Differential display RT-PCR analysis of ECV304 endothelial-like cells infected with dengue virus type 2 reveals messenger RNA expression profiles of multiple human genes involved in known and novel roles.
Differential display (DD)-RT-PCR was employed to analyze mRNAs from ECV304 human endothelial-like cells undergoing apoptosis following infection with the virulent New Guinea C strain of dengue virus type 2 in order to elucidate the cellular gene responses to dengue viral infection at the transcriptional level. We isolated, sequenced, and identified 203 differentially expressed and overlapping cDNA fragments, all of which were of human origin except 1 that was of viral origin. Out of these, 78 were individual distinct clones comprising 46 and 32 expressed sequence tags (ESTs) that exhibited upregulated and downregulated trends, respectively. Of the 78 differentially expressed mRNAs, 16 did not match any characterized genes or ESTs. The remaining 62 mRNAs modified by dengue virus infection matched known genes, including those encoding components of the cell cycle (Anillin, CDC27), cytoskeleton (epsilon-tubulin), signal transduction (OPHN1, PPP2R2A, TIRAP), protein translation and modification (EIF3S10, IF2, TMEM1), transcriptional regulation (alpha-NAC, C20orf104, EGR1, ELP2), apoptotic cell death (RICK), membrane (BPAG1), and mitochondrial-related proteins. Semiquantitative RT-PCR and real-time RT-PCR authenticated further the altered expression patterns of selected genes of interest. These data demonstrate the feasibility of mRNA DD in providing insights into the complex responses of the transcriptional machinery of permissive and apoptotic human endothelial-like cells in the pathogenesis of dengue and/or its complications induced by the virulent dengue virus type 2.
Human Genome Laboratory, Department of Microbiology, Faculty of Medicine, National University of Singapore, Kent Ridge, Singapore.
Kingsley J L
KJ
Liew
Vincent T K
VT
Chow
D017209:Apoptosis / Q000235:genetics; D002453:Cell Cycle / Q000235:genetics; D002460:Cell Line; D003599:Cytoskeleton / Q000235:genetics; D003716:Dengue Virus / Q000254:growth & development*; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism* / Q000821:virology*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D005796:Genes / Q000235:genetics / Q000502:physiology; D005814:Genes, Viral / Q000235:genetics / Q000502:physiology; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics; D024101:Mitochondrial Proteins / Q000235:genetics; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D015398:Signal Transduction / Q000235:genetics; D014158:Transcription, Genetic
D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D012333:RNA, Messenger