Modulation of Natural Killer cell response by human cytomegalovirus

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Sugrue, Daniel Martyn;
  • Subject: QR | R1
    mesheuropmc: viruses

The Natural Killer (NK) cell activating receptor DNAM-1 (CD226) is stimulated through recognition of CD112 (nectin-2) and CD155 (nectin-like molecule 5; PVR) on target cells. HCMV UL141 elicits protection from NK-cells by down-regulating CD155 from the cell surface and ... View more
  • References (36)
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    3.0. ANALYSIS OF CD112 EXPRESSION DURING HCMV INFECTION. ................113 3.1. Developing assays for the detection of CD112. ....................................................113 3.1.1. Flow cytometry detected the cell surface expression of CD112 on a range of cell lines. .........................................................................................................113 3.1.2. Immunofluorescence detected CD112 at inter-cellular junctions. ............117 3.1.3. Enrichment of CD112 for immunoblotting. ..............................................117 3.2. CD112 is a glycoprotein with two isoforms. .........................................................120 3.3. The product of UL141 is essential but not sufficient for the down-regulation of CD112 during HCMV infection. ..................................................................................121 3.4. UL141-GFP cell line does not down-regulate CD112...........................................124 3.5. UL141 was not essential for viral growth/replication. ..........................................124 3.6. CD112 is notably down-regulated at 2 days p.i. during HCMV infection. ...........126 3.7. CD112 transcription is not negatively affected during HCMV infection..............130 3.8. Conclusion .............................................................................................................133

    4.0. SCREENING STRATEGY FOR THE IDENTIFICATION OF GENE(S)

    RESPONSBILE FOR CD112 DOWN-REGULATION...................................................134 4.1. Identification of screening strategy........................................................................134

    5.0. CHARACTERISATION OF THE INTRACELLULAR LOCAISATION OF HCMV

    PROTEINS EXPRESSED FROM RAD BY IMMUNOFLUORESCENCE ...................147 5.1. Transgene detection ...............................................................................................148 5.2. Categorisation of localisation ................................................................................168 5.3. Comparison of protein localisation to the literature: Differences in localisation ..171 5.3.1. Genes that showed partial matches with descriptions in the literature......171 5.3.2. Genes that did not match descriptions in the literature .............................175 5.3.3. Potential causes for difference in localisation described in the literature compared to localisation in this study. ................................................................178 5.4. Undetectable proteins ...................................................................................181 5.5. Conclusion .............................................................................................................183

    6.0. SCREENING OF THE RECOMBINANT ADENOVIRUS LIBRARY FOR CD112

    DOWN-REGULATION....................................................................................................184 6.1 Confirming CD112 down-regulation in HFFF-CAR's...........................................184 6.2. Screening RAd library by flow cytometry.............................................................186 6.3. RAd-HCMV-ORF library screening results: CD155 ............................................222 6.4. RAd-HCMV-ORF library screening results: MHC-1 ...........................................223 6.5. RAd-HCMV-ORF library screening results: US2 appeared to be involved in CD112 down-regulation ...............................................................................................223 6.6. Conclusion .............................................................................................................226

    7.0. INVESTIGATION INTO THE US1-11 REGION OF HCMV FOR INVOLVEMENT

    IN CD112 DOWN-REGULATION..................................................................................227 7.1. Generating HCMV deletion mutants using the technique of Recombineering .....227 7.2. A HCMVΔUS1-11 mutant did not down-regulate CD112 in infected cells. ........228 7.3. Recombineering using the GALK selection cassette generated HCMVΔUS11 but not HCMVΔUS2...........................................................................................................232 7.4. Recombineering using the Strep selection cassette did not generate HCMVΔUS2. ......................................................................................................................................236 7.5. Co-infection of RCMVΔUS1-11 with RAd expressing one of the US1-11 genes did not rescue the Merlin phenotype of CD112 down-regulation. .....................................238 7.6. Infecting with multiple RAd's in the US1-11 region did not reproduce the Merlin phenotype......................................................................................................................240 7.7. CD112 was down-regulated in RAd-UL141 infected HELA-US2 cells, but not - UL141 infected Hela-US11. .........................................................................................240

    8.0. DISCUSSION.............................................................................................................249 8.1. Use of RAd-HCMV-ORF library in screening for HCMV function.....................252 8.2. Building further screening tools ............................................................................253 8.3. Potential future work: CD112 in NK and DC cell interaction...............................254 8.4. Conclusion .............................................................................................................256

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