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Title:	Kajian ke atas jujukan dalaman terpulihara dalam promoter pemimpin virus nipah (NiV) serta peranannya dalam transkripsi dan replikasi NiV
Authors:	Pong Lian Yih (P40981)
Supervisor:	Amir Rabu, Dr.
Keywords:	Nipah virus (NiV) Chloramphenicol acetyltransferase (CAT ELISA) Transkripsi virus Dissertations, Academic -- Malaysia Universiti Kebangsaan Malaysia -- Dissertations
Issue Date:	1-Aug-2014
Description:	Asai replikasi secara in vitro dengan menggunakan sistem minigenom virus Nipah (NiV) yang mengandungi kedua-dua promoter pemimpin serta pengekor NiV telah digunakan untuk mengkaji replikasi NiV. Objektif kajian ini adalah untuk mengenalpasti dan mentakrif jujukan dalaman terpulihara dalam kawasan promoter pemimpin serta peranannya dalam replikasi NiV. Analisis secara mutasi telah dilakukan terhadap nukleotida pada kedudukan 73 hingga 108 yang berada pada kawasan 5' bahagian tak tertranslasi (NTR) mRNA gen nukleokapsid (N) dalam promoter pemimpin NiV. Mutasi delesi dalaman, mutasi titik dan mutasi penukargantian bes-bes telah dijana dengan menggunakan kaedah mutagenesis berasaskan PCR bertindih. Bagi menilai kesan mutasi terhadap transkripsi dan replikasi genom virus, plasmid yang membawa minigenom NiV terubah suai telah ditransfeksi ke dalam titisan sel BHK-21 dan ekstrak sel serta RNA yang dipencilkan masing-masing telah dianalisis melalui asai imunojerapan berpaut enzim chloramphenicol acetyltransferase (CAT ELISA) dan PCR masa nyata. Selain itu, transkrip RNA minigenom NiV yang mengandungi mutasi tertentu dan protein rekombinan N NiV telah dihasilkan dan dianalisis melalui asai pengikatan proteinRNA secara in vitro. Penyingkiran kawasan nukleotida 73 – 90 dan nukleotida 103 – 108 daripada promoter pemimpin NiV masing-masing didapati menindas sintesis RNA antigenom terenkapsidasi NiV, namun ia tidak menindas transkripsi virus. Didapati nukleotida di kedudukan 73 hingga 91 penting tetapi tidak kritikal bagi transkripsi virus, sebaliknya ia kritikal bagi replikasi genom NiV. Nukleotidanukleotida ini berinteraksi antara satu sama lain dan bekerjasama bagi mendorong aktiviti promoter replikasi NiV. Hasil mutasi pada nukleotida di kedudukan 92 hingga 102 didapati kurang penting kerana tiada penindasan terhadap transkripsi dan replikasi genom NiV. Mutasi juga dilakukan pada kesemua sembilan nukleotida di tiga kedudukan yang terakhir dalam heksamer 13, 14 dan 15 (kedudukan 76, 77, 78, 82, 83, 84, 88, 89 dan 90) dan didapati ia merencat transkripsi virus sepenuhnya di samping juga menindas replikasi genom NiV. Ini mencadangkan bahawa nukleotida-nukleotida tersebut adalah kritikal untuk aktiviti promoter dalam langkah pertama replikasi NiV khususnya dalam sintesis RNA bererti-positif. Didapati mutasi-mutasi yang boleh menindas penghasilan RNA antigenom terenkapsidasi dikaitkan dengan pengikatan protein N NiV yang lemah pada transkrip RNA antigenom terubah suai NiV. Kawasan 5' NTR mRNA gen N dalam promoter pemimpin NiV mengandungi tapak nukleasi dalam bentuk jujukan motif berulang yang sangat terpulihara. Kajian ini mencadangkan jujukan motif yang berulang sebanyak tiga kali – (3'-NNNAAC-5')3 dalam kawasan nukleotida 73 – 90 dan isyarat tindakan-cis ini diperlukan untuk promoter RNA NiV bagi mencapai sintesis RNA yang cekap.,In vitro replication assays with Nipah virus (NiV) minigenome carrying both leader and trailer promoters of NiV was used to study the replication of NiV. The objective of this study is to determine and define the conserved internal sequence in the leader promoter and its roles in replication of NiV. The mutational analysis was performed on nucleotides 73 to 108 within the 5' non-translated region (NTR) of the nucleocapsids (N) gene mRNA region which is located in the leader promoter of NiV. The internal deletion mutations, point mutations and bases substitution mutations were generated by using overlapping PCR-mediated mutagenesis method. To evaluate the effects of mutations upon viral transcription and genome replication, the plasmids carrying altered NiV minigenome were transfected into BHK-21 cell line and the isolated cell extracts and RNAs were analysed via chloramphenicol acetyltransferase enzyme-linked immunosorbent assay (CAT ELISA) and real-time PCR assay respectively. Besides, the NiV minigenome RNA transcripts containing certain mutation and recombinant N protein of NiV were produced and analysed through in vitro protein-RNA binding assay. Deletion of nucleotides 73 – 90 and 103 – 108 regions from the leader promoter of NiV respectively were found suppress the synthesis of viral encapsidated antigenome RNA, but it did not suppress the viral transcription. It was found that the nucleotides at positions 73 to 91 appeared to be important but not critical in viral transcription, instead they are critical in genome replication of NiV. These nucleotides interact with each other and enhance the promoter activity of viral replication cooperatively. Mutations on nucleotides at positions 92 to 102 were less important since no suppression in viral transcription and genome replication was observed. The mutation was also generated in all nine nucleotides at the last three positions of hexamers 13, 14 and 15 (positions 76, 77, 78, 82, 83, 84, 88, 89 and 90) and it was found to completely abrogate the viral transcription and also suppress the genome replication of NiV. It is suggested that these nucleotides were critical for promoter activity in the first step of viral replication, especially in the synthesis of positive-sense RNA. It was found that the mutations which could suppress the production of encapsidated antigenome RNA were associated with the weak binding of N protein to the altered antigenome RNA transcripts of NiV. The 5' NTR of the N gene mRNA region in leader promoter of NiV contains nucleation site in the form of highly conserved repeated motifs sequence. This study propose the three times repeated motif sequence – (3’-NNNAAC-5’)3 in nucleotides 73 – 90 region and this cis-acting signal is essential for NiV RNA promoter to achieve efficient RNA synthesis.
Pages:	343
Call Number:	QR404.2.N55 P644 2014 tesis
Publisher:	UKM, Bangi
Appears in Collections:	Faculty of Science and Technology / Fakulti Sains dan Teknologi

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