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 paramyxovirus infection in humans.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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.
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
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.
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.
The airway epithelium constitutes an essential immunological and cytoprotective barrier to inhaled insults, such as cigarette smoke, environmental particles, or viruses. Although bronchial epithelial ...integrity is crucial for airway homeostasis, defective epithelial barrier function contributes to chronic obstructive pulmonary disease (COPD). Tight junctions at the apical side of epithelial cell-cell contacts determine epithelial permeability. Cigarette smoke exposure, the major risk factor for COPD, is suggested to impair tight junction integrity; however, detailed mechanisms thereof remain elusive. We investigated whether cigarette smoke extract (CSE) and transforming growth factor (TGF)-β1 affected tight junction integrity. Exposure of human bronchial epithelial cells (16HBE14o(-)) and differentiated primary human bronchial epithelial cells (pHBECs) to CSE significantly disrupted tight junction integrity and barrier function. Specifically, CSE decreased transepithelial electrical resistance (TEER) and tight junction-associated protein levels. Zonula occludens (ZO)-1 and ZO-2 protein levels were significantly reduced and dislocated from the cell membrane, as observed by fractionation and immunofluorescence analysis. These findings were reproduced in isolated bronchi exposed to CSE ex vivo, as detected by real-time quantitative reverse-transcriptase PCR and immunohistochemistry. Combined treatment of 16HBE14o(-) cells or pHBECs with CSE and TGF-β1 restored ZO-1 and ZO-2 levels. TGF-β1 cotreatment restored membrane localization of ZO-1 and ZO-2 protein and prevented CSE-mediated TEER decrease. In conclusion, CSE led to the disruption of tight junctions of human bronchial epithelial cells, and TGF-β1 counteracted this CSE-induced effect. Thus, TGF-β1 may serve as a protective factor for bronchial epithelial cell homeostasis in diseases such as COPD.
Defective viral genomes (DVGs) are generated during viral replication and are unable to carry out a full replication cycle unless coinfected with a full-length virus. DVGs are produced by many ...viruses, and their presence correlates with alterations in infection outcomes. Historically, DVGs were studied for their ability to interfere with standard virus replication as well as for their association with viral persistence. More recently, a critical role for DVGs in inducing the innate immune response during infection was appreciated. Here we review the role of DVGs of RNA viruses in shaping outcomes of experimental as well as natural infections and explore the mechanisms by which DVGs impact infection outcome.
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.
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-Lambda1 over IFN-Beta 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 paramyxovirus infection in humans.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The airway epithelium constitutes an essential immunological and cytoprotective barrier to inhaled insults, such as cigarette smoke, environmental particles, or viruses. Although bronchial epithelial ...integrity is crucial for airway homeostasis, defective epithelial barrier function contributes to chronic obstructive pulmonary disease (COPD). Tight junctions at the apical side of epithelial cell-cell contacts determine epithelial permeability. Cigarette smoke exposure, the major risk factor for COPD is suggested to impair tight junction integrity; however, detailed mechanisms thereof remain elusive. The authors have investigated whether cigarette smoke extract (CSE) and transforming growth factor (TGF)-b1 affected tight junction integrity. Exposure of human bronchial epithelial cells (16HBE14o2) and differentiated primary human bronchial epithelial cells (pHBECs) to CSE significantly disrupted tight junction integrity and barrier function. These findings were reproduced in isolated bronchi exposed to CSE ex vivo, as detected by real-time quantitative reverse-transcriptase PCR and immunohistochemistry. In conclusion, CSE led to the disruption of tight junctions of human bronchial epithelial cells, and TGF-b1 counteracted this CSE induced effect.
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