Measles Virus Infect Insect Cells With The Receptor SLAM And The Mechanism Of MV Induced Apoptosis

Signaling lymphocytic activation molecule (SLAM; also known as CDw150) has been reported as the receptor of measles virus (MV) interacting with MV hemagglutinin (MVH). In this study, we developed a baculovirus-derived vector, the Bacmid-egfp, containing a reporter gene encoding the enhanced green fluorescent protein (EGFP) under the control of the promoter of very late polyhedrin gene from Autographa californica Multiple Nucleopolyhedrovirus (AcMNPV), and employed the recombinant baculovirus to express SLAM in Sf9 (Spodoptera frugiperda) cells and investigate SLAM function. The result showed that the integration of the EGFP expression cassette in the Bac to Bac system facilitated research with the system without introducing compromises due to its use. SLAM protein fused to His-tag was expressed in Sf9 cells through the modified Bac to Bac system. The expressed SLAM was identified as approximately 46 kD and presented on the cell surface, as revealed by fluorescent immunochemical staining and confocal microscopic analysis. The pull down assay proved that SLAM protein expressed in this system could interact with MVH protein. After incubating with MV vaccine strain S191, cell fusion was only observed in the Sf9 cells expressing both EGFP and SLAM from recombinant baculovirus rather than those expressing EGFP only from the modified viral vector. Furthermore, MV replicated and induced apoptosis in the Sf9 cells with SLAM expression.DNA ladder analysis, hoechst33342 staining and flow cytometry analysis demonstrated that apparent apoptosis characteristics exhibit in HeLa cells infected with measles virus (SI91). A wealth of information supports the notion that apoptosis is controlled via two major apoptotic pathways, one that originates at the membrane and another that involves the mitochondria. Most of them are ultimately coupled to the activation of effector caspases and Bcl-2 classes. However, endoplasmic reticulum (ER), an organelle whose critical contributions to apoptosis is only now becoming apparent. Mobilization of ER calcium stores can initiate the activation of cytoplasmic death pathways as well as sensitize mitochondria to directly initiate pathways to apoptosis However, Measles virus vaccine strain SI 91 induced apoptosis of HeLa cells and the mechanism is not well known.In our research, mRNA and protein level of endogenous HAP/RTN3 were upregulated in HeLa cells infected with measles virus vaccine strain SI91 at 48 h p.i.. Our previous work demonstrated that the overexpression of HAP (RTN3, homolgue of ASY/RTN4) dramatically induced apoptosis with the depletion of ER (endoplasmic reticulum) Ca + stores. The sublocalization of endogenous HAP and forming homopolymers with the disulfide bond on ER membrane were important for the apoptosis inducing function of HAP. Real time PCR results demonstrated that MV infection specifically up-regulated BIP and CHOP of the HeLa cell, which control ER stress of HeLa cells. When Bcl-2 was upregulated after MV infection at 24 h p.i. , HAP was decreased.lt demonstrated that since the induction of apoptosis early after infection would severely limit virus production and reduce or even eliminate the spread of progeny virus in the host, most viruses have evolved strategies to evade or delay early apoptosis in an attempt to allow production of high yields of progeny virus. Therefore, viruses are also known to encode products that directly block the apoptotic signal cascade.There were two activities: 1) the increasing intensity of [Ca2+]i by depletion of ER (endoplasmic reticulum) Ca2+ stores; 2) TRAIL upregulation induced by measles viral infection . In contrast, suppression of HAP using RNAi technology, the infected cells abrogated these two activities. And MV-induced apoptosis can be regulated by HAP throughforming polymers with the disulfide bond on ER membrane. TRAIL dependent pathway suggests that HAP/RTN3 plays a pivotal role in apoptosis induced with MV infection. There were the cross-talking between ER and mitochondrion with the change of [Ca +]i oThe extent of apoptotic cell death was examined in central nervous system (CNS) tissues from cases of subacute sclerosing panencephalitis (SSPE) induced by measles virus. Live attenuated measles virus (MV-Edm) has potent oncolytic activity against myeloma xenografts in mice. The apoptosis pathway involved HAP which was induced by MV infection was described, and HAP was highly expressed in adult brain. It maybe also helpful to the study the subacute sclerosing panencephalitis (SSPE) induced by measles virus in central nervous system (CNS).Moreover, measles virus and the tH protein were used to screen a 15-mer phage display peptide library in this study. After 4 rounds of screening and sequence analysis, the deduced amino acid sequence of screened peptides GCLVSLGL*WVV*FW and MSKNIHFVVTL GWSL and showed highly homologous with amino acid 56-70 of SLAM (MNKSIHIVVT MAKSL). To further determine the importance of the sequence in SLAM protein during the interaction between H and SLAM, it showed that amino acid changes in 59 Ser or 61 His and 69 Ser were important for SLAM protein to interact with H based on the software ANTHEPROT 4.3 analysis above. And also the peptides GCLVSLGL*WVV*FW showed highly homologous with amino acid of NFkB and IkBoi complex ELLVSLGADVNAQEP.