDNMT2 in complex with S-adenosyl-L-homocysteine (SAH) at 2.7 Å resolution (Supplementary Table S1). SfDNMT2 adopts a typical class I MTase fold (Cheng et al., 1993) and comprises a catalytic domain and a TRD domain (Figure 1E). The catalytic domain is composed of a central eight-stranded b-sheet flanked by two layers of a-helices on each side (Figure 1E). The cofactor SAH is bound at the active site with well-defined electron density and forms extensive interactions with the surrounding residues (Figure 1E, Supplementary Figure S2A and B). The overall structure of SfDNMT2 is very similar to that of human DNMT2 (an RMSD of 1.2 Å for 298 Ca atoms), although the two enzymes share only 36% sequence identity (Figure 1F and Supplementary Figure S1). Nevertheless, there are notable differences in two regions. Specifically, the catalytic loop (motif IV) and the linker between motif VIII and the TRD are well defined in SfDNMT2 (Figure 1F), whereas the corresponding regions are disordered in human DNMT2 (Dong et al., 2001). Sequence comparison shows that this linker in SfDNMT2 and DNMT2s from other insects contains only several residues which is much shorter than that in DNMT2s from human and other high mammals ( 60 residues) (Supplementary Figure S1). The variation in the length of this linker suggests that DNMT2s in mammals might gain new function(s) and/ or different regulation mechanism through acquisition of this insertion. In the structure of SfDNMT2, the catalytic loop protrudes out from the catalytic domain with a positively charged surface, indicative of an ability to bind negatively charged nucleic acids (Figure 1G). The conformation of the catalytic loop in SfDNMT2 is between those in the free form and the DNA-bound M.HhaI, a classic bacterial DNA MTase (Cheng et al., 1993; Klimasauskas et al., 1994) (Figure 1H). However, the catalytic residue Cys78 points its side chain away from the active site which is similar to that in the free form 64 | Journal of Molecular Cell Biology (2013), 5, 64–66 doi:10.1093/jmcb/mjs057 Published online October 25, 2012

Inhibition of nasopharyngeal carcinoma growth by RTA-expressing baculovirus vectors containing oriP.

Nasopharyngeal carcinoma (NPC) is closely associated with latent Epstein–Barr virus (EBV) infection. Activation of latent EBV into lytic replication by introducing viral lytic gene BRLF1 (RTA) has been shown to be a potential approach to suppress the growth of EBV‐associated NPC tumor.

The activation of lytic replication of Epstein-Barr virus by baculovirus-mediated gene transduction.

Summary.A baculoviral mammalian-cell vector was constructed to express Rta, a protein of Epstein-Barr virus (EBV) responsible for the transition from latent infection to lytic replication. EBV lytic replication and cell-growth inhibition was observed in infected D98/HR1 cells. The baculovirus caused little cytotoxicity in the non-targeted HeLa cells, compared to an adenovirus vector. It is concluded that recombinant baculovirus might have the potential as a vector for the therapy of EBV-related cancer.

DNA methyltransferase inhibitors increase baculovirus-mediated gene expression in mammalian cells when applied before infection

The application of baculovirus as a gene delivery vector for mammalian cells is limited by gene silencing in host cells. Although histone deacetylation inhibitors were known to improve baculovirus-mediated gene expression in mammalian cells, another group of chemicals that induces epigenetic changes, DNA methyltransferase inhibitors (DNMTis), were reported to have no such effect. In the current work, we found that, when added prior to virus inoculation, DNMTis improved baculovirus-mediated gene expression by fourfold or more in all four mammalian cell lines tested. The presence of inhibitors during the period of virus inoculation was important for them to be effective.

Characterization of the immune responses elicited by baculovirus-based vector vaccines against influenza virus hemagglutinin

Aim:To compare the specific immune responses elicited by different baculovirus vectors in immunized mice.Methods:We constructed and characterized two recombinant baculoviruses carrying the expression cassette for the H5N1 influenza virus hemagglutinin (HA) gene driven by either an insect cell promoter (vAc-HA) or a dual-promoter active both in insect and mammalian cells (vAc-HA-DUAL). Virus without the HA gene (vAc-EGFP) was used as a control. These viruses were used to immunize mice subcutaneously and intraperitoneally. The production of total and specific antibodies was determined by ELISA and competitive ELISA. Cytokine production by the spleen cells of immunized mice was studied using the ELISPOT assay.Results:Both the vAc-HA and vAc-HA-DUAL vectors expressed HA proteins in insect Sf9 cells, and HA antigen was present in progeny virions. The vAc-HA-DUAL vector also mediated HA expression in virus-transduced mammalian cell lines (BHK and A547). Both vAc-HA and vAc-HA-DUAL exhibited higher transduction efficiencies than vAc-EGFP in mammalian cells, as shown by the expression of the reporter gene egfp. Additionally, both vAc-HA and vAc-HA-DUAL induced high levels of HA-specific antibody production in immunized mice; vAc-HA-DUAL was more efficient in inducing IFN-γ and IL-2 upon stimulation with specific antigen, whereas vAc-HA was more efficient in inducing IL-4 and IL-6.Conclusion:Baculovirus vectors elicited efficient, specific immune responses in immunized mice. The vector displaying the HA antigen on the virion surface (vAc-HA) elicited a Th2-biased immune response, whereas the vector displaying HA and mediating HA expression in the cell (vAc-HA-DUAL) elicited a Th1-biased immune response.

Identification and Characterization of the Spodoptera Su(var) 3-9 Histone H3K9 trimethyltransferase and Its Effect in AcMNPV Infection

Histone H3-lysine9 (H3K9) trimethyltransferase gene Su(var) 3-9 was cloned and identified in three Spodoptera insects, Spodoptera frugiperda ( S . frugiperda ), S . exigua and S . litura . Sequence analysis showed that Spodoptera Su(var) 3-9 is highly conserved evolutionarily. Su(var) 3-9 protein was found to be localized in the nucleus in Sf9 cells, and interact with histone H3, and the heterochromatin protein 1a (HP1a) and HP1b. A dose-dependent enzymatic activity was found at both 27 °C and 37 °C in vitro, with higher activity at 27 °C. Addition of specific inhibitor chaetocin resulted in decreased histone methylation level and host chromatin relaxation. In contrast, overexpression of Su(var) 3-9 caused increased histone methylation level and cellular genome compaction. In AcMNV-infected Sf9 cells, the transcription of Su(var) 3-9 increased at late time of infection, although the mRNA levels of most cellular genes decreased. Pre-treatment of Sf9 cells with chaetocin speeded up viral DNA replication, and increased the transcription level of a variety of virus genes, whereas in Sf9 cells pre-transformed with Su(var) 3-9 expression vector, viral DNA replication slow down slightly. These findings suggest that Su(var) 3-9 might participate in the viral genes expression an genome replication repression during AcMNPV infection. It provided a new insight for the understanding virus–host interaction mechanism.

Enhanced transgene expression in mammalian cells by recombinant baculovirus vector containing bovine papillomavirus type 1 replication elements

The baculovirus Autographa californica multiple nucleopolyhedrovirus has been widely explored as a transgene expression vector. Further improvement of the expression of the transgene is important for its application.