臺灣博碩士論文加值系統

中文摘要
A型流感病毒（IAV）是一種具有模套的RNA病毒。其基因組由八個單股負鏈的RNA所組成，共帶有12個病毒蛋白編碼。每個病毒RNA片段都與病毒核蛋白（nucleoprotein, NP）以及病毒RNA聚合酶（PB1，PB2和PA）所包覆，形成病毒核蛋白(viral ribonucleoprotein, vRNP）複合體。我們先前的研究顯示，NP是單泛素化的蛋白質(mono-ubiquitinated protein)，其可透過細胞去泛素酶USP11專一性的去泛素化(deubiquitination)。NP的泛素化穩定了其與病毒RNA之間的相互作用，另USP11可藉由NP的去泛素化來降低病毒RNA的複製效率。鑑於這些研究結果，我們將從RNAi pooled screening中得出的候選基因再進行小規模RNAi篩選，試圖找出哪些E3連接酶是負責NP泛素化的功能；一個名為CNOT4的E3泛素連接酶被篩選出，並接續做進一步研究。我們發現，抑制CNOT4表現之A549細胞感染IAV後，NP RNA的表現量降低；另外，在抑制CNOT4表現之293T細胞中，利用微複製子報導基因檢測法(minireplicon reporter assay)我們發現病毒RNA聚合酶的活性也受到抑制。這些結果表明，CNOT4可能參與病毒RNA的轉錄和複製作用。在in vitro泛素化試驗中，抑制CNOT4表現也使得NP泛素化降低；當過度表現CNOT4時，NP泛素化和病毒複製酶的活性都能在被抑制的細胞中再次恢復。此外，當USP11與CNOT4共同表現時，NP的泛素化會再次的減少、並比CNOT4單獨表現時更低。結果表明，CNOT4可能會增加病毒NP的泛素化、進而提高病毒RNA聚合酶之活性；在IAV複製過程中，CNOT4與USP11具有相反的功能。根據目前的研究結果，我們推測CNOT4是NP的E3連接酶。

Abstract
Influenza A virus (IAV) is an enveloped RNA virus. Its genome consists of eight single-stranded negative-sense RNAs that encode 12 viral proteins. Each viral RNA segment is packaged with viral nucleoprotein (NP) and RNA-dependent RNA polymerase complex (PB1, PB2 and PA) to form viral ribonucleoprotein (vRNP) complexes. Our previous report showed that NP is a mono-ubiquitinated protein and can be specifically deubiquitinated by cellular deubiquitinase USP11. Ubiquitination of NP could alter the interaction of NP with viral RNA, and USP11 can cleave ubiquitin from NP, thereby reducing the RNA replication efficiency. Given these findings, we attempted to determine which E3 ligase(s) are responsible for NP ubiquitination; we used a small-scale RNAi screen based on candidates derived from RNAi pooled screening. An E3 ubiquitin ligase termed CNOT4 was picked from that screening for follow-up study. We found that expression of virus NP was decreased in a CNOT4 knockdown A549 cell line upon IAV infection. In addition, using CNOT4 knockdown 293T cells, we determined that viral RdRp activity was also inhibited as demonstrated by the minireplicon reporter assay. These findings suggest that CNOT4 may play a role in viral RNA transcription and replication. Interestingly, NP ubiquitination was decreased as evaluated by an in vitro ubiquitin assay in CNOT4 knockdown cells. When overexpressed wobble CNOT4, both NP ubiquitination and viral RdRp activity ware rescued in knockdown cells. Furthermore, when USP11 was co-expressed with CNOT4, the level of ubiquitination of NP was lower as compared with CNOT4 expressed alone. The results indicate that CNOT4 may increase ubiquitination of viral NP protein and enhance viral RdRp activity, and CNOT4 has opposite function with USP11 for IAV replication. Based on current findings, we hypothesize that CNOT4 is an E3 ligase of NP protein.

CNOTENT
中文摘要…………………………………………………………………… I
Abstract………………………………………………………………… II
Chapter1. Introduction……………………………………… 1
Chapter2. Result……………………………………………………… 4
2.1 Search for E3 ligase genes by secondary small-scale RNAi screening.……………………………………………………… 4
2.2 Knockdown of CNOT4 reduces IAV replication.…………… 6
2.3 Knockdown CNOT4 reduces NP ubiquitination and viral RdRp activity.………………………………………………………… 7
2.4 Expression of a wobble mutant of CNOT4 increases NP ubiquitination and enhances viral RdRp activity in knockdown cells.…………………………………………………………… 8
2.5 CNOT4 and USP11 can co-regulate NP ubiquitination.…… 9
Chapter3. Discussion………………………………………………… 11
Chapter4. Conclusion………………………………………………… 15
Chapter5. Materials and Methods…………………… 16
Chapter6. Figures………………………………………………………… 22
Chapter7. Tables…………………………………………………………… 38
References…………………………………………………………………………… 40

LIST of FIGURES
Figure 1. A proposed model for the regulation of influenza A virus RNA replication by ubiquitination/deubiquitination of NP (Liao et al. 2010)……………………23
Figure 2. Search for E3 ligase genes by secondary small-scale RNAi screen.……………………25
Figure 3. Knockdown of CNOT4 inhibits IAV replication.…………27
Figure 4. Knockdown of CNOT4 reduces NP ubiquitination and viral RdRp activity.……………………29
Figure 5. Construction of CNOT4-expressing plasmids and wobble mutants.……………………31
Figure 6. Overexpression of wobble mutant of CNOT4 increases NP ubiquitination and enhances viral RdRp activity in knockdown cells.……………………33
Figure 7. CNOT4 and USP11 can co-regulate NP ubiquitination.……………………34
Figure 8. The postulated mechanism of NP ubiquitination andregulation of RNA synthesis by CNOT4 and USP11.……………………35
Figure 9. The CNOT4 and USP11 RNA expression level during IAV infection.……………………37

LIST of TABLES
Page
Table 1. Candidate genes of secondary small-scale RNAi screen.…………………38
Table 2. Hit genes form secondary small-scale RNAi screen.…………………39
Table 3. Knockdown efficiency and IAV inhibition effect of shCNOT4.…………………39
Table 4. The information of CNOT4 isoforms (from NCBI reference sequence database).…………………39

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