scholarly journals Phosphorylation of Human Metapneumovirus M2-1 Protein Upregulates Viral Replication and Pathogenesis

2016 ◽  
Vol 90 (16) ◽  
pp. 7323-7338 ◽  
Author(s):  
Hui Cai ◽  
Yu Zhang ◽  
Mijia Lu ◽  
Xueya Liang ◽  
Ryan Jennings ◽  
...  
Keyword(s):  
Respiratory Tract ◽  
Viral Replication ◽  
Lower Respiratory Tract ◽  
Rna Synthesis ◽  
Binding Activity ◽  
Human Metapneumovirus ◽  
Zinc Binding ◽  
Recombinant Viruses ◽  
Cotton Rats

ABSTRACTHuman metapneumovirus (hMPV) is a major causative agent of upper- and lower-respiratory-tract infections in infants, the elderly, and immunocompromised individuals worldwide. Like all pneumoviruses, hMPV encodes the zinc binding protein M2-1, which plays important regulatory roles in RNA synthesis. The M2-1 protein is phosphorylated, but the specific role(s) of the phosphorylation in viral replication and pathogenesis remains unknown. In this study, we found that hMPV M2-1 is phosphorylated at amino acid residues S57 and S60. Subsequent mutagenesis found that phosphorylation is not essential for zinc binding activity and oligomerization, whereas inhibition of zinc binding activity abolished the phosphorylation and oligomerization of the M2-1 protein. Using a reverse genetics system, recombinant hMPVs (rhMPVs) lacking either one or both phosphorylation sites in the M2-1 protein were recovered. These recombinant viruses had a significant decrease in both genomic RNA replication and mRNA transcription. In addition, these recombinant viruses were highly attenuated in cell culture and cotton rats. Importantly, rhMPVs lacking phosphorylation in the M2-1 protein triggered high levels of neutralizing antibody and provided complete protection against challenge with wild-type hMPV. Collectively, these data demonstrated that phosphorylation of the M2-1 protein upregulates hMPV RNA synthesis, replication, and pathogenesisin vivo.IMPORTANCEThe pneumoviruses include many important human and animal pathogens, such as human respiratory syncytial virus (hRSV), hMPV, bovine RSV, and avian metapneumovirus (aMPV). Among these viruses, hRSV and hMPV are the leading causes of acute respiratory tract infection in infants and children. Currently, there is no antiviral or vaccine to combat these diseases. All known pneumoviruses encode a zinc binding protein, M2-1, which is a transcriptional antitermination factor. In this work, we found that phosphorylation of M2-1 is essential for virus replication and pathogenesisin vivo. Recombinant hMPVs lacking phosphorylation in M2-1 exhibited limited replication in the upper and lower respiratory tract and triggered strong protective immunity in cotton rats. This work highlights the important role of M2-1 phosphorylation in viral replication and that inhibition of M2-1 phosphorylation may serve as a novel approach to develop live attenuated vaccines as well as antiviral drugs for pneumoviruses.

scholarly journals Zinc Binding Activity of Human Metapneumovirus M2-1 Protein Is Indispensable for Viral Replication and PathogenesisIn Vivo

2015 ◽  
Vol 89 (12) ◽  
pp. 6391-6405 ◽  
Author(s):  
Hui Cai ◽  
Yu Zhang ◽  
Yuanmei Ma ◽  
Jing Sun ◽  
Xueya Liang ◽  
...  
Keyword(s):  
Respiratory Tract ◽  
Viral Replication ◽  
Binding Activity ◽  
Human Metapneumovirus ◽  
Zinc Binding ◽  
Binding Motif ◽  
Content Type ◽  
Cotton Rats ◽  
Zinc Binding Motif

ABSTRACTHuman metapneumovirus (hMPV) is a member of thePneumovirinaesubfamily in theParamyxoviridaefamily that causes respiratory tract infections in humans. Unlike members of theParamyxovirinaesubfamily, the polymerase complex of pneumoviruses requires an additional cofactor, the M2-1 protein, which functions as a transcriptional antitermination factor. The M2-1 protein was found to incorporate zinc ions, although the specific role(s) of the zinc binding activity in viral replication and pathogenesis remains unknown. In this study, we found that the third cysteine (C21) and the last histidine (H25) in the zinc binding motif (CCCH) of hMPV M2-1 were essential for zinc binding activity, whereas the first two cysteines (C7 and C15) play only minor or redundant roles in zinc binding. In addition, the zinc binding motif is essential for the oligomerization of M2-1. Subsequently, recombinant hMPVs (rhMPVs) carrying mutations in the zinc binding motif were recovered. Interestingly, rhMPV-C21S and -H25L mutants, which lacked zinc binding activity, had delayed replication in cell culture and were highly attenuated in cotton rats. In contrast, rhMPV-C7S and -C15S strains, which retained 60% of the zinc binding activity, replicated as efficiently as rhMPV in cotton rats. Importantly, rhMPVs that lacked zinc binding activity triggered high levels of neutralizing antibody and provided complete protection against challenge with rhMPV. Taken together, these results demonstrate that zinc binding activity is indispensable for viral replication and pathogenesisin vivo. These results also suggest that inhibition of zinc binding activity may serve as a novel approach to rationally attenuate hMPV and perhaps other pneumoviruses for vaccine purposes.IMPORTANCEThe pneumoviruses include many important human and animal pathogens, such as human respiratory syncytial virus (hRSV), hMPV, bovine RSV, and avian metapneumovirus (aMPV). Among these viruses, hRSV and hMPV are the leading causes of acute respiratory tract infection in infants and children. Despite major efforts, there is no antiviral or vaccine to combat these diseases. All known pneumoviruses encode a zinc binding protein, M2-1, which is a transcriptional antitermination factor. In this work, we found that the zinc binding activity of M2-1 is essential for virus replication and pathogenesisin vivo. Recombinant hMPVs that lacked zinc binding activity were not only defective in replication in the upper and lower respiratory tract but also triggered a strong protective immunity in cotton rats. Thus, inhibition of M2-1 zinc binding activity can lead to the development of novel, live attenuated vaccines, as well as antiviral drugs for pneumoviruses.

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

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.

scholarly journals The NS Segment of H1N1pdm09 Enhances H5N1 Pathogenicity in a Mouse Model of Influenza Virus Infections

Viruses ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 504
Author(s):  
Olivier Ferraris ◽  
Jean-Sébastien Casalegno ◽  
Emilie Frobert ◽  
Maude Bouscambert Duchamp ◽  
Martine Valette ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Respiratory Tract ◽  
Lower Respiratory Tract ◽  
Target Cells ◽  
Virus Infections ◽  
Reassortment Event ◽  
Highly Pathogenic ◽  
2009 H1n1 ◽  
Reassortant Viruses

In 2009, the co-circulation of H5N1 and H1N1pdm09 raised concerns that a reassortment event may lead to highly pathogenic influenza strains. H1N1pdm09 and H5N1 are able to infect the same target cells of the lower respiratory tract. To investigate the capacity of the emergence of reassortant viruses, we characterized viruses obtained from the co-infection of cells with H5N1 (A/Turkey/13/2006) and H1N1pdm09 (A/Lyon/969/2009 H1N1). In our analysis, all the screened reassortants possessed the PB2, HA, and NP segments from H5N1 and acquired one or two of the H1N1pdm09 segments. Moreover, the in vivo infections showed that the acquisition of the NS segment from H1N1pdm09 increased the virulence of H5N1 in mice. We conclude, therefore, that reassortment can occur between these two viruses, even if this process has never been detected in nature.

Methodology for the measurement of mucociliary function in the mouse by scintigraphy

2001 ◽  
Vol 90 (3) ◽  
pp. 1111-1118 ◽  
Author(s):  
W. Michael Foster ◽  
Dianne M. Walters ◽  
Malinda Longphre ◽  
Kristin Macri ◽  
Laura M. Miller
Keyword(s):  
Respiratory Tract ◽  
Lower Respiratory Tract ◽  
Particle Deposition ◽  
Time Course ◽  
Colloidal Particles ◽  
Intratracheal Instillation ◽  
Aerosol Inhalation ◽  
Mucociliary Function

The objective of the study was to develop a scintigraphic method for measurement of airway mucociliary clearance in small laboratory rodents such as the mouse. Previous investigations have characterized the secretory cell types present in the mouse airway, but analysis of the mucus transport system has been limited to in vitro examination of tissue explants or invasive in vivo measures of a single airway, the trachea. Three methods were used to deposit insoluble, radioisotopic colloidal particles: oropharyngeal aspiration, intratracheal instillation, and nose-only aerosol inhalation. The initial distribution of particles within the lower respiratory tract was visualized by γ-camera, and clearance of particles was followed intermittently over 6 h and at the conclusion, 24 h postdelivery. Subsets of mice underwent lavage for evidence of tissue inflammation, and others were restudied for reproducibility of the methods. The aspiration and instillation methods of delivery led to greater distributions of deposited activity within the lungs, i.e., ∼60–80% of the total respiratory tract radioactivity, whereas the nose-only aerosol technique attained a distribution of 32% to the lungs. However, the aerosol technique maximized the fraction of particles that cleared the airway over a 24-h period, i.e, deposited onto airway epithelial surfaces and cleared by mucociliary function such that lung retention at 24 h averaged 57% for delivery by aerosol inhalation and ≥80% for the aspiration or intratracheal instillation techniques. Particle delivery methods did not cause lung inflammation/injury with use of inflammatory cells and chemoattractant cytokines as criteria. Scintigraphy can discern particle deposition and clearance from the lower respiratory tract in the mouse, is noninvasive and reproducible, and includes the capability for restudy and lung lavage when time course or chronic treatments are being considered.

scholarly journals Severe acute respiratory syndrome vaccine efficacy in ferrets: whole killed virus and adenovirus-vectored vaccines

2008 ◽  
Vol 89 (9) ◽  
pp. 2136-2146 ◽  
Author(s):  
Raymond H. See ◽  
Martin Petric ◽  
David J. Lawrence ◽  
Catherine P. Y. Mok ◽  
Thomas Rowe ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Respiratory Tract ◽  
Viral Replication ◽  
Lower Respiratory Tract ◽  
Clinical Signs ◽  
Neutralizing Antibody ◽  
Lung Damage ◽  
Upper Respiratory Tract ◽  
Administration Routes ◽  
Antibody Titres

Although the 2003 severe acute respiratory syndrome (SARS) outbreak was controlled, repeated transmission of SARS coronavirus (CoV) over several years makes the development of a SARS vaccine desirable. We performed a comparative evaluation of two SARS vaccines for their ability to protect against live SARS-CoV intranasal challenge in ferrets. Both the whole killed SARS-CoV vaccine (with and without alum) and adenovirus-based vectors encoding the nucleocapsid (N) and spike (S) protein induced neutralizing antibody responses and reduced viral replication and shedding in the upper respiratory tract and progression of virus to the lower respiratory tract. The vaccines also diminished haemorrhage in the thymus and reduced the severity and extent of pneumonia and damage to lung epithelium. However, despite high neutralizing antibody titres, protection was incomplete for all vaccine preparations and administration routes. Our data suggest that a combination of vaccine strategies may be required for effective protection from this pathogen. The ferret may be a good model for SARS-CoV infection because it is the only model that replicates the fever seen in human patients, as well as replicating other SARS disease features including infection by the respiratory route, clinical signs, viral replication in upper and lower respiratory tract and lung damage.

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