RNA binding specificity of Hepatitis C virus core protein
| Project/Area Number |
12670527
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Gastroenterology
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| Research Institution | Kanazawa Medical University |
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Principal Investigator |
DATE Takayasu Kanazawa Medical University, Department of Medicine, Professor, 医学部, 教授 (50019676)
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| Co-Investigator(Kenkyū-buntansha) |
MATSUI Tadashi Kanazawa Medical University, Department of Medicine, Assistant Researcher, 医学部, 助手 (60288272)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2001: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2000: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Keywords | Hepatitis C virus )HCV) / Core protein / RNA binding / ERA / EMSA / 5'-noncoding region)3'-NCR) / PKR / 3'-noncoding region)3'-NCR) / 3'-非翻訳領域(5'-NCR) / ゲルシフトアッセイ(EMSA) / 酵母three-hybrid system / HCVゲノム / RNA / RNAライブラリー |
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| Research Abstract |
1. We developed an activity gel method named electrophoretic assay (ERA), by which immobilized RNA-bindig protein in the gel was able to trap RNA in electrophoresis. ERA by a number of Glutathione S-transferase (GST)-truncated HCV core fusion proteins revealed that RNA binding regions were mapped at amino acids 4 -18 and 46-80, which were named RBRI, and RBR II, respectively. RBR I shared homologies with other RNA/DNA binding proteins, such as ribosomal protein S6. We proposed a consensus sequence PKPXRK(S/T) (R/K)R as a RNA/DNA binding motif.
2. On the basis of this result, HCV core protein between 1 and 90 amino acid residues was used for a subsequent electrophoretic gel mobility shift assay (EMSA). The core protein showed a high affinity to double-stranded (ds) RNA irrespective of its sequence and low affinity to most single-stranded (ss) RNA.
3. Activation of interferon inducible dsRNA activated protein kinase (PKR) was strongly inhibited by core protein by sequestered dsRNA, in vitro.
4. The affinities of the core protein to HCV RNA region was determined by competitive EMSA with ^<32>P-labeled dsRNA. In 5'-NCR, sense (+) RNA strands of loops 3a (193-209 nt) and 3d (288-321 nt) showed strong competition with dsRNA in contrast other fragments. In 3'-NCR, core protein showed higher affinity to both sense and antisense strands of loops 1 and 3 in 3'X tail than the rest regions including loop 2. When loops 1 or 3 was joined to loop 2, sense strand loop 1-2 or loop 2-3 showed strong competition with dsRNA in contrast to their antisense RNAs, indicating that the loop 2 in 3'X tail plays an important role in enhancement of sense strand-binding.
5. We propose that the complex of core, 5'-NCR and 3'-NCR plays an important role in initiation of encapsidation.
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Report
(3 results)
Research Products
(8 results)
