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. 2013 May;87(9):4798-807.
doi: 10.1128/JVI.03383-12. Epub 2013 Feb 28.

Deep Sequencing Analysis of Defective Genomes of Parainfluenza Virus 5 and Their Role in Interferon Induction

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Free PMC article

Deep Sequencing Analysis of Defective Genomes of Parainfluenza Virus 5 and Their Role in Interferon Induction

M J Killip et al. J Virol. .
Free PMC article

Abstract

Preparations of parainfluenza virus 5 (PIV5) that are potent activators of the interferon (IFN) induction cascade were generated by high-multiplicity passage in order to accumulate defective interfering virus genomes (DIs). Nucleocapsid RNA from these virus preparations was extracted and subjected to deep sequencing. Sequencing data were analyzed using methods designed to detect internal deletion and "copyback" DIs in order to identify and characterize the different DIs present and to approximately quantify the ratio of defective to nondefective genomes. Trailer copybacks dominated the DI populations in IFN-inducing preparations of both the PIV5 wild type (wt) and PIV5-VΔC (a recombinant virus that does not encode a functional V protein). Although the PIV5 V protein is an efficient inhibitor of the IFN induction cascade, we show that nondefective PIV5 wt is unable to prevent activation of the IFN response by coinfecting copyback DIs due to the interfering effects of copyback DIs on nondefective virus protein expression. As a result, copyback DIs are able to very rapidly activate the IFN induction cascade prior to the expression of detectable levels of V protein by coinfecting nondefective virus.

Figures

Fig 1
Fig 1
Activation of the IFN response by PIV5 preparations generated by high-multiplicity passage. A549/pr(IFN-β).GFP reporter cells were infected with equivalent dilutions of PIV5-VΔC vM0 or vM2 (A) or (B) PIV5 wt vM0, vM3, vM6, vM9, or vM12 (B). Sixteen hours later, the culture media were harvested and the cells were fixed and immunostained for viral NP expression. Expression of GFP and virus proteins was analyzed by fluorescence microscopy (left panel). Multiplicity (PFU/cell) for each virus is indicated by the number shown in the NP panel. The amount of IFN present in the culture media was estimated by cell-based CPE reduction assay and is shown by the number in the GFP panel. Duplicate monolayers were trypsinized, fixed, and subjected to FACS analysis of GFP expression (right panel). n.d., not determined. (C) A549 cells were left uninfected (UI) or were infected with an equivalent multiplicity of PIV5 wt vM0, vM6, or vM12. Cell lysates were prepared 24 h later and subjected to SDS-PAGE and immunoblotting for phospho-IRF3 (p-IRF3), GFP, ISG56, MxA, viral NP, and actin.
Fig 2
Fig 2
Deep sequencing analyses of RNA isolated from PIV5-infected cells. Viral RNPs were extracted from Vero cells infected with PIV5-VΔC (A) or PIV5 wt (B). Associated RNA was subjected to deep sequencing using the Illumina GA2x platform, and sequencing reads were mapped to PIV5-VΔC (A) or PIV5 wt (B) reference genomes. The frequency of reads at each nucleotide is shown in red for vM0 virus preparations and in black for the DI-rich virus preparations PIV5-VΔC vM2 (A) and PIV5 wt vM12 (B). Coverage from nt 14000 to 15246 is shown as an inset in order to highlight the peaks at the 5′ end of the genome.
Fig 3
Fig 3
PIV5 wt does not prevent activation of the IFN-β promoter by coinfecting copyback DIs, despite encoding an efficient IFN antagonist. (A) A549/pr(IFN-β).GFP cells were infected at an MOI of 10 PFU/cell with PIV5 wt vM0, PIV5 wt vM12, or a 50:50 mixture thereof. The cells were fixed at 20 h p.i., and GFP-positive cells and the distribution of NP (red), following immunostaining, were visualized by fluorescence microscopy. (B) A549/pr(IFN-β).GFP cells were treated as described above. At 18 h p.i., the cells were trypsinized, fixed, and immunostained for viral NP. Expression of GFP and NP was analyzed by FACS. Uninfected cells were included as a negative control. (C) Uninfected (UI) A549/pr(IFN-β).GFP cells or cells that had been preinfected with PIV5 wt vM0 (at 5 PFU/cell) for 24 h were infected with PIV5-VΔC vM2. The cells were fixed at 16 h p.i., and GFP was visualized by fluorescence microscopy.
Fig 4
Fig 4
Copyback DIs impair the ability of PIV5 to limit IFN signaling. (A) Monolayers of A549/pr(IFN-β).GFP cells were infected with 1 PFU/cell of PIV5 wt vM0 or vM9. Cells were fixed at 18 h p.i. and immunostained for MxA and virus NP expression. GFP (green), MxA (blue), and NP (red) were visualized by confocal microscopy. (B) A549/pr(IFN-β).GFP cells were infected with 20 PFU/cell of either PIV5 wt vM0, PIV5-VΔC vM2, or a mixture of PIV5 wt vM0 and PIV5-VΔC vM2. At 8 h p.i., IFN-α (1,000 IU/ml) was added to the culture medium and the cells were fixed at 24 h p.i. Following immunostaining, GFP (green), MxA (blue), and NP (red) were visualized by confocal microscopy.
Fig 5
Fig 5
Coinfecting copyback DIs limit expression of the V protein by nondefective PIV5. Replicates of A549/pr(IFN-β).GFP cells were infected with PIV5 wt vM0 and PIV5-VΔC vM2 (5 PFU/cell of each virus; top panel) or PIV5 wt vM0 alone (5 PFU/cell; bottom panel). Cell lysates were prepared at various times p.i. (as indicated) and subjected to immunoblotting for phospho-IRF3 (p-IRF3), GFP, PIV5 V (C terminus [C-term.]), and actin.
Fig 6
Fig 6
Inhibition of nondefective virus replication by copyback DIs is independent of the antiviral response. (A) A549, A549/V, and A549/NPro cells were treated with IFN-α (1,000 IU/ml), infected with PIV5-VΔC vM2, or left untreated. Cell lysates were prepared after 16 h and subjected to immunoblotting for IRF3, ISG56, STAT1, PIV5 P, and actin. (B) A549 (-), A549/NPro (N), and A549/V (V) monolayers were infected at an MOI of 5 PFU/cell with PIV5 wt vM0, PIV5-VΔC vM0, or PIV5-VΔC vM2 or were uninfected. At 20 h p.i., the monolayers were radioactively labeled with [35S]methionine for 1 h. Labeled polypeptides in total cell extracts were immunoprecipitated using monoclonal antibodies to viral NP, P, M, HN, and L proteins and visualized by SDS-PAGE analysis and autoradiography. (C) Autoradiogram of total cell extracts used in panel B. (D) Coomassie staining of total cell extracts used in panel B. The positions of the viral proteins are indicated.

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