Functional characterization of murine gammaherpesvirus 68 ORF20 in vitro and in vivo
- 주제(키워드) gammaherpesvirus , immune modulation , interferon-beta
- 발행기관 고려대학교 대학원
- 지도교수 송문정
- 발행년도 2010
- 학위수여년월 2010. 8
- 학위구분 석사
- 학과 일반대학원 생명공학과
- 세부전공 분자생물공학
- 원문페이지 58 p
- 실제URI http://www.dcollection.net/handler/korea/000000023640
- 본문언어 한국어
- 제출원본 000045610984
초록/요약
While interferons (IFNs) play a central role in limiting the spread of herpesviruses in the host, herpesviruses in turn modulate the host interferon responses for their replication. Murine gammaherpesvirus 68 (MHV-68 or gammaHV-68) provides an experimental model system to study dynamic equilibrium between virus and host immune responses. Our previous functional screening of a MHV-68 mutant virus library in 5xISRE-Luc/NIH3T3 cells identified an ORF20null virus that failed to down-regulate the activation of the ISRE-containing promoter in contrast to the wild-type. In this study, we further characterized functional properties of ORF20 in vitro and in vivo. Transiently-expressed ORF20 was localized in the nucleolus. The ORF20 protein inhibited TBK1-mediated transactivation of the IFN-？ promoter as well as the 5xISRE promoter but had little effect on IRF3-5D-mediated transactivation. Consistent with this result, infection of ORF20 deficient MHV-68 (ORF20STOP) increased the level of STAT1 phosphorylation. When an ORF20STOP virus expressing the firefly luciferase driven by a viral lytic promoter was intranasally infected into mice, in vivo bio-imaging results suggest that the lack of ORF20 protein may alter the systemic spread of the virus from the site of infection to the site of persistency. While acute replication of the ORF20STOP virus was similar to that of the wild type in the lung of the infected mice, its establishment of latency was significantly impaired in the spleen. These results suggest a potential link of viral immune modulation to establishment of persistent infection in vivo.
more목차
1. Abstract 1
2. Introduction 3
3. Materials and Methods 8
3.1 Cells and Viruses 8
3.2 Plaque assay 9
3.3 Luciferase reporter assay 9
3.4 Immunofluorescence assay 10
3.5 Western blot analysis 11
3.6 Mouse experiments 12
3.7 Bioluminescence optical imaging 12
3.8 Titration of lytic viruses in the lung tissues 13
3.9 Ex vivo reactivation assay ？ Limiting dilution 13
3.10 Quantitative real time PCR 14
4. Results 15
4.1Expression of MHV-68 ORF20 expression 15
4.2 ORF20 protein reduces IFN-？ promoter activity that was activated by TBK1 and IRF3, but not by IRF3-5D 21
4.3 ORF20 suppresses interferon signaling of the host 27
4.4 ORF20 is required for establishment of latency but not for lytic replication 31
5. Discussion 40
6. References 42
