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Defective Viral Genomes Alter How Sendai Virus Interacts With Cellular Trafficking Machinery, Leading to Heterogeneity in the Production of Viral Particles Among Infected Cells

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Defective Viral Genomes Alter How Sendai Virus Interacts With Cellular Trafficking Machinery, Leading to Heterogeneity in the Production of Viral Particles Among Infected Cells

Emmanuelle Genoyer et al. J Virol.

Abstract

Defective viral genomes (DVGs) generated during RNA virus replication determine infection outcome by triggering innate immunity, diminishing virulence, and, in many cases, facilitating the establishment of persistent infections. Despite their critical role during virus-host interactions, the mechanisms regulating the production and propagation of DVGs are poorly understood. Visualization of viral genomes using RNA fluorescent in situ hybridization revealed a striking difference in the intracellular localization of DVGs and full-length viral genomes during infections with the paramyxovirus Sendai virus. In cells enriched in full-length virus, viral genomes clustered in a perinuclear region and associated with cellular trafficking machinery, including microtubules and the GTPase Rab11a. However, in cells enriched in DVGs, defective genomes distributed diffusely throughout the cytoplasm and failed to interact with this cellular machinery. Consequently, cells enriched in full-length genomes produced both DVG- and full-length-genome-containing viral particles, while DVG-high cells poorly produced viral particles yet strongly stimulated antiviral immunity. These findings reveal the selective production of both standard and DVG-containing particles by a subpopulation of infected cells that can be differentiated by the intracellular localization of DVGs. This study highlights the importance of considering this functional heterogeneity in analyses of virus-host interactions during infection.IMPORTANCE Defective viral genomes (DVGs) generated during Sendai virus infections accumulate in the cytoplasm of some infected cells and stimulate antiviral immunity and cell survival. DVGs are packaged and released as defective particles and have a significant impact on infection outcome. We show that the subpopulation of DVG-high cells poorly engages the virus packaging and budding machinery and do not effectively produce viral particles. In contrast, cells enriched in full-length genomes are the primary producers of both standard and defective viral particles during infection. This study demonstrates heterogeneity in the molecular interactions occurring within infected cells and highlights distinct functional roles for cells as either initiators of immunity or producers and perpetuators of viral particles depending on their content of viral genomes and their intracellular localization.

Keywords: defective interfering particles; defective viral genomes; infection heterogeneity; paramyxovirus; particle production.

Figures

FIG 1
FIG 1
Defective viral genomes alter viral nucleoprotein distribution within infected cells. (A) A549 cells infected with SeV-LD at an MOI of 1.5 TCID50/cell supplemented with purified DPs (pDP) at the indicated HAU for 24 h and stained for SeV NP (gray). Wide-field images were captured with a 40× objective. Images are representative of results from 3 independent experiments. Bar = 100 μm. (B) RT-qPCR of SeV (+)DVG-546 relative to GAPDH. (C) Distribution of NP by pixel area within individual cells at 24 h postinfection. (D and E) Quantification of SeV NP amounts by fluorescence intensity (D) and percentage of cells per field that are NP positive (NP+) as determined by the level of fluorescence above the background (E). Results show the sum of data from 3 independent experiments with >250 individual cells analyzed under each condition. Individual cells are plotted with a line at the mean, and error bars represent standard errors of the means (SEM). ****, P < 0.0001 by a Kruskal-Wallis test with Dunn’s multiple-comparison test. ns, not significant. (F and G) A549 cells infected with SeV-LD (F) and SeV-HD (G) for the indicated times and stained for SeV NP (gray). Wide-field images were captured with a 63× objective. Images are representative of data from four independent experiments. Bar = 100 μm. (H) RT-qPCR for SeV NP, (+)gSeV, and (+)DVG-546 relative to GAPDH. Data are represented as means ± SEM from three independent experiments, **, P < 0.005; *, P < 0.05 by two-way analysis of variance (ANOVA), with significance indicated between viral infections.
FIG 2
FIG 2
FL genomes accumulate in a perinuclear region, while DVGs are distributed throughout the cytoplasm. (A) Schematic of positive- and negative-sense probe sets binding to SeV genomes and antigenomes. For positive-sense FISH, probes targeting the 5′ end of the positive-sense genome are shown in red, and probes targeting the 3′ end of the positive-sense genome are shown in green, interpreted as +gSeV (orange) and +DVG (green). For negative-sense FISH, probes targeting the 3′ end of the negative-sense genome are shown in red, and probes targeting the 5′ end of the negative-sense genome are shown in green, interpreted as −gSeV (orange) and −DVG (green). (B) A549 cells infected with SeV-LD at an MOI of 1.5 TCID50/cell supplemented with purified DPs at the indicated HAU for 24 h followed by positive-sense viral RNA-FISH. Wide-field images were captured with a 40×, objective. Images are representative of results from 3 independent experiments. Deconvolved, extended focus images are shown. Bar = 100 μm. (C) A549 cells infected with SeV-HD for 24 h and then subjected to positive-sense viral RNA-FISH or negative-sense viral RNA-FISH. A 63× wide-field, deconvolved, extended focus is shown. Images are representative of results from three independent experiments. Bar = 100 μm.
FIG 3
FIG 3
Different species of SeV DVGs are cytoplasmically distributed. A549 cells were infected with the indicated virus for 24 h. (A) Agarose gel electrophoresis of PCR amplification of DVGs using common primers targeting all copyback DVGs. (B) A549 cells infected with SeV-52-LD or -HD and then subjected to negative-sense viral RNA-FISH. Wide-field images were captured with a 63× objective. Deconvolved, extended focus is shown. Images are representative of results from three independent experiments. Bar = 50 μm.
FIG 4
FIG 4
SeV NP colocalizes with Rab11a and microtubules in FL-high cells but not in DVG-high cells. A549 cells were infected with SeV-LD or SeV-HD for 24 h. Cells were fixed and subjected to immunofluorescence analysis for the viral protein SeV NP (red) and Rab11a (recycling endosomes), α-tubulin (microtubules), TOM20 (mitochondria), GM130 (cis-Golgi network), or calnexin (endoplasmic reticulum) (all in green). Confocal images were captured with a 100× objective. A single plane is shown. SeV-LD infection was used to capture FL-high cells, and DVG-high cells were captured from SeV-HD infection focusing on fields with cells containing a majority cytoplasmically distributed SeV NP. Bar = 50 μm. Global Pearson’s correlation was quantified by field for 3 independent experiments, with >5 fields per experiment. **, P < 0.005; ****, P < 0.0001 by Student’s t test.
FIG 5
FIG 5
DVG-high cells show less colocalization with Rab11a than FL-high cells. (A) Immunofluorescence for Rab11a (magenta) with negative-sense viral RNA-FISH of A549 cells infected with SeV-HD for 24 h. Confocal images were captured with a 63× objective. Deconvolved, extended-focus images are shown. Bar = 50 μm. Single-channel images of Rab11a (bottom) and RNA-FISH (top) are shown to the right. (B) Global Pearson’s colocalization between Rab11a and the 5′ end of the genome quantified per cell for 3 independent experiments, with 5 fields per experiment. Cells were binned as DVG+ or FL+ based on the ratio of the 5′/3′ probe intensity. Individual cells are plotted with a line at the mean, and error bars represent SEM. ****, P < 0.0001 by a Mann-Whitney nonparametric U test.
FIG 6
FIG 6
Cytoplasmic distribution of DVGs in DVG-high cells is independent of microtubule integrity and Rab11a expression. (A) A549 cells infected with SeV-HD and treated with nocodazole at 4 h postinfection. Positive-sense viral RNA-FISH was performed at 24 h postinfection. Wide-field images were captured with a 63× objective. Deconvolved, extended focus is shown. Bar = 100 μm. Images are representative of results from four independent experiments. DMSO, dimethyl sulfoxide. (B to D) RT-qPCR for SeV NP (B), (+)gSeV (C), and (+)DVG-546 (D) transcripts relative to GAPDH at 24 h postinfection. Data are represented as means ± SEM of results from three independent experiments. *, P < 0.05 by a paired t test. (C) A549 cells transfected with siRNA targeting Rab11a or scrambled control siRNA (siCtrl) prior to infection, infected with SeV-HD for 24 h, and then subjected to positive-sense viral RNA-FISH. Wide-field images were captured with a 63× objective. Deconvolved, extended focus is shown. Bar = 100 μm. Images are representative of results from four independent experiments. (F to I) RT-qPCR for Rab11a (F), SeV NP (G), (+)gSeV (H), and (+)DVG-546 (I) transcripts relative to GAPDH at 24 h postinfection. Data are represented as means ± SEM of results from seven independent experiments. *, P < 0.05; **, P < 0.005 by a paired t test. (J) Western blotting for Rab11a protein with a GAPDH loading control in mock (M), SeV-LD (LD), and SeV-HD (HD) infections. (K) Relative levels of infectious virus by TCID50 normalized to an siRNA control. Data are represented as means ± SEM of results from three independent experiments. *, P < 0.05 by a paired t test. The supernatant from control siRNA and Rab11a siRNA KD (knockdown) cells was adjusted to an MOI of 1 TCID50/cell with SeV-LD, and LLCMK2 cells were infected for 24 h. (L to N) RT-qPCR for SeV Cantell-specific DVG-546 (L), (+)gSeV (M), and SeV NP (N) mRNAs relative to GAPDH. Data are represented as means ± SEM of results from three independent experiments normalized to values for SeV-LD alone. **, P <0.005 by one-way ANOVA followed by a Bonferroni post hoc test.
FIG 7
FIG 7
Infectious virus is produced by FL-high cells and not by DVG-high cells. (A) Representative flow cytometry plot of SeV-LD-infected LLCMK2 cells at 24 h postinfection. The HN-low gate is shown in green, and the HN-high gate is shown in red. (B) TCID50/25 μl of the supernatant from the indicated cell populations. (C) Representative flow cytometry plot of SeV-HD-infected LLCMK2 cells at 24 h postinfection. The DVG-high gate is shown in green, and the FL-high gate is shown in red. (D and E) Cytospin of the indicated populations after sorting (D) and unsorted SeV-HD-infected LLCMK2 cells as a control (E) subjected to positive-sense RNA-FISH, (wide field, 20× objective). (F) Relative ratio of DVGs to genomes calculated by RT-qPCR for DVG-546 and positive-sense genomes in each population, relative to GAPDH (n = 4). *, P < 0.05 by a Mann-Whitney nonparametric U test. (G) TCID50/25 μl of the supernatant from the indicated cell populations (n = 3). **, P < 0.005 by Student’s t test.
FIG 8
FIG 8
Defective particles are produced by FL virus-high cells and not by DVG-high cells. (A and B) Purified defective particles added to SeV-52, and levels of (+)DVG-546 (A) and (+)gSeV (B) were measured at 6 and 24 h postinfection. Data from three independent experiments are shown. *, P < 0.05; ***, P < 0.001 by two-way ANOVA with Sidak’s multiple-comparisons test. (C to F) The supernatant (sup) from sorted cells was mixed with SeV-52 at an MOI of 1 TCID50/cell, and LLCMK2 cells were infected for 24 h. Shown are RT-qPCR data for Cantell-specific DVG-546 (C), (+)gSeV (D), IFNB (E), and IFIT1 (F) mRNAs relative to GAPDH (n = 3). **, P <0.005 by one-way ANOVA followed by a Bonferroni post hoc test.
FIG 9
FIG 9
Differential distribution of viral genomes is independent of antiviral signaling. (A and B) RT-qPCR for IFNB (A) and IFIT1 (B) mRNAs relative to GAPDH (n = 3). **, P < 0.005 by one-way ANOVA followed by a Bonferroni post hoc test. (C to E) CRISPR control A549 (C), MAVS KO (D), and IFNAR KO (E) cell lines were infected with SeV-HD for 24 h and then subjected to positive-sense viral RNA-FISH. Wide-field images were captured with a 63× objective. Deconvolved, extended focus is shown. Images are representative of results from three independent experiments. Bar = 50 μm.

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