Differential display RT-PCR analysis of enterovirus-71-infected rhabdomyosarcoma cells reveals mRNA expression responses of multiple human genes with known and novel functions.
In order to better understand cellular responses to viral infection at the transcriptional level, we employed differential display RT-PCR to analyze mRNAs from RD rhabdomyosarcoma cells following infection with a neurovirulent enterovirus 71 (EV71) strain, compared with mRNAs from uninfected cells. Of 250 expressed sequence tags (ESTs) isolated, sequenced, and identified, all were of cellular origin except 1 that was of viral origin. Of these, 156 were individual distinctive clones, comprising 45 mRNAs showing unaltered expression and 111 mRNAs exhibiting upregulation or downregulation. Of the 45 uniformly expressed mRNAs, 14 represented unknown genes. Of the 111 differentially expressed mRNAs, 63 did not match any known genes. Forty-eight of the 111 mRNAs modified by EV71 infection matched known genes, including those encoding components of cell cycle, cytoskeleton, and cell death mediators; protein degradation mediators; mitochondrial-related proteins; components of protein translation and modification; and cellular transport proteins. The altered expression profiles of representative genes were authenticated by semiquantitative RT-PCR and real-time RT-PCR. We also identified a novel alternatively spliced transcript of TRIP7 thyroid receptor interactor protein; the putative human homolog of murine mc7 mRNA predominantly expressed in the brain; and a novel mRNA similar to that encoding vacuolar protein 8 involved in protein targeting. These results underscore the applicability of the mRNA differential display technique for elucidating the expression profiles of known and even novel genes in response to cellular infection with pathogenic viruses.
Leong
Peter W F
PW
Human Genome Laboratory, National University of Singapore, Kent Ridge, 117597, Singapore.
Liew
Kingsley
K
Lim
William
W
Chow
Vincent T K
VT
D000595:Amino Acid Sequence; D001483:Base Sequence; D000075686:COP9 Signalosome Complex; D002352:Carrier Proteins / Q000235:genetics; D002453:Cell Cycle / Q000235:genetics; D016923:Cell Death / Q000235:genetics; D003599:Cytoskeleton / Q000235:genetics; D004770:Enterovirus / Q000235:genetics / Q000502:physiology*; D020224:Expressed Sequence Tags; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics; D009687:Nuclear Proteins; D021381:Protein Transport / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis*; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D011988:Receptors, Thyroid Hormone; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012208:Rhabdomyosarcoma; D014157:Transcription Factors; D000070584:Tripartite Motif Proteins; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; C118919:Cops2 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; D011988:Receptors, Thyroid Hormone; D014157:Transcription Factors; C425297:Trim44 protein, mouse; D000070584:Tripartite Motif Proteins; D000075686:COP9 Signalosome Complex