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fbtwitterlinkedinvimeoflicker grey 14rssslideshare1
Sugrue, Daniel Martyn
Languages: English
Types: Doctoral thesis
Subjects: QR, R1

Classified by OpenAIRE into

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 sequestering it in the ER (Tomasec, 2005). Here, HCMV UL141 was shown to be involved in the down-regulation of CD112. Interestingly, UL141 appeared necessary but not sufficient to modulate CD112 expression. This thesis therefore focused on a hypothesis whereby UL141 was acting with one or more additional HCMV genes to target CD112 for degradation.\ud This project was the first to utilise an entire recombinant adenovirus (RAd) library expressing individual HCMV ORFs (RAd-HCMV-ORF library) to screen for function. The RAd-HCMV-ORF library clearly provided an extremely powerful tool for the screening of HCMV gene function as results were highly repeatable and robust. The co-infection of RAd-UL141 and RAd-US2 resulted in a single, clear, positive hit in the final screening process. This hit was further verified by immunoblot where CD112 appeared to be down-regulated in cells infected with both RAd-UL141 and RAd-US2, compared to controls. While a Hela-US2 cell line which stably expressed US2 also down-regulated CD112 when infected with RAd-UL141. A RCMVΔUS1-11 virus was constructed, which failed to down-regulate CD112 from the cell surface of RCMVΔSU1-11 infected cells.\ud The addition of proteasome inhibitors was able to partially restore CD112 expression in HCMV infected cells (Prod'homme et al., 2010). It therefore appeared that US2 and UL141 act to degrade CD112 via the proteasome during HCMV infection. CD112 downregulation may have the potential to prevent DNAM-1:CD112 interaction between HCMV infected targets and effector cells of the immune system, providing another facet to HCMV’s ability to avoid the human immune response.
  • The results below are discovered through our pilot algorithms. Let us know how we are doing!

    • 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
    • Figure 1.1. The HCMV virion.........................................................................................2
    • Figure 1.2. Comparison of human herpesviridae family genome size.............................5
    • Figure 1.3. HCMV capsid assembly model.....................................................................9
    • Figure 1.4. HCMV strain AD169 and Toledo genome structures..................................16
    • Figure 1.5. Genomic map of HCMV strain Merlin........................................................18
    • Figure 1.6. Cascade of HCMV gene expression............................................................20
    • Figure 1.7. Overview of HCMV immunomodulation of infected cells.........................33
    • Figure 1.8. Summary of HCMV inhibition of IFN signaling through the Jak/STAT pathway........................................................................................................38
    • Figure 1.9. Summary of HCMV mediated MHC-1 degradation....................................42
    • Figure 1.10. Selection of NK cell activatory and inhibitory receptors and their cognate ligands..........................................................................................................48
    • Figure 1.11. Nectin structure and proposed model for intercellular adhesion activity....56
    • Figure 1.12. Interactions between the Nectin family and IGSF.......................................57
    • Figure 1.13. Nectin intracellular signaling pathway.......................................................59
    • Figure 7.1. The construction of RCMV ΔUS1-11 using the recombineering technique and the AmpR/SacB/LacZ selection cassette..............................................229
    • Figure 7.2. RCMV ΔUS1-11 does not downregulate CD112 or MHC-1....................231
    • Figure 7.3. The construction of RCMV ΔUS11 using the recombineering technique and the GalK selection cassette..................................................................233
    • Figure 7.4. RCMV ΔUS11 is still capable of down-regulating CD112.......................235
    • Figure 7.5. RCMV ΔUS2 could not be generated using the recombineering technique and the RpsL-Neo-LacZ selection cassette.................................................237
    • Figure 7.6. Co-infection of RAd with RCMVΔUS1-11 did not rescue the Merlin phenotype...................................................................................................239
    • Figure 7.7. Flow cytometry detection of CD112 in HFFF-CARs infected with three RAds: US2, UL141 and one RAd covering the US1-11 region................241
    • Figure 7.8. Flow cytometry detection of CD155 in HFFF-CARs infected with three RAds: US2, UL141 and one RAd covering the US1-11 region................242
    • Figure 7.9. Flow cytometry detection of MHC-1 in HFFF-CARs infected with three RAds: US2, UL141 and one RAd covering the US1-11 region................243
    • Figure 7.10. CD112 is down-regulated in RAd-UL141 infected Hela-US2 cells.........244
    • Figure 7.11. Proteasome inhibitors prevent the degradation of CD112 in HCMV infection of HFFF's....................................................................................246
    • Appendix I: Immunofluorescent staining of cells infected with a RAd from the RAdHCMV-ORF library to determine transgene expression and subcellular location: A. RL1-UL1...................................................................................................291 B. UL2-UL11.................................................................................................292 C. UL13-UL21A............................................................................................293 D. UL22A-UL30............................................................................................294 E. UL31-UL38...............................................................................................295 F. UL40 and UL112-UL120.........................................................................296 G. UL121-UL136...........................................................................................297 H. UL138-UL148...........................................................................................298 I. UL148A-IRS1...........................................................................................299
    • Appendix II. Screening RAd-HCMV-ORF library for CD112 down-regulation: A. RAd-RL1, RAd-RL5A, RAd-RL6 and RAd-RL8A..............................301 B. RAd-RL9, RAd-RL10, RAd-RL11 and RAd-RL12..............................302 C. RAd-RL13, RAd-UL1, RAd-UL2 and RAd-UL4..................................303 D. RAd-UL5, RAd-UL6, RAd-UL7 and RAd-UL7/8................................304 E. RAd-UL8, RAd-UL9, RAd-UL10 and RAd-UL11................................305 F. RAd-UL22A, RAd-UL23, RAd-UL24 and RAd-UL25.........................306
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