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

ABSTRACT: 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 purpose of this study was to biologically characterize this protein and its immunogenicity compared to that of native pp65. Unlike the native CMV pp65, following either DNA- or recombinant adeno-associated virus-based transduction of CMV pp65mIINLSKO into cells in vitro, the first observation of pp65mIINLSKO expression was in the cytoplasm and pp65mIINLSKO was expressed at higher levels than the native protein. The CMV pp65mIINLSKO mRNA was more abundant earlier than CMV pp65 mRNA (at 4 h and 8 h, respectively), but the half-lives of the proteins were the same. This modification altered the antigenic processing of CMV pp65 in vitro, as measured by the improved efficiency of cytotoxic killing in a pp65mIINLSKO-transduced human HLA A*0201 target cell line. In HHDII mice expressing HLA A*0201, pp65mIINLSKO was as immunogenic as CMV pp65. By RNA microarray analysis, expression of the CMV pp65mIINLSKO had less of an effect on cell cycle pathways than the native CMV pp65 did and a greater effect on cell surface signaling pathways involving immune activity. It is concluded that the removal of the primary NLS motif from pp65 does not impair its immunogenicity and should be considered in the design of a vaccine.

SUBMITTER: Zaia JA

PROVIDER: S-EPMC2691050 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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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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Project description: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.

Project description:Abstract: We have identified a cytomegalovirus virion protein capable of modulating the rapid induction of an interferon-like response in cells that follows virus binding and penetration. Functional genomics revealed a role for the major cytomegalovirus structural protein, pp65 (ppUL83), in counteracting this response. The underlying mechanism involves a differential impact of this structural protein on the regulation of interferon response factor 3 (IRF-3). In contrast, NF-{kappa}B is activated independent of pp65, and neither STAT1 nor STAT3 becomes activated by either virus. pp65 is sufficient to prevent the activation of IRF-3 when introduced alone into cells. pp65 acts by inhibiting nuclear accumulation of IRF-3 and is associated with a reduced IRF-3 phosphorylation state. Thus, this investigation shows that the major structural protein of cytomegalovirus is committed to the modulation of the IRF-3 response, a primary mediator of the type I interferon response. By subverting IRF-3, the virus escapes throwing a central alarm devoted to both immediate antiviral control and regulation of the immune response. Polyadenylated RNA from 107 confluent HFs was purified by using Oligotex (QIAGEN, Valencia, Calif.) from Trizol (Invitrogen)-extracted total RNA. This RNA was prepared, reverse transcribed, labeled with Cy3-dUTP or Cy5-dUTP (Amersham, Little Chalfont, Buckinghamshire, United Kingdom) by random primed synthesis with DNA pol I Klenow (Amersham Life Science, Inc., Cleveland, Ohio), and hybridized to spotted, human cDNA microarrays as previously described. Sequence-verified human cDNA microarrays (HE and HG series, 31,000 spots; HD51 series, 17,000 spots) were produced at Stanford. Images were collected by using a GenePix 4000B microarray scanner, manually flagged to eliminate poor spots and analyzed by GenePix Pro 2.0 (Axon, Union City, Calif.). A replicate experimental design type is where a series of replicates are performed to evaluate reproducibility or as a pilot study to determine the appropriate number of replicates for a subsequent experiments. Keywords: disease_state_design, pathogenicity_design

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

Publications

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

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