Respiratory syncytial virus M2-1 protein associates non-specifically with viral messenger RNA and with specific cellular messenger RNA transcripts.

Molly R Braun,W Evan Johnson,Sarah L Noton,Emmeline L Blanchard,Philip J Santangelo,Afzaal Shareef,Rachel Fearns

doi:10.1371/journal.ppat.1009589

Abstract

Respiratory syncytial virus (RSV) is a major cause of respiratory disease in infants and the elderly. RSV is a non-segmented negative strand RNA virus. The viral M2-1 protein plays a key role in viral transcription, serving as an elongation factor to enable synthesis of full-length mRNAs. M2-1 contains an unusual CCCH zinc-finger motif that is conserved in the related human metapneumovirus M2-1 protein and filovirus VP30 proteins. Previous biochemical studies have suggested that RSV M2-1 might bind to specific virus RNA sequences, such as the transcription gene end signals or poly A tails, but there was no clear consensus on what RSV sequences it binds. To determine if M2-1 binds to specific RSV RNA sequences during infection, we mapped points of M2-1:RNA interactions in RSV-infected cells at 8 and 18 hours post infection using crosslinking immunoprecipitation with RNA sequencing (CLIP-Seq). This analysis revealed that M2-1 interacts specifically with positive sense RSV RNA, but not negative sense genome RNA. It also showed that M2-1 makes contacts along the length of each viral mRNA, indicating that M2-1 functions as a component of the transcriptase complex, transiently associating with nascent mRNA being extruded from the polymerase. In addition, we found that M2-1 binds specific cellular mRNAs. In contrast to the situation with RSV mRNA, M2-1 binds discrete sites within cellular mRNAs, with a preference for A/U rich sequences. These results suggest that in addition to its previously described role in transcription elongation, M2-1 might have an additional role involving cellular RNA interactions.

Highlights

Human respiratory syncytial virus (RSV), recently renamed as human orthopneumovirus [1,2] is the leading cause of pediatric acute lower respiratory tract infection
Our results show that M2-1 associates with viral mRNA across the entire length of each gene, consistent with M2-1 contacting each nucleotide of nascent mRNA being extruded from the polymerase
For the CLIP-seq analysis we employed single-end enhanced CLIP-seq [48], a technique that can resolve protein:RNA binding at individual nucleotide resolution

Summary

Introduction

Human respiratory syncytial virus (RSV), recently renamed as human orthopneumovirus [1,2] is the leading cause of pediatric acute lower respiratory tract infection. RSV transcription and genome replication occur in the cytoplasm of the cell [9]. These processes are performed by the viral RNA dependent RNA polymerase, which is a complex of at least two viral proteins, the large polymerase subunit, L, and the phosphoprotein, P [10,11]. The polymerase scans to the gene start sequence to transcribe the gene [21] In this way, the polymerase sequentially transcribes monocistronic viral mRNAs. During replication, the polymerase initiates at the 3 ́ end of the genome and disregards the gene start and gene end signals to synthesize the antigenome [22]. The antigenome and genome RNAs are encapsidated as they are synthesized [26] and this is thought to increase polymerase processivity, enabling it to synthesize the full-length antigenome and genome RNAs

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