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Series GSE14347 Query DataSets for GSE14347
Status Public on Jan 31, 2009
Title Knock-out of the nuclear localization in pp65 protein of Cytomegalovirus: biologic and immunologic effects
Organism Homo sapiens
Experiment type Expression profiling by array
Summary The CMVpp65 protein contains 2 bipartite nuclear localization signals (NLS) at 415-438aa and 537-561aa near the carboxy terminus of CMVpp65 and a phosphate binding site related to kinase activity at lysine-436. A mutation of pp65 having K436N (CMVpp65mII) and further deletion of aa537-561 resulted in a novel protein (pp65mIINLSKO) that is kinase-less and has markedly reduced nuclear localization. The purpose of this report was to study the biologic characterization of this protein and its immunogenicity compared to native pp65.Using RNA microarray analysis, expression of the CMVpp65mIINLSKO had less effect on cell cycle pathways than did the native CMVpp65 and a greater effect on cell surface signalling pathways involving immune activity. It is concluded that the removal of the primary NLS motif from pp65 does not impair its immunogenicity and may actually be advantageous in the design of a vaccine.

Keywords: comparison of cellular response to wild-type gene expression vs a mutated gene.
 
Overall design The MRC-5 cells were transduced in triplicate, with rAAVintA, rAAVpp65N, rAAVpp65NLSKO for 48hrs and the total RNA was purified with RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. One µg of each sample was processed using the Affymetrix GeneChip Whole Transcript Sense Target Labeling Assay. After ribosomal RNA reduction (Invitrogen, Carlsbad, CA) to remove most of the 18S and 28S rRNAs, the GeneChip WT (whole transcript) cDNA Synthesis Kit, WT cDNA Amplification Kit, and WT Terminal Labeling Kit (Affymetrix, Inc., Santa Clara, CA) were used for target preparation. Ten µg of cRNA (antisense RNA) were added to the second-cycle cDNA reaction followed by fragmentation and Terminal Labeling process. The hybridization cocktails containing 2.5 ug of the fragmented, end-labeled cDNA were applied to the GeneChip® Human Gene 1.0 ST arrays. The Genechip® Human Gene 1.0 ST array was chosen because it offers the whole transcript coverage consisting of 28,869 genes on the array by approximately 26 probes spread across the full length of the gene.
 
Contributor(s) Zaia JA, Li X, Franck AE, Wu X, Thao L, Gallez-Hawkins G
Citation(s) 19369477
Submission date Jan 08, 2009
Last update date Jul 26, 2018
Contact name Xiwei Wu
E-mail(s) xwu@coh.org
Organization name City of Hope National Medical Center
Department Computational and Quantitative Medicine
Street address 1500 E. Duarte Rd.
City Duarte
State/province CA
ZIP/Postal code 91010
Country USA
 
Platforms (1)
GPL6244 [HuGene-1_0-st] Affymetrix Human Gene 1.0 ST Array [transcript (gene) version]
Samples (9)
GSM358849 rAAVpp65N-rep1
GSM358850 rAAVintA-rep1
GSM358851 rAAVpp65mIINLSKO-rep1
Relations
BioProject PRJNA111533

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Supplementary file Size Download File type/resource
GSE14347_RAW.tar 38.7 Mb (http)(custom) TAR (of CEL)
Processed data included within Sample table

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