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
Diserahkan:
7 Jun 2010 / Diterima: 14 Disember 2010
ABSTRACT
Nipah virus has been
identified as the causative agent responsible for an outbreak of fatal human
viral encephalitis in Malaysia and Singapore during 1998 – 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 that 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 resulted 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.
The suggestions have surfaced that the substitution of G at positions 79 and 85 may decrease 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 – 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 bahawa ia 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 yang kesan mutasi
transversi dari segi kesan penahanan ke atas aktiviti transkripsi minigenom
virus.
Kata kunci: Jujukan pemimpin; minigenom; mutagenesis; virus Nipah
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*Pengarang
untuk surat-menyurat; email: amirrabu@gmail.com
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