Analysis of gene expression in mouse fibroblast cells during infection with minute virus of mice

• Main Author: Williams, Warren Perry
• Format: Others
• Language: English
• Published: 2009
• Online Access: http://hdl.handle.net/2429/15023

Minute Virus of Mice (MVM), a mouse parvovirus, has served as a model for understanding parvovirus infection. To further understand how parvoviruses replicate within the cell and cause disease, the effect of MVM infection on host cell gene expression in mouse fibroblast cells (LA9 cells) was studied. This was accomplished through three techniques: Differential display, Clontech macroarrays, and Affymetrix microarray analysis. The RT/PCR technique differential display was used to studied altered gene expression in unsynchronized infected mouse fibroblast LA9 cells at 12, 24 and 36 hours post-infection. Twenty-four primer pair combinations were used, representing ~ 15% of actively transcribed RNA. Surprisingly, few genes were found to change, although the technique did detect MVM NS1 transcript. In addition, the rodent retroelements, B1 & B2 SINEs and the L l LINE were all found to be increased as a result of MVMp infection. Further gene expression studies on synchronized MVM infected LA9 cells at 12 and 24 hours post-block were undertaken with Clontechs macroarrays (~1000 genes per array). Overall, the signal from these arrays were low and there were problems with background. Again, very few transcripts appeared to change as a result of infection, with only 3 genes being altered at the 12 hour time point, and another 7 reproducibly (by independent arrays) at the 24 hour time point. These included the murine B2 SINE (2.8 fold increase, confirming the differential display results), RNA exchange factor binding protein 1 (two fold increase) and three cyclins. A preliminary study was also done with the superior Affymetrix oligonucleotide microarrays (12,000 different genes, 16 oligonucleotides/gene). Of the 5347 genes and ESTs that were detected in the screen, 74 were found to be altered in unsynchronized LA9 cells infected with MVMp. These included genes involved in promoting cell growth (epiregulin, 13.9 fold increase), genes that inhibited growth (nuclear protein p8, 3.3 fold decrease), immune/inflammatory genes, specifically targeting transforming growth factor (3 (2.5 fold decrease), a number of transcription factors (C/EBP, 2.5 fold decrease) and genes involved in cholesterol synthesis and transport (farnesyl diphosphate sythetase, 2 fold decrease) as well as a number of unknown ESTs. The SINE response to MVM infection was further investigated. Primer extension assays confirmed that the murine B l and B2 SfNEs are up-regulated in LA9 cells throughout MVM infection. These studies also demonstrated that the SINE response was due to RNA polymerase III transcription and not contaminating DNA or RNA polymerase II transcription. Furthermore, expression of MVM NS1 in LA9 cells by transient transfection also leads to an increase in both murine SINEs. This is the first time that the B l and B2 SINEs have been shown to be altered by viral infection. The increase in the SINE transcripts does not appear to be due to an increase in either of the basal transcription factors TFIIIC110 or 220, as these proteins do not increase during MVMp infection. However, nuclear run-on experiments to determine if increased SINE expression was due to increased SINE RNA transcription were inconclusive. Protein levels of some 75 different protein kinases were also investigated in synchronized, MVMp infected LA9 cells. Very few changes were observed. === Medicine, Faculty of === Biochemistry and Molecular Biology, Department of === Graduate