Neutral sphingomyelinase 2 is required for HIV-1 maturation.

HIV-1 assembly occurs at the inner leaflet of the plasma membrane (PM) in highly ordered membrane microdomains. The size and stability of membrane microdomains is regulated by activity of the sphingomyelin hydrolase neutral sphingomyelinase 2 (nSMase2) that is localized primarily to the inner leaflet of the PM. In this study, we demonstrate that pharmacological inhibition or depletion of nSMase2 in HIV-1-producer cells results in a block in the processing of the major viral structural polyprotein Gag and the production of morphologically aberrant, immature HIV-1 particles with severely impaired infectivity. We find that disruption of nSMase2 also severely inhibits the maturation and infectivity of other primate lentiviruses HIV-2 and simian immunodeficiency virus, has a modest or no effect on nonprimate lentiviruses equine infectious anemia virus and feline immunodeficiency virus, and has no effect on the gammaretrovirus murine leukemia virus. These studies demonstrate a key role for nSMase2 in HIV-1 particle morphogenesis and maturation.

Inhibition of SARS-CoV-2 infection in human airway epithelium with a xeno-nucleic acid aptamer.

BACKGROUND: SARS-CoV-2, the agent responsible for the COVID-19 pandemic, enters cells through viral spike glycoprotein binding to the cellular receptor, angiotensin-converting enzyme 2 (ACE2). Given the lack of effective antivirals targeting SARS-CoV-2, we previously utilized systematic evolution of ligands by exponential enrichment (SELEX) and selected fluoro-arabino nucleic acid (FANA) aptamer R8-9 that was able to block the interaction between the viral receptor-binding domain and ACE2. METHODS: Here, we further assessed FANA-R8-9 as an entry inhibitor in contexts that recapitulate infection in vivo. RESULTS: We demonstrate that FANA-R8-9 inhibits spike-bearing pseudovirus particle uptake in cell lines. Then, using an in-vitro model of human airway epithelium (HAE) and SARS-CoV-2 virus, we show that FANA-R8-9 significantly reduces viral infection when added either at the time of inoculation, or several hours later. These results were specific to the R8-9 sequence, not the xeno-nucleic acid utilized to make the aptamer. Importantly, we also show that FANA-R8-9 is stable in HAE culture secretions and has no overt cytotoxic effects. CONCLUSIONS: Together, these results suggest that FANA-R8-9 effectively prevents infection by specific SARS-CoV-2 variants and indicate that aptamer technology could be utilized to target other clinically-relevant viruses in the respiratory mucosa.

Inhibition of SARS-CoV-2 Infection in Human Airway Epithelium with a Xeno-Nucleic Acid Aptamer.

Background: SARS-CoV-2, the agent responsible for the COVID-19 pandemic, enters cells through viral spike glycoprotein binding to the cellular receptor, angiotensin-converting enzyme 2 (ACE2). Given the lack of effective antivirals targeting SARS-CoV-2, we previously utilized systematic evolution of ligands by exponential enrichment (SELEX) and selected fluoro-arabino nucleic acid (FANA) aptamer R8-9 that was able to block the interaction between the viral receptor-binding domain and ACE2. Methods: Here, we further assessed FANA-R8-9 as an entry inhibitor in contexts that recapitulate infection in vivo. Results: We demonstrate that FANA-R8-9 inhibits spike-bearing pseudovirus particle uptake in cell lines. Then, using an in-vitro model of human airway epithelium (HAE) and SARS-CoV-2 virus, we show that FANA-R8-9 significantly reduces viral infection when added either at the time of inoculation, or several hours later. These results were specific to the R8-9 sequence, not the xeno-nucleic acid utilized to make the aptamer. Importantly, we also show that FANA-R8-9 is stable in HAE culture secretions and has no overt cytotoxic effects. Conclusions: Together, these results suggest that FANA-R8-9 effectively prevents infection by specific SARS-CoV-2 variants and indicate that aptamer technology could be utilized to target other clinically-relevant viruses in the respiratory mucosa.

Leveraging 3D Model Systems to Understand Viral Interactions with the Respiratory Mucosa.

Respiratory viruses remain a significant cause of morbidity and mortality in the human population, underscoring the importance of ongoing basic research into virus-host interactions. However, many critical aspects of infection are difficult, if not impossible, to probe using standard cell lines, 2D culture formats, or even animal models. In vitro systems such as airway epithelial cultures at air-liquid interface, organoids, or 'on-chip' technologies allow interrogation in human cells and recapitulate emergent properties of the airway epithelium-the primary target for respiratory virus infection. While some of these models have been used for over thirty years, ongoing advancements in both culture techniques and analytical tools continue to provide new opportunities to investigate airway epithelial biology and viral infection phenotypes in both normal and diseased host backgrounds. Here we review these models and their application to studying respiratory viruses. Furthermore, given the ability of these systems to recapitulate the extracellular microenvironment, we evaluate their potential to serve as a platform for studies specifically addressing viral interactions at the mucosal surface and detail techniques that can be employed to expand our understanding.