The critical region for viral RNA encapsidation in leader promoter of Nipah virus
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ORIGINAL ARTICLE
The critical region for viral RNA encapsidation in leader promoter of Nipah virus Lian Yih Pong1,2 · Amir Rabu1 · Nazlina Ibrahim1 Received: 23 December 2019 / Accepted: 25 July 2020 © Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract Encapsidation by nucleocapsid (N) protein is crucial for viral RNA to serve as a functional template for virus replication. However, the potential region that is vital for RNA encapsidation of Nipah virus (NiV) is still unknown. Thus, this study was aimed to identify these regions using a NiV minireplicon system. A series of broad range internal deletion mutations was generated in the 5′ non-translated region (NTR) of the N gene mRNA region of NiV leader promoter via site-directed overlapping PCR-mediated mutagenesis. The mutation effects on synthesis and encapsidation of antigenome RNA, transcription, and RNA binding affinity of N protein were evaluated. The deletions of nucleotides 73–108, 79–108, and 85–108 from NiV leader promoter inhibited the encapsidation of antigenome RNA, while the deletion of nucleotides 103–108 suppressed the synthesis and encapsidation of antigenome RNA, implying that these regions are required for genome replication. Surprisingly, none of the mutations had detrimental effect on viral transcription. Using isothermal titration calorimetry, the binding of NiV N protein to genome or antigenome RNA transcript lacking of nucleotides 73–108 was found to be suppressed. Additionally, in silico analysis on secondary structure of genome RNA further supported the plausible cause of inefficient encapsidation of antigenome RNA by the loss of encapsidation signal in genome template. In conclusion, this study suggests that the nucleotides 73–90 within 5′ NTR of the N gene mRNA region in NiV leader promoter contain cis-acting RNA element that is important for efficient encapsidation of antigenome RNA. Keywords Paramyxovirus · Reverse genetics · Minigenome system · Site-directed mutagenesis · Non-translated region · Cis-acting element
Introduction Communicated by Stefan Hohmann. Amir Rabu is Deceased. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00438-020-01716-3) contains supplementary material, which is available to authorized users. * Nazlina Ibrahim [email protected] Lian Yih Pong [email protected] 1
Department of Biological Sciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia
Present Address: Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Jalan Lagoon Selatan, 47500 Bandar Sunway, Selangor Darul Ehsan, Malaysia
2
The essential sequences for directing viral genome replication and transcription activity have been reported in the nucleotides resided in terminal regions of genome and antigenome RNA of the paramyxoviruses including Nipah virus (NiV) (Halpin et al. 2004; Lamb and Parks 2007). In virus replication, only the viral RNA that is e
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