Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Knock-out of the nuclear localization in pp65 protein of Cytomegalovirus: biologic and immunologic effects

ABSTRACT: 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. 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.

ORGANISM(S): Homo sapiens

SUBMITTER: Xiwei Wu

PROVIDER: E-GEOD-14347 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Biologic and immunologic effects of knockout of human cytomegalovirus pp65 nuclear localization signal.

Zaia John A JA Li Xiuli X Franck Anne E AE Wu Xiwei X Thao Lia L Gallez-Hawkins Ghislaine G

Clinical and vaccine immunology : CVI 20090415 6

The human cytomegalovirus (CMV) pp65 protein contains two bipartite nuclear localization signals (NLSs) at amino acids (aa) 415 to 438 and aa 537 to 561 near the carboxy terminus of CMV pp65 and a phosphate binding site related to kinase activity at lysine-436. A mutation of pp65 with K436N (CMV pp65mII) and further deletion of aa 537 to 561 resulted in a novel protein (pp65mIINLSKO, where NLSKO indicate NLS knockout) that is kinaseless and that has markedly reduced nuclear localization. The pur ...[more]

PMID: 19369477

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Dataset Information

Knock-out of the nuclear localization in pp65 protein of Cytomegalovirus: biologic and immunologic effects

Publications

Biologic and immunologic effects of knockout of human cytomegalovirus pp65 nuclear localization signal.

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