| Summary: | 碩士 === 國立陽明大學 === 生命科學系暨基因體科學研究所 === 102 === 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.
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