Viral N6-methyladenosine upregulates replication and pathogenesis of human respiratory syncytial virus

Abstract N6-methyladenosine (m6A) is the most prevalent internal modification of mRNAs in most eukaryotes. Here we show that RNAs of human respiratory syncytial virus (RSV) are modified by m6A within discreet regions and that these modifications enhance viral replication and pathogenesis. Knockdown of m6A methyltransferases decreases RSV replication and gene expression whereas knockdown of m6A demethylases has the opposite effect. The G gene transcript contains the most m6A modifications. Recombinant RSV variants expressing G transcripts that lack particular clusters of m6A display reduced replication in A549 cells, primary well differentiated human airway epithelial cultures, and respiratory tracts of cotton rats. One of the m6A-deficient variants is highly attenuated yet retains high immunogenicity in cotton rats. Collectively, our results demonstrate that viral m6A methylation upregulates RSV replication and pathogenesis and identify viral m6A methylation as a target for rational design of live attenuated vaccine candidates for RSV and perhaps other pneumoviruses.

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Respiratory syncytial virus increases IL-8 gene expression and protein release in A549 cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1995.269.6.l865 ◽

1995 ◽

Vol 269 (6) ◽

pp. L865-L872 ◽

Cited By ~ 13

Author(s):

M. A. Fiedler ◽

K. Wernke-Dollries ◽

J. M. Stark

Keyword(s):

Gene Expression ◽

Respiratory Syncytial Virus ◽

Viral Replication ◽

Mrna Expression ◽

A549 Cells ◽

Protein Release ◽

Tnf Alpha ◽

Epithelial Cell Line ◽

Airway Epithelial ◽

Syncytial Virus

The mechanism of respiratory syncytial virus (RSV)-induced inflammation in the airways of infants and children is not fully understood. We hypothesized that RSV directly induces interleukin (IL)-8 gene expression in airway epithelial cells, independent of IL-1 beta and tumor necrosis factor-alpha (TNF-alpha) production. Exposure of A549 cells (an airway epithelial cell line) to RSV resulted in increased IL-8 mRNA expression and IL-8 protein release from the cells as early as 2 h after treatment. Neither IL-1 beta nor TNF-alpha (mRNA or protein) were detected. Viral replication was not necessary for the effects of RSV on IL-8 mRNA expression and protein release early in the infectious process. However, sustained levels of increased IL-8 production required RSV replication. A dose-response relationship was observed between the multiplicity of infection and IL-8 production with both active and nonreplicative RSV at the 2-h time point. Both active RSV and nonreplicative RSV increased the transcriptional activity of the 1.6-kb 5' flanking region of the IL-8 gene. Neither active RSV nor nonreplicative RSV increased the stability of the IL-8 mRNA in A549 cells. We conclude that RSV increases IL-8 gene expression in A549 cells in a biphasic pattern independent of viral replication early (2 h) but dependent on viral replication late (24 h).

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Recombinant subtype A and B human respiratory syncytial virus clinical isolates co-infect the respiratory tract of cotton rats

Journal of General Virology ◽

10.1099/jgv.0.001471 ◽

2020 ◽

Vol 101 (10) ◽

pp. 1056-1068

Author(s):

Linda J. Rennick ◽

Sham Nambulli ◽

Ken Lemon ◽

Grace Y. Olinger ◽

Nicholas A. Crossland ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Respiratory Tract ◽

Reverse Genetics ◽

Fluorescent Protein ◽

Human Respiratory Syncytial Virus ◽

Subtype B ◽

Cotton Rats ◽

Syncytial Virus ◽

Subtype A

Human respiratory syncytial virus (HRSV) is an important respiratory pathogen causing a spectrum of illness, from common cold-like symptoms, to bronchiolitis and pneumonia requiring hospitalization in infants, the immunocompromised and the elderly. HRSV exists as two antigenic subtypes, A and B, which typically cycle biannually in separate seasons. There are many unresolved questions in HRSV biology regarding the interactions and interplay of the two subtypes. Therefore, we generated a reverse genetics system for a subtype A HRSV from the 2011 season (A11) to complement our existing subtype B reverse genetics system. We obtained the sequence (HRSVA11) directly from an unpassaged clinical sample and generated the recombinant (r) HRSVA11. A version of the virus expressing enhanced green fluorescent protein (EGFP) from an additional transcription unit in the fifth (5) position of the genome, rHRSVA11EGFP(5), was also generated. rHRSVA11 and rHRSVA11EGFP(5) grew comparably in cell culture. To facilitate animal co-infection studies, we derivatized our subtype B clinical isolate using reverse genetics toexpress the red fluorescent protein (dTom)-expressing rHRSVB05dTom(5). These viruses were then used to study simultaneous in vivo co-infection of the respiratory tract. Following intranasal infection, both rHRSVA11EGFP(5) and rHRSVB05dTom(5) infected cotton rats targeting the same cell populations and demonstrating that co-infection occurs in vivo. The implications of this finding on viral evolution are important since it shows that inter-subtype cooperativity and/or competition is feasible in vivo during the natural course of the infection.

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Primary airway epithelial cultures from children are highly permissive to respiratory syncytial virus infection

Thorax ◽

10.1136/thoraxjnl-2011-200131 ◽

2011 ◽

Vol 67 (1) ◽

pp. 42-48 ◽

Cited By ~ 21

Author(s):

A M Fonceca ◽

B F Flanagan ◽

R Trinick ◽

R L Smyth ◽

P S McNamara

Keyword(s):

Respiratory Syncytial Virus ◽

Virus Infection ◽

Respiratory Syncytial Virus Infection ◽

Airway Epithelial ◽

Epithelial Cultures ◽

Syncytial Virus

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Infection-enhancing lipopeptides do not improve intranasal immunization of cotton rats with a delta-G candidate live-attenuated human respiratory syncytial virus vaccine

Human Vaccines & Immunotherapeutics ◽

10.4161/hv.26096 ◽

2013 ◽

Vol 9 (12) ◽

pp. 2578-2583 ◽

Cited By ~ 1

Author(s):

D Tien Nguyen ◽

Jolande Boes ◽

Geert van Amerongen ◽

Yvonne van Remmerden ◽

Selma Yüksel ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Virus Vaccine ◽

Human Respiratory Syncytial Virus ◽

Intranasal Immunization ◽

Cotton Rats ◽

Syncytial Virus ◽

Delta G ◽

Respiratory Syncytial Virus Vaccine

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Faculty Opinions recommendation of Respiratory syncytial virus uses CX3CR1 as a receptor on primary human airway epithelial cultures.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726008363.793526355 ◽

2016 ◽

Cited By ~ 1

Author(s):

Cecilia Johansson

Keyword(s):

Respiratory Syncytial Virus ◽

Airway Epithelial ◽

Human Airway ◽

Epithelial Cultures ◽

Syncytial Virus

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Distinct gene subsets are induced at different time points after human respiratory syncytial virus infection of A549 cells

Journal of General Virology ◽

10.1099/vir.0.82187-0 ◽

2007 ◽

Vol 88 (2) ◽

pp. 570-581 ◽

Cited By ~ 49

Author(s):

Isidoro Martínez ◽

Luis Lombardía ◽

Blanca García-Barreno ◽

Orlando Domínguez ◽

José A. Melero

Keyword(s):

Respiratory Syncytial Virus ◽

Time Course ◽

A549 Cells ◽

Cellular Protein ◽

Human Respiratory Syncytial Virus ◽

Virus Infectivity ◽

Dependent Manner ◽

Down Regulation ◽

Cytoskeleton Organization ◽

Syncytial Virus

cDNA microarray technology was applied to time course analysis of differentially expressed genes in A549 cells following human respiratory syncytial virus (HRSV) infection. Both up- and down-regulation of cellular genes were observed in a time-dependent manner. However, gene up-regulation prevailed over gene down-regulation. Virus infectivity was required as UV-inactivated virus failed to up-regulate/down-regulate those genes. At early times post-infection (0–6 h p.i.) 85 genes were up-regulated. Some of those genes were involved in cell growth/proliferation, cellular protein metabolism and cytoskeleton organization. Among the most strongly up-regulated genes at that time were the urokinase plasminogen activator (PLAU) and its receptor (PLAUR), a pleiotropic system involved in many biological processes, including chemotaxis and inflammation. Functionally related genes encoding the α- and β-chains of several integrins were also up-regulated within the first 12 h of infection. Genes up-regulated between 6 and 12 h p.i. included interferon-stimulated genes (ISGs), genes related to oxidative stress and genes of the non-canonical NF-κB pathway. At later times, genes involved in the immune response became predominant among the up-regulated genes, most of them being ISGs. Different up-regulation kinetics of cytokine and cytokine-signalling-related genes were also observed. These results highlight the dynamic interplay between the virus and the host cell and provide a general picture of changes in cellular gene expression along the HRSV replicative cycle.

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Human respiratory syncytial virus affects nonadrenergic noncholinergic inhibition in cotton rat airways

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1995.268.6.l1006 ◽

1995 ◽

Vol 268 (6) ◽

pp. L1006-L1011 ◽

Cited By ~ 6

Author(s):

G. N. Colasurdo ◽

V. G. Hemming ◽

G. A. Prince ◽

J. E. Loader ◽

J. P. Graves ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Human Respiratory Syncytial Virus ◽

Control Group ◽

Neurokinin A ◽

Contractile Responses ◽

Cotton Rat ◽

Airway Function ◽

Cotton Rats ◽

Syncytial Virus

A dysfunction of the nonadrenergic noncholinergic inhibitory (NANCi) system has been invoked as a possible mechanism underlying or contributing to altered airway function. In the present study we assessed whether human respiratory syncytial virus (HRSV) infection affects the airways' neurally mediated contractile and relaxant (NANCi) responses in vitro. NANCi responses were studied on tracheal smooth muscle (TSM) segments obtained from young adult cotton rats, a well-established model for HRSV infection. To assess NANCi responses, TSM segments were removed and placed in tissue baths containing modified Krebs-Henseleit, atropine (1 x 10(-6) M) and propranolol (5 x 10(-6) M). After contraction with neurokinin A (1 x 10(-5) M), electrical field stimulation (EFS) was applied at stimulation frequencies ranging from 5 to 30 Hz. The NANCi responses were measured and expressed as the mean (+/- SE) percent relaxation. To evaluate neurally mediated contractile responses, full frequency response curves (0.5-30 Hz) to EFS were also performed. We found significantly decreased NANCi responses in TSM segments obtained from infected cotton rats (n = 12) compared with control animals (n = 9) (P < 0.002). Furthermore, the contractile responses to EFS were increased in infected animals compared with the control group (P = 0.0001). These findings demonstrate that HRSV infection leads to an enhanced contractile response to EFS and a significant decrease in NANCi response in cotton rat airways in vitro. This disruption of the neural control of airways may lead to the development of altered airway function.

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Mutation of Respiratory Syncytial Virus G Protein’s CX3C Motif Attenuates Infection in Cotton Rats and Primary Human Airway Epithelial Cells

Vaccines ◽

10.3390/vaccines7030069 ◽

2019 ◽

Vol 7 (3) ◽

pp. 69 ◽

Cited By ~ 1

Author(s):

Binh Ha ◽

Tatiana Chirkova ◽

Marina S. Boukhvalova ◽

He Ying Sun ◽

Edward E. Walsh ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Epithelial Cells ◽

Airway Epithelial Cells ◽

Host Responses ◽

Airway Epithelial ◽

Live Virus ◽

Human Airway ◽

Cotton Rats ◽

Syncytial Virus ◽

Human Airway Epithelial Cells

Despite being a high priority for vaccine development, no vaccine is yet available for respiratory syncytial virus (RSV). A live virus vaccine is the primary type of vaccine being developed for young children. In this report, we describe our studies of infected cotton rats and primary human airway epithelial cells (pHAECs) using an RSV r19F with a mutation in the CX3C chemokine motif in the RSV G protein (CX4C). Through this CX3C motif, RSV binds to the corresponding chemokine receptor, CX3CR1, and this binding contributes to RSV infection of pHAECs and virus induced host responses that contribute to disease. In both the cotton rat and pHAECs, the CX4C mutation decreased virus replication and disease and/or host responses to infection. Thus, this mutation, or other mutations that block binding to CX3CR1, has the potential to improve a live attenuated RSV vaccine by attenuating both infection and disease pathogenesis.

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