Tiếp theo, em mong đựơc thảo luận cùng các anh chị về vấn đề miễn dịch và cấu trúc gene của PRRSV với anh/chị quan tâm. Em sẽ up lên một đoạn nghiên cứu về một đoạn ORF7 (qui định Nucleocapsid protein) của PRRSV. Em đã khuếch đại đựơc các đoạn gene của PRRSV nên bây h cần hiểu và các motif, vùng chức năng,... của các đoạn ORF để phân tích. Nhờ các anh chị nào hiểu rộng các thuật ngữ chuyên ngành giúp em. Thuật ngữ tiếng ANh thì OK, dễ hiểu, nhưng để chuyển sang tiếng vịêt thì...nhờ mấy anh chị chuyên về miễn dịch và sinh học phân tử giúp em.
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The 15-kDa nonglycosylated N protein does not possess a signal peptide and is translated and maintained as mature 123 and 128 amino acid proteins, for VR-2332 and Lelystad viruses, respectively . Even though the North American and European PRRSV N proteins are only about 63% identical at the amino acid level, they share a common antigenic region between amino acids 52 and 69. During virus replication, the N protein localizes to the cytoplasm and nucleolus of cultured porcine macro phages and MARC-145 cells infected with either North American or European PRRSV genotypes . An identical nucleolar–cytoplasmic distribution pattern is bserved for the N protein of another arterivirus,EAV , and for the N proteins of group I,II, and III coronaviruses . The arterivirus and coronavirus N proteins, when expressed alone or fused to the red-shifted enhanced green fluorescent protein (EGFP), localize to the nucleolus, demonstrating that translocation across the nuclear pore complex (NPC) and accumulation in the nucleolus are independent of other viral proteins .
Within a population of infected cells, the intracellular distribution of arterivirus and coronavirus N proteins is highly variable. For example, a small percentage of coronavirus-infected cells possess N in the nucleolus. In a similar study using PRRSV, we observed that the percentage of infected cells containing N in the nucleolus was dependent on when observations were made. In addition, cells were identified that contained N in thenucleoplasmic region surrounding the nucleoli. These observations suggest that the localization of N to the nucleolus is a complex and highly regulated process.
The translocation of a protein from the cytoplasm, across the NPC, and into the nucleus is initiated by the binding of a nuclear localization signal (NLS) on the cargo protein to the importin-alpha shuttle protein. The cargo/importin-alpha complex binds to a second shuttle protein, importin-alpha, which is then targeted to the NPC. In the presence of additional accessory proteins, including Ran-GDP, and ATP as a source of energy, the cargo/importin complex is transported across the NPC and released into the nucleoplasm . Classical NLS sequences in- corporate regions enriched in basic amino acids and generally conform to one of three types . The “pat4” NLS consists of a continuous stretch of four basic amino acids (lysine or arginine) or three basic amino acids associated with histi dine or proline. The “pat7” NLS starts with a proline and is followed within three residues by a segment containing three basic residues out of four. The third type of NLS, known as a “bipartite” motif, consists of two basic amino acids, a 10-amino-acid spacer, and a 5-amino-acid segment containing at least three basic residues. There are exceptions to these established rules. Proteins that lack an NLS can localize to the nucleus as a result of cotransport with other nuclear proteins. Proteins which possess an NLS may remain cytoplasmic, especially if the NLS is not exposed on the protein’s surface. And finally, small proteins less than 50–70 kDa can passively diffuse through the nuclear pore complex . The addition of an NLS to a small protein that passively diffuses through the NPC confers to that protein all of the properties of NLS-dependent transport .
Unlike the nucleus, the nucleolus is not a membrane bound organelle. Therefore, the targeting of a protein to the nucleolus occurs through the diffusion of the protein through the nucleoplasm and accumulation in the nucleolus. Localization to the nucleolus is mediated by a nucleolar targeting sequence (NoTS), which functions by forming an interaction with a nucleolar component, such as a nucleolar protein, rRNA, or rDNA, or small nucleolar RNAs. By definition, the NoTS by itself cannot translocate a protein across the NPC, but once in the nucleoplasm is capable of targeting the protein to the nucleolus. Another route to the nucleolus is the direct translocation of a protein from the cytoplasm to the nucleolus by cotransport with a nucleolar shuttle protein, such as nucleolin, fribrillarin, and B-23, which normally shuttle between the cytoplasm and the nucleolus. The purpose of this study was to gain insight into the mechanism of PRRSV N protein localization to the nucleolus by characterizing the individual oligopeptides domains, which participated in the translocation of the N protein across the NPC and localization to thenucleolus.
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Võ Khánh Hưng
Võ Khánh Hưng
