Innate Intracellular Antiviral Responses Restrict the Amplification of Defective Virus Genomes of Parainfluenza Virus 5.

Susana López,Elizabeth B Wignall-Fleming,Andri Vasou,John A L Short,David J Hughes,Richard E Randall,Dan Young,Steve Goodbourn

doi:10.1128/jvi.00246-20

Susana López, Elizabeth B Wignall-Fleming + Show 6 more

Open Access

https://doi.org/10.1128/jvi.00246-20

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Abstract

During the replication of parainfluenza virus 5 (PIV5), copyback defective virus genomes (DVGs) are erroneously produced and are packaged into "infectious" virus particles. Copyback DVGs are the primary inducers of innate intracellular responses, including the interferon (IFN) response. While DVGs can interfere with the replication of nondefective (ND) virus genomes and activate the IFN-induction cascade before ND PIV5 can block the production of IFN, we demonstrate that the converse is also true, i.e., high levels of ND virus can block the ability of DVGs to activate the IFN-induction cascade. By following the replication and amplification of DVGs in A549 cells that are deficient in a variety of innate intracellular antiviral responses, we show that DVGs induce an uncharacterized IFN-independent innate response(s) that limits their replication. High-throughput sequencing was used to characterize the molecular structure of copyback DVGs. While there appears to be no sequence-specific break or rejoining points for the generation of copyback DVGs, our findings suggest there are region, size, and/or structural preferences selected for during for their amplification.IMPORTANCE Copyback defective virus genomes (DVGs) are powerful inducers of innate immune responses both in vitro and in vivo They impact the outcome of natural infections, may help drive virus-host coevolution, and promote virus persistence. Due to their potent interfering and immunostimulatory properties, DVGs may also be used therapeutically as antivirals and vaccine adjuvants. However, little is known of the host cell restrictions which limit their amplification. We show here that the generation of copyback DVGs readily occurs during parainfluenza virus 5 (PIV5) replication, but that their subsequent amplification is restricted by the induction of innate intracellular responses. Molecular characterization of PIV5 copyback DVGs suggests that while there are no genome sequence-specific breaks or rejoin points for the generation of copyback DVGs, genome region, size, and structural preferences are selected for during their evolution and amplification.

Highlights

During the replication of parainfluenza virus 5 (PIV5), copyback defective virus genomes (DVGs) are erroneously produced and are packaged into “infectious” virus particles
We have suggested that during replication of nondefective (ND) paramyxoviruses, DVGs are produced which subsequently activate the IFN induction cascade in a minority of cells as the virus spreads during plaque development [34]
A549/pr(IFN-␤)GFP reporter cells were infected with PIV5-W3 at a multiplicity of infection (MOI) of 0.001 and at 2 days postinfection (p.i.) the cells were trypsinized, fixed, stained for NP and the number of GFP-positive (GFP ϩve) cells was compared to the number of cells positive for NP by fluorescence-activated cell sorting (FACS) analysis (Fig. 1b)

Summary

Introduction

During the replication of parainfluenza virus 5 (PIV5), copyback defective virus genomes (DVGs) are erroneously produced and are packaged into “infectious” virus particles. IMPORTANCE Copyback defective virus genomes (DVGs) are powerful inducers of innate immune responses both in vitro and in vivo. They impact the outcome of natural infections, may help drive virus᎑host coevolution, and promote virus persistence. Due to their potent interfering and immunostimulatory properties, DVGs may be used therapeutically as antivirals and vaccine adjuvants. We show here that the generation of copyback DVGs readily occurs during parainfluenza virus 5 (PIV5) replication, but that their subsequent amplification is restricted by the induction of innate intracellular responses. Efficient viral replication requires that virus genomes follow the “rule of six,” meaning that the virus genome must be a multiple of six, presumably because 6 nt are associated with one encapsidating NP during the formation of the RNPs [15]

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