Sains Malaysiana 40(10)(2011): 1139–1146

Effects of Point Mutations at Positions 79, 85 and 91 of the Nipah

Virus Leader Sequence to Its Minigenome Expression

(Kesan Mutasi Titik pada Posisi 79, 85 dan 91 dalam Jujukan Pemimpin

ke Atas Pengekspresan Minigenom Virus Nipah)

Lian-Yih Pong

Makmal Biologi Molekul, Faculty of Science and Technology, Universiti Kebangsaan Malaysia

43600 Bangi, Selangor D.E. Malaysia

Zulkeflie Zamrod

Inno Biologics’ Biopharmaceutical Complex, Lot 1, Persiaran Negeri BBN, Putra Nilai,

71800 Nilai, Negeri Sembilan Darul Khusus, Malaysia

Amir Rabu*

School of Biosciences and Biotechnology, Faculty of Science and Technology

Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor D.E. Malaysia

Received: 7 June 2010/Accepted: 14 December 2010

ABSTRACT

Nipah virus has been identified as the causative agent responsible for an outbreak of fatal human viral encephalitis in Malaysia and Singapore in 1998 and 1999. In vitro replication assays with Nipah virus minigenome carrying CAT gene (chloramphenicol acetyltransferase) has been developed without the use of infectious virus to allow further study of the replication of Nipah virus in vitro. It has been reported that the viral RNA replication and transcription activity of paramyxoviruses are controlled by essential sequences located in the terminal regions of viral genomic and antigenomic RNAs. In this study, single base substitution was carried out on the Nipah virus minigenome separately at the three guanine residues (G) located in positions 79, 85 and 91 of the leader promoter within the 5’ non-translated region (NTR) of the nucleocapsid gene (N) mRNA region. The guanine residues of these positions were substituted with the cytosine (C) or adenine (A) residue, respectively by using the overlapping PCR-mediated mutagenesis method. The resultant mutants containing the desired point mutation were confirmed by sequencing. The mutants were analyzed to determine the effect of substitution mutation on the viral transcription activity of the minigenome. It was found that the substitution of G at positions 79 and 85 decreased the efficiency of transcription of Nipah virus minigenome while the substitution of G at position 91 did not. Our findings also showed that the effect of transition mutation gave more impact than the transversion mutation in term of suppression effect upon the transcription activity of minigenome.

Keywords: Leader sequence; minigenome; mutagenesis; Nipah virus

ABSTRAK

Virus Nipah telah dikenalpasti sebagai agen yang menyebabkan letusan jangkitan ensefalitis yang boleh membawa maut di kalangan manusia di Malaysia dan Singapura dalam tahun 1998 dan 1999. Asai replikasi secara in vitro yang terdiri daripada minigenom virus Nipah yang mengandungi penanda gen CAT (chloramphenicol acetyltransferase) telah dibangunkan tanpa melibatkan virus berjangkit, dengan tujuan untuk mengkaji replikasi virus Nipah secara in vitro. Aktiviti replikasi RNA dan transkripsi paramyxovirus telah dilaporkan dikawal oleh jujukan penting yang terkandung dalam kawasan hujung RNA genom dan antigenom virus. Dalam kajian ini, mutagenesis secara penggantian bes tunggal telah dilakukan ke atas minigenom virus Nipah secara berasingan dalam kawasan promoter pemimpin, yakni tiga residu guanina (G) yang terletak di posisi 79, 85 dan 91 dalam kawasan 5’ NTR mRNA gen nukleokapsid (N). Residu G tersebut telah digantikan dengan residu sitosina (C) dan adenina (A) masing-masing dengan menggunakan kaedah mutagenesis berasaskan tindak balas berantai polimerase (PCR) bertindih. Mutan-mutan terhasil yang mengandungi mutasi titik yang dikehendaki telah dikenalpastikan melalui penjujukan DNA. Mutan-mutan tersebut telah dianalisis untuk menentu kesan mutasi penukargantian tersebut ke atas aktiviti transkripsi minigenom virus Nipah. Didapati bahawa penukargantian residu G pada posisi 79 dan 85 menurunkan kecekapan aktiviti transkripsi minigenom virus Nipah manakala penukargantian residu G pada posisi 91 tidak memberikan kesan tersebut. Penemuan ini juga menunjukkan kesan mutasi transisi memberikan kesan yang lebih ketara daripada kesan mutasi transversi daripada segi kesan penahanan ke atas aktiviti transkripsi minigenom virus.

Kata kunci: Jujukan pemimpin; minigenom; mutagenesis; virus Nipah

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*Corresponding author; email; amirrabu@gmail.com