探討蛋白質PTBP1對C型肝炎病毒核糖體內結合位依賴性轉譯之調控

Abstract

C型肝炎起因於C型肝炎病毒(HCV)，且為造成肝癌的主因。約有30%的C型肝炎病毒感染者在沒有治療的情況下會自發性的痊癒，剩下70%的患者則會逐漸演變為慢性C型肝炎。根據估計，世界上約有7100萬人有慢性C型肝炎，然而現今並無有效的疫苗足以對抗C型肝炎病毒。C型肝炎病毒的基因體為一條單股、正股的RNA，並能夠直接被轉譯成蛋白質。過去研究顯示，宿主蛋白質例如聚嘧啶束結合蛋白(PTBP1)可能調控C型肝炎病毒的蛋白轉譯及RNA複製。選擇性剪接(alternative splicing)造成的三種PTBP1同功型蛋白(isoform)在人類細胞中被發現，且所有的PTBP1同功型蛋白都具有4個RNA 辨認模體(RNA recognition motifs, RRMs)，其可以結合到特定具有多個尿嘧啶/胞嘧啶的RNA序列。最短的PTBP1同功型蛋白c，具有531個胺基酸。而同功型蛋白b及c則在RRM2及RRM3之間分別多了19及26個胺基酸。此外，PTBP1曾被報導能夠在細胞核、細胞質之間穿梭，細胞質中的PTBP1能夠調控細胞RNA及病毒RNA的內核醣體結合位(internal ribosome binding site, IRES)依賴性轉譯。當C型肝炎病毒感染時，細胞中的PTBP1會結合當C型肝炎病毒內核醣體結合位，進而影響了內核醣體結合位依賴性蛋白轉譯。在此，我們聚焦於PTBP1是如何調控C型肝炎病毒的內核醣體結合位依賴性蛋白轉譯，以及哪個/哪些PTBP1的RRM對於C型肝炎病毒的內核醣體結合位依賴性蛋白轉譯是中必要的。為了監控PTBP1對於C型肝炎病毒轉譯的調控，我們建構了具有兩個順反子的報導質體，並利用髮夾環圈相隔。本篇研究中，PTBP1 同功型蛋白a及c都能夠降低C型肝炎病毒的內核醣體結合位依賴性蛋白轉譯；且只保有N端或是C端的PTBP1不能有抑制的作用。過去的研究指出，PTBP1會受到脊髓灰白質炎病毒(小兒麻痺病毒；poliovirus)及A型肝炎病毒(hepatitis A virus, HAV)的蛋白酶切割，進而抑制病毒的內核醣體結合位依賴性蛋白轉譯。從過去文獻得知，較小片段的PTBP1會在含有HCV複製原(replicon)細胞中被引發。在本篇研究中，我們得知PTBP1會與HCV 蛋白酶NS3/4A有交互作用，且PTBP1蛋白量可能受到NS3/4A的負調控。總結而言，PTBP1 同功型蛋白a及c都會抑制C型肝炎病毒的內核醣體結合位依賴性蛋白轉譯，且僅有全長PTBP1而非N端PTBP1或C端PTBP1能夠抑制C型肝炎病毒的內核醣體結合位依賴性蛋白轉譯。我們的研究提供了細胞中的蛋白質PTBP1可能與C型肝炎病毒蛋白質NS3/4A 共同調控C型肝炎病毒的內核醣體結合位依賴性蛋白轉譯的可能性。

Hepatitis C is a major cause of liver cancer and is caused by hepatitis C virus (HCV). About 30% of HCV infection can be cleared without any treatments. The remaining 70% of cases will develop chronic hepatitis C. It is estimated that 71 million people have chronic hepatitis C worldwide. Currently there is no effective vaccine for HCV. The HCV genome is a positive-single-stranded RNA, which can be directly translated into viral proteins. Previous studies have shown that host proteins such as polypyrimidine tract-binding protein 1 (PTBP1) may regulate HCV RNA translation and replication. Three different alternative spliced isoforms of PTBP1 were found in human cells, and all isoforms of PTBP1 contains 4 RNA recognition motifs (RRMs), which bind to a specific RNA sequence with a poly U/UC tract. The shortest PTBP1, isoform c, contains 531 amino acids. While the isoform a and isoform b have additional 26 and 19 amino acid residues between RRM2 and RRM3. PTBP1 has been reported to shuttle between nucleus and cytoplasm. Cytosolic PTBP1 can regulate internal ribosome binding site (IRES)-dependent translation initiation of both cellular and viral RNA. PTBP1 binds to HCV IRES and influences HCV IRES-dependent translation. Here, we focus on how PTBP1 regulates HCV IRES-dependent translation and which RRM(s) of PTBP1 is/are important for HCV IRES-dependent translation. To monitor the PTBP1 effect on HCV translation activity, we constructed a bicistronic reporter plasmid separated by a hairpin loop. In our study, both expression of isoform a and c of PTBP1 decreased HCV IRES translation activity, but both halves of PTBP1 which were truncated at Theronine residue 261 could not inhibit HCV IRES-dependent translation. It has been shown that PTBP1 can be cleaved by proteases of the poliovirus and hepatitis A Virus (HAV), thereby inhibiting the viral IRES-dependent translation. Previous studies have also shown that small fragment of PTBP1 was induced in HCV replicon cells. It is intriguing whether HCV IRES-mediated translation and/or replication of HCV RNA would be influenced by the fragment of PTBP1. In this study, we found that the PTBP1 interacted with the HCV NS3/4A and the protein level of PTBP1 was downregulated by increasing doses of NS3/4A. In conclusion, both PTBP1 isoform a and c could inhibit HCV IRES-dependent translation, and only the full-length PTBP1 but not the N-PTBP1 nor the C-PTBP1 could inhibit the HCV IRES-dependent translation. Our study provides new insights into how the host protein PTBP1 and the viral protein NS3/4A may together regulate HCV IRES-dependent translation.