A specific sequence in the genome of respiratory syncytial virus regulates the generation of copy-back defective viral genomes.

Yan Sun,Carolina B López,Gregory R Grant,Divyansh Agarwal,Louis J Taylor,Sébastien A Felt,Eun Ji Kim,Marco Vignuzzi

doi:10.1371/journal.ppat.1007707

Abstract

Defective viral genomes of the copy-back type (cbDVGs) are the primary initiators of the antiviral immune response during infection with respiratory syncytial virus (RSV) both in vitro and in vivo. However, the mechanism governing cbDVG generation remains unknown, thereby limiting our ability to manipulate cbDVG content in order to modulate the host response to infection. Here we report a specific genomic signal that mediates the generation of a subset of RSV cbDVG species. Using a customized bioinformatics tool, we identified regions in the RSV genome frequently used to generate cbDVGs during infection. We then created a minigenome system to validate the function of one of these sequences and to determine if specific nucleotides were essential for cbDVG generation at that position. Further, we created a recombinant virus unable to produce a subset of cbDVGs due to mutations introduced in this sequence. The identified sequence was also found as a site for cbDVG generation during natural RSV infections, and common cbDVGs originated at this sequence were found among samples from various infected patients. These data demonstrate that sequences encoded in the viral genome determine the location of cbDVG formation and, therefore, the generation of cbDVGs is not a stochastic process. These findings open the possibility of genetically manipulating cbDVG formation to modulate infection outcome.

Highlights

Defective viral genomes (DVGs), which are generated during the replication of most RNA viruses, potentiate the host innate immune response [1,2,3,4,5] and attenuate the infection in vitro and in vivo [4, 6,7,8,9]
Copy-back defective viral genomes regulate infection and pathogenesis of Mononegavirales. cbDVGs are believed to arise from random errors that occur during virus replication and the predominant hypothesis is that the viral polymerase is the main driver of cbDVG generation
To acquire a comprehensive view of the population of cbDVGs generated during infection, we developed an algorithm to identify cbDVG junction regions within RNA-seq datasets with high sensitivity

Summary

Introduction

Defective viral genomes (DVGs), which are generated during the replication of most RNA viruses, potentiate the host innate immune response [1,2,3,4,5] and attenuate the infection in vitro and in vivo [4, 6,7,8,9]. Deletion DVGs, common in influenza virus and positive strand RNA viruses, retain the 3’ and 5’ ends of the viral genomes but carry an internal deletion [14,15,16] These types of DVGs are believed to arise from recombination events [17, 18] and can strongly interfere with the standard virus [19]. CbDVGs arise when the viral polymerase detaches from its template at a “break point” and resumes elongation at a downstream “rejoin point” by copying the 5’ end of the nascent daughter strand [12, 22] This process results in the formation of a new junction sequence and a truncated genome flanked by reverse complementary ends [23]. No pattern or specific sequences for the break and rejoin points of cbDVGs have been reported so far

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Discussion

Conclusion