Virus-Inducible Gene Expression Changes In Mouse Brain : Studies With Japanese Encephalitis & Rabies Viruses
One of the key events in a virus-infected host cell is the activation and repression of a large number of host genes. In recent years, such differentially expressed host genes have been identified for several viruses, bacteria and parasites. Such studies indicate that reprogramming of host transcriptome during infection by a pathogen is a major component of host response and many of the reprogrammed genes may promote or prevent pathogen infection or may contribute to pathogen-induced pathological changes. Host gene expression changes have been studied for a number of viruses in cell lines. However, in case of neurotropic viruses which infect nonrenewable populations of central nervous system (CNS), changes in the host gene expression need to be studied in the intact host rather than cells grown in culture. Since such studies are reported only for a few neurotropic viruses, an attempt has been made in this thesis to identify and characterize genes that are differentially expressed in the mouse brain during infection by Japanese encephalitis virus (JEV) and rabies virus. Using subtraction hybridization technique, subtraction cDNA libraries were generated representing mRNAs that are induced or repressed in the mouse brain during JEV infection. Sequence analysis of the 350 isolated clones resulted in the identification of 73 unique genes. Out of these, 66 were of forward library clones (upregulated genes) and 7 of reverse library clones (downregulated genes). The forward library clones was clustered in different functional groups such as, proteins involved in immune response and interferon-inducible proteins, GTPase and GTP binding proteins, transcriptional regulators, enzymes, ribosomal proteins, neuronal proteins, carrier proteins, DNA-binding proteins, miscellaneous and proteins of unknown function. The differential expression of all these genes was further validated by northern blot analysis of brain RNA isolated from normal and JEV-infected mice, which indicate that out of 66 forward library clones 33 were genuinely upregulated in JEV-infected mouse brain, whereas all 7 reverse library clones were repressed in JEV infection. Since vaccination is known to prevent virus replication in the brain, host gene expression changes in mice immunized with BIKEN JE vaccine was also examined. There was a good correlation between inhibition of JEV replication and reduced expression of JEV-inducible CNS genes in the vaccinated mice. To check whether JEV-induced CNS genes identified in this study are specific to JEV or can be induced by any other neurotropic virus, expression patterns of 15 randomly chosen genes were checked in RV infected mouse brain. Results indicated that all the chosen genes are modulated in the same way during RV infection as well. Comparison of JEV-induced gene expression changes with those induced by other neurotropic viruses indicated that 83% of the JEV-inducible mouse CNS genes are also induced by Sindbis virus, a neurotropic virus of the family alphaviridae, indicating that despite diverse life cycles, these two viruses may activate common host signaling pathways. This study also led to the identification of 9 unique JEV-inducible genes (LRG-21, VHSV induced gene1, Tpt1, SLC25A3, Olfm1, Ina/NF-66, Dst/Bpag1, Mdm2 and Gbp5) which are not reported to be activated by any other neurotropic virus. Since it is beyond the scope of this study to characterize all the JEV-induced and repressed genes, two genes were chosen for a detailed analysis. These are: JEV-inducible gene encoding GARG-39 protein which is a member of the glucocorticoid attenuated response gene family and an unannotated, JEV-repressible gene designated in this study as clone # 137. The gene encoding GARG-39 identified as a JEV-inducible gene in this study was originally discovered as lipopolysaccharide- and interferon-inducible gene in macrophages. This protein contains tetratricopeptide repeat (TPR) motifs that are known to be involved in protein-protein interactions. However, the function of this protein remains unknown till date. Therefore the gene was cloned and over-expressed in E. coli and antibodies were raised against the recombinant protein. Western blot analysis revealed that GARG-39 protein is detectable only in JEV-infected but not in the normal mouse brain. Surprisingly, immunoflourescence studies carried out in NIH3T3 cells revealed that GARG-39 is localized in the cytosol of normal cells and it colocalizes with α-tubulin in the mitotic spindle in a small fraction of cells which are in the mitotic stage. Further, in an in vitro assay, GARG-39 was found to interact with taxol-stabilized tubulin polymers. Since microtubules are known to play an important role in virus assembly, it is possible that GARG-39 may have a role in virus assembly and maturation. Alternatively, microtubule-associated proteins are implicated in several neurodegenerative disorders including Parkinson’s, Alzheimer’s and mental retardation and therefore, a role for GARG-39 in virus-induced neuropathogenesis cannot be ruled out. In addition, the expression of GARG-39 in normal dividing cells in the culture indicates a role for this protein in mitosis. In a normal mouse brain, mitotically active cells are very low in number and hence GARG-39 expression (both at the RNA and protein levels) is below the detection limits. JEV infection may trigger mitotic activity in brain leading to increased expression of GARG-39. One of the cDNA clones identified in this study, designated as clone # 137, hybridized to a ~2.6 kb transcript which was found to be down regulated in the mouse brain by JEV as well as rabies virus. A series of investigations led to the conclusion that clone #137 corresponds to the 3′ end of a ~2.6 kb transcript encoding mouse calcium calmodulin kinase inhibitor II α (mCaMKIINα). Interestingly, only the α isoform but not the β isoform of mCaMKIINα mRNA is down regulated in the mouse brain during JEV infection. Since the physiological function of mCaMKIINα is not known, the gene encoding 8 kDa mouse mCaMKIINα open reading frame was cloned into an E. coli expression vector and antibodies were raised against the purified recombinant protein. Surprisingly, antibodies raised against the ~8 kDa recombinant mouse CaMKIINα reacted with a ∼37 kDa mouse brain protein. This protein designated as CaMKIINα-immunoreactive protein (CaMKIINα-IRP) is also down regulated during JEV infection and is localized in the post synaptic density (PSD) of normal mouse brain. In addition, distinct changes are also observed in the subcellular localization and phosphorylation of CaMKIIα leading to an increase in cytosolic CaMKII activity in JEV-infected mouse brain. The differential regulation of CaMKIIα and CaMKIINα during JEV infection suggests a possible role for CaMKII signaling pathway in JEV infection and/or JEV-induced neuropathogenesis in the CNS. Conclusions: • A number of host genes whose expression is modulated in the mouse brain during JEV and/or rabies virus infection have been identified. • One of the JEV-inducible genes encoding the GARG-39 protein was shown to be a microtubule-associated protein with a possible role in mitosis. • One of the JEV-repressible genes was found to encode the mouse CaMKIINα mRNA. • A novel JEV-repressible ∼37 kDa protein immunoreactive to antibodies raised against the recombinant CaMKIINα was identified in the post synaptic density of the mouse brain.
- Biochemistry (BC)