Impacts of Intracellular Localizations of Full-Length and Defective Viral Genomes on Paramyxovirus Particle Production

Paramyxoviruses are negative-sense single-stranded RNA viruses that comprise many important human and animal pathogens. During viral replication, paramyxoviruses produce defective viral genomes (DVGs), truncated genomic products that are unable to replicate in the absence of standard virus. DVGs influence the outcomes of infection through interference with standard viral replication and by inducing antiviral immunity. Using the model paramyxovirus, Sendai virus (SeV), we found that full-length (FL) and DVG viral RNA (vRNA) accumulated heterogeneously in cells during infection, with some cells accumulating predominantly full-length genomes (FL-high) and some accumulating predominantly DVGs (DVG-high). Interestingly, in FL-high cells genomes accumulated in a perinuclear region while viral genomes in DVG-high cells remained diffusely distributed throughout the cytoplasm. We sought to address the mechanisms and consequences of the differential intracellular distributions of viral RNA in the presence of DVGs. We found that vRNA in FL-high cells interacts with the host GTPase Rab11a and uses the recycling endosome system for particle production, while viral RNA in DVG-high cells does not interact with the host cell in this way. Consequently, FL-high cells produce both standard virions and defective particles, while DVG-high cells do not produce virions. We next addressed the determinants of this distinct intracellular localization. We reasoned that DVG-high cells, which robustly replicate vRNA but do not progress to virion assembly, fail to accumulate the viral proteins required for interaction between vRNA and Rab11a. We found that neither SeV matrix nor nucleoproteins are sufficient to drive this interaction. We identified the viral polymerase protein L and the accessory protein C as differentiating factors in cells that engage with Rab11a, and found C proteins to be the most enriched proteins in Rab11a immunoprecipitation followed by mass spectrometry. These data suggest that the polymerase complex proteins L and its cofactor C are critical in regulating initial steps in SeV assembly. Overall, this work investigated the intracellular distributions of viral genomes in the presence of DVGs to understand the impact of DVGs on the dynamics of full length and defective particle production, as well as to gain insights into viral proteins required to initiate viral assembly

Impacts Of Intracellular Localizations Of Full-Length And Defective Viral Genomes On Paramyxovirus Particle Production

Paramyxoviruses are negative-sense single-stranded RNA viruses that comprise many important human and animal pathogens. During viral replication, paramyxoviruses produce defective viral genomes (DVGs), truncated genomic products that are unable to replicate in the absence of standard virus. DVGs influence the outcomes of infection through interference with standard viral replication and by inducing antiviral immunity. Using the model paramyxovirus, Sendai virus (SeV), we found that full-length (FL) and DVG viral RNA (vRNA) accumulated heterogeneously in cells during infection, with some cells accumulating predominantly full-length genomes (FL-high) and some accumulating predominantly DVGs (DVG-high). Interestingly, in FL-high cells genomes accumulated in a perinuclear region while viral genomes in DVG-high cells remained diffusely distributed throughout the cytoplasm. We sought to address the mechanisms and consequences of the differential intracellular distributions of viral RNA in the presence of DVGs. We found that vRNA in FL-high cells interacts with the host GTPase Rab11a and uses the recycling endosome system for particle production, while viral RNA in DVG-high cells does not interact with the host cell in this way. Consequently, FL-high cells produce both standard virions and defective particles, while DVG-high cells do not produce virions. We next addressed the determinants of this distinct intracellular localization. We reasoned that DVG-high cells, which robustly replicate vRNA but do not progress to virion assembly, fail to accumulate the viral proteins required for interaction between vRNA and Rab11a. We found that neither SeV matrix nor nucleoproteins are sufficient to drive this interaction. We identified the viral polymerase protein L and the accessory protein C as differentiating factors in cells that engage with Rab11a, and found C proteins to be the most enriched proteins in Rab11a immunoprecipitation followed by mass spectrometry. These data suggest that the polymerase complex proteins L and its cofactor C are critical in regulating initial steps in SeV assembly. Overall, this work investigated the intracellular distributions of viral genomes in the presence of DVGs to understand the impact of DVGs on the dynamics of full length and defective particle production, as well as to gain insights into viral proteins required to initiate viral assembly

Impacts of Intracellular Localizations of Full-Length and Defective Viral Genomes on Paramyxovirus Particle Production

Paramyxoviruses are negative-sense single-stranded RNA viruses that comprise many important human and animal pathogens. During viral replication, paramyxoviruses produce defective viral genomes (DVGs), truncated genomic products that are unable to replicate in the absence of standard virus. DVGs influence the outcomes of infection through interference with standard viral replication and by inducing antiviral immunity. Using the model paramyxovirus, Sendai virus (SeV), we found that full-length (FL) and DVG viral RNA (vRNA) accumulated heterogeneously in cells during infection, with some cells accumulating predominantly full-length genomes (FL-high) and some accumulating predominantly DVGs (DVG-high). Interestingly, in FL-high cells genomes accumulated in a perinuclear region while viral genomes in DVG-high cells remained diffusely distributed throughout the cytoplasm. We sought to address the mechanisms and consequences of the differential intracellular distributions of viral RNA in the presence of DVGs. We found that vRNA in FL-high cells interacts with the host GTPase Rab11a and uses the recycling endosome system for particle production, while viral RNA in DVG-high cells does not interact with the host cell in this way. Consequently, FL-high cells produce both standard virions and defective particles, while DVG-high cells do not produce virions. We next addressed the determinants of this distinct intracellular localization. We reasoned that DVG-high cells, which robustly replicate vRNA but do not progress to virion assembly, fail to accumulate the viral proteins required for interaction between vRNA and Rab11a. We found that neither SeV matrix nor nucleoproteins are sufficient to drive this interaction. We identified the viral polymerase protein L and the accessory protein C as differentiating factors in cells that engage with Rab11a, and found C proteins to be the most enriched proteins in Rab11a immunoprecipitation followed by mass spectrometry. These data suggest that the polymerase complex proteins L and its cofactor C are critical in regulating initial steps in SeV assembly. Overall, this work investigated the intracellular distributions of viral genomes in the presence of DVGs to understand the impact of DVGs on the dynamics of full length and defective particle production, as well as to gain insights into viral proteins required to initiate viral assembly

The Viral Polymerase Complex Mediates the Interaction of Viral Ribonucleoprotein Complexes with Recycling Endosomes during Sendai Virus Assembly

Paramyxoviruses are members of a family of viruses that include a number of pathogens imposing significant burdens on human health. In particular, human parainfluenza viruses are an important cause of pneumonia and bronchiolitis in children for which there are no vaccines or directly acting antivirals. These cytoplasmic replicating viruses bud from the plasma membrane and co-opt cellular endosomal recycling pathways to traffic viral ribonucleoprotein complexes from the cytoplasm to the membrane of infected cells. The viral proteins required for viral engagement with the recycling endosome pathway are still not known. Here, we used the model paramyxovirus Sendai virus, or murine parainfluenza virus 1, to investigate the role of viral proteins in this initial step of viral assembly. We found that the viral polymerase components large protein L and accessory protein C are necessary for engagement with recycling endosomes. These findings are important in identifying viral proteins as potential targets for development of antivirals.Paramyxoviruses are negative-sense single-stranded RNA viruses that comprise many important human and animal pathogens, including human parainfluenza viruses. These viruses bud from the plasma membrane of infected cells after the viral ribonucleoprotein complex (vRNP) is transported from the cytoplasm to the cell membrane via Rab11a-marked recycling endosomes. The viral proteins that are critical for mediating this important initial step in viral assembly are unknown. Here, we used the model paramyxovirus, murine parainfluenza virus 1, or Sendai virus (SeV), to investigate the roles of viral proteins in Rab11a-driven virion assembly. We previously reported that infection with SeV containing high levels of copy-back defective viral genomes (DVGs) (DVG-high SeV) generates heterogenous populations of cells. Cells enriched in full-length (FL) virus produce viral particles containing standard or defective viral genomes, while cells enriched in DVGs do not, despite high levels of defective viral genome replication. Here, we took advantage of this heterogenous cell phenotype to identify proteins that mediate interaction of vRNPs with Rab11a. We examined the roles of matrix protein and nucleoprotein and determined that their presence is not sufficient to drive interaction of vRNPs with recycling endosomes. Using a combination of mass spectrometry and comparative analyses of protein abundance and localization in DVG-high and FL-virus-high (FL-high) cells, we identified viral polymerase complex component protein L and, specifically, its cofactor C as interactors with Rab11a. We found that accumulation of L and C proteins within the cell is the defining feature that differentiates cells that proceed to viral egress from cells containing viruses that remain in replication phases

Immunostimulatory Defective Viral Genomes from Respiratory Syncytial Virus Promote a Strong Innate Antiviral Response during Infection in Mice and Humans

<div><p>Human respiratory syncytial virus (RSV) is a major cause of severe respiratory illness in children and susceptible adults. RSV blocks the development of the innate antiviral immune response and can grow to high titers in the respiratory tract. Here we demonstrate that immunostimulatory defective viral genomes (iDVGs) that are naturally generated during RSV replication are strong inducers of the innate antiviral response to RSV in mice and humans. In mice, RSV iDVGs stimulated the expression of antiviral genes, restricted viral replication, and prevented weight loss and lung inflammation. In human cells, the antiviral response to RSV iDVGs was dominated by the expression of IFN-λ1 over IFN-β and was driven by rapid intranuclear accumulation of the transcription factor IRF1. RSV iDVGs were detected in respiratory secretions of hospitalized patients, and their amount positively correlated with the level of expression of antiviral genes in the samples. Infection of explanted human lung tissue from different donors revealed that most humans can respond to RSV iDVGs and that the rate of accumulation of iDVGs during infection directly correlates with the quality of the antiviral response. Taken together, our data establish iDVGs as primary triggers of robust antiviral responses to RSV and provide the first evidence for an important biological role for naturally occurring iDVGs during a <i>paramyxovirus</i> infection in humans.</p></div

RSV iDVGs stimulate an IRF1/IFNL1-mediated antiviral response.

<p>A549 cells were infected with RSV-LD or RSV-HD at a moi of 1.5 TCID₅₀/cell. Expression of (A) RSV G and antiviral genes mRNA, and (B) protein level of IFNB1 and IFNL1/3 in the cultures supernatants at 24 h post infection (*p<0.05 by one way unpaired t-test). (C, D) Cells were lysed or fixed at 2, 6, 12, and 24 h post infection for western blot (WB) and IFA. (C) For WB, nuclear and cytosolic fractions were immunoblotted for IRF1, GAPDH, and Histone 3. (D) For IFA, cells were co-stained for IRF1 (green, left panel), RSV F + G proteins (red in merged panel), and nuclei (blue). (E) Quantification of nuclear IRF1 upon iDVGs stimulation at designated time points post RSV-HD infection based on IFA images. (F, G) D54 control cells and D54 cells overexpressing IRF1 (D54-IRF1) were infected with RSV-HD at a moi of 1.5 TCID₅₀/cell for 6 h. (F) IRF1 and IRF3 protein detected by WB from whole cell lysates. (G) Expression of RSV G and IFNL1 mRNA. (H, I) A549 cells were mock transfected (WT) or transfected with control siRNA (si-C), or IRF1 siRNA (si-1). After 40 h, the cells were mock infected or infected with RSV-HD at moi of 1.5 TCID₅₀/cell. (H) WB for IRF3 and IRF1 was performed to confirm specific knockdown of IRF1 protein. (I) Expression of RSV G and other antiviral genes at 10 h post infection. Gene expression is shown as copy number relative to a house keeping gene expression index determined from the expression of ACTB (β-actin) and GAPDH. All error bars indicate mean ± SEM of at least three independent experiments (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-way ANOVA with Bonferroni post hoc test).</p

RSV DVGs prevent viral pathogenesis in vivo.

<p>(A) Hep2 cells were infected with RSV-LD or HD at a moi of 1.5 TCID₅₀/cell and DVGs were detected by PCR at the indicated times. Details of the PCR assay can be seen in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005122#ppat.1005122.s001" target="_blank">S1 Fig</a> and sequences of the amplicons labeled with a star can be found in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005122#ppat.1005122.s002" target="_blank">S2 Fig</a> Base pair size references are indicated in the gel. (B) Mice weight loss was monitored overtime. Error bars indicate standard deviation of data pooled from two independent experiments (n = 7–9 mice per group total; **p<0.01, ****p<0.0001 by two-way ANOVA with Bonferroni post hoc test. Variance was not significantly different between groups as per Bartlett’s test). (C) Representative H&E staining for lung sections from mock, RSV-LD, or RSV-HD-infected mice on day 2 post infection. Picture magnification: 10X; insert is a digital amplification. Red arrows indicate alveolar cellular infiltrate. (D) Pathology score for alveolar infiltration in the lung (n = 6 mice per group; **p<0.01, by two-tailed Mann Whitney test). (E) Differential counts from cytospins from mice bronchoalveolar lavage (BAL) on day 2 post infection (Mono: monocytes and macrophages, PMNs: polymorphonuclear cells; ***p<0.001 by two-way ANOVA with Bonferroni’s post hoc test, n = 5–8 mice per group). (F) Representative cytospin images (20X). (G) Representative flow cytometry plots from whole lung single cell suspensions on day 2 post infection. Plots are pre-gated in singlets, live, CD45⁺CD11b⁺ cells. (H) Quantification of different cell types in the lung of infected mice on day 2 post infection (****p<0.0001 by two-way ANOVA with Bonferroni’s post hoc test, n = 5–8 mice per group, Alv. Macs: alveolar macrophages). (I) Expression of pro-inflammatory genes in whole lung tissue on day 2, 5, and 8 post infection. (n = 3–5 mice per group, *p<0.05, **p<0.01 by one-way ANOVA with Bonferroni’s post hoc test).</p

Host-intrinsic factors determine the response to iDVGs in the human lung.

<p>(A) Images of precision cut lung slices from human lungs (hPCLS) infected for 24 h with 10⁶ RSV-GFP TCID₅₀/slice. Top: Overlay of bright field and fluorescence channels; Bottom: fluorescence channel alone. (B) Gene expression from hPCLS infected with 10⁷ TCID₅₀/slice of RSV-LD or HD for up to 5 days (n = 7–8, grey numbers indicate individual lung donor). Results show paired data, numbers correspond to different donors. (*p<0.05, **p<0.01 by one-tailed Wilcoxon matched-pairs signed rank test). (C) Ratio of gene expression in RSV-HD and RSV-LD infected tissue from different donors (P3-P9). Ratio>1: HD induced a higher gene expression than LD. (D) PCR for DVGs in hPCLS infected with 10⁶ TCID₅₀/slice of RSV-LD. (E) Gene expression from (D). Error bars indicate mean ± SEM of three slices from the same patient.</p

iDVGs associate with high expression of antiviral genes in respiratory secretions from patients infected with RSV.

<p>(A) Representative PCR results for gRSV and DVGs in human nasopharyngeal control samples infected with adenovirus (A1-A5) and samples infected with RSV (R1-R4). (B) Gene expression determined by RT-qPCR shown as copy number relative to house keeping genes (*p<0.05, **p<0.01, by two-tailed Mann Whitney test). (C) Samples were scored based on the intensity of the DVG amplicons (1–4, absent to highest intensity) and correlated with the level of expression of antiviral genes. (r = correlation coefficient, p<0.0001 for slope deviation from 0).</p