VLP-based model for the study of airborne viral pathogens.

The recent COVID-19 pandemic has underscored the danger of airborne viral pathogens. The lack of model systems to study airborne pathogens limits the understanding of airborne pathogen distribution as well as potential surveillance and mitigation strategies. In this work, we develop a novel model system to study airborne pathogens using virus-like particles (VLPs). Specifically, we demonstrate the ability to aerosolize VLP and detect and quantify aerosolized VLP RNA by reverse transcription-loop-mediated isothermal amplification in real-time fluorescent and colorimetric assays. Importantly, the VLP model presents many advantages for the study of airborne viral pathogens: (i) similarity in size and surface components; (ii) ease of generation and noninfectious nature enabling the study of biosafety level 3 and biosafety level 4 viruses; (iii) facile characterization of aerosolization parameters; (iv) ability to adapt the system to other viral envelope proteins, including those of newly discovered pathogens and mutant variants; and (v) the ability to introduce viral sequences to develop nucleic acid amplification assays. IMPORTANCE: The study and detection of airborne pathogens are hampered by the lack of appropriate model systems. In this work, we demonstrate that noninfectious virus-like particles (VLPs) represent attractive models to study airborne viral pathogens. Specifically, VLPs are readily prepared, are similar in size and composition to infectious viruses, and are amenable to highly sensitive nucleic acid amplification techniques.

VLP-Based Model for Study of Airborne Viral Pathogens.

The recent COVID-19 pandemic has underscored the danger of airborne viral pathogens. The lack of model systems to study airborne pathogens limits the understanding of airborne pathogen distribution, as well as potential surveillance and mitigation strategies. In this work, we develop a novel model system to study airborne pathogens using virus like particles (VLP). Specifically, we demonstrate the ability to aerosolize VLP and detect and quantify aerosolized VLP RNA by Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) in real-time fluorescent and colorimetric assays. Importantly, the VLP model presents many advantages for the study of airborne viral pathogens: (i) similarity in size and surface components; (ii) ease of generation and noninfectious nature enabling study of BSL3 and BSL4 viruses; (iii) facile characterization of aerosolization parameters; (iv) ability to adapt the system to other viral envelope proteins including those of newly discovered pathogens and mutant variants; (v) the ability to introduce viral sequences to develop nucleic acid amplification assays.

Thy-1 mRNA destabilization by norepinephrine a 3' UTR cAMP responsive decay element and involves RNA binding proteins.

Thy-1 is a cell surface protein important in immunologic and neurologic processes, including T cell activation and proliferation, and neuronal outgrowth. In murine thymocytes, Thy-1 is downregulated in response to norepinephrine (NE) through posttranscriptional destabilization of its mRNA mediated by ßAR/AC/cAMP/PKA signaling. In this study we investigated factors involved in NE/cAMP-mediated Thy-1 mRNA destabilization in S49 thymoma cells, and identified a region containing two copies of the AUUUA regulatory element (ARE), a motif commonly associated with mRNA decay, in the Thy-1 mRNA 3' UTR. Insertion of the Thy-1 ARE region into a reporter gene, resulted in cAMP induced destabilization of the reporter gene mRNA. RNA-protein binding studies revealed multiple Thy-1 ARE binding proteins, including AUF1, HuR, and TIAR. RNA silencing of HuR enhanced cAMP-mediated downregulation of Thy-1 mRNA, in contrast, silencing AUF1 had no effect. Immunoblotting revealed multiple proteins phosphorylated by PKA as a result of NE or cAMP signaling. These results reveal that the machinery of NE/cAMP modulation of Thy-1 mRNA decay involves a cAMP responsive ARE in its 3' UTR and multiple site specific ARE binding proteins. These findings add to our knowledge of Thy-1 mRNA regulation and provide insight into the regulation of ARE containing mRNAs, which impacts stress-related immunosuppression.