Self-assembling short immunostimulatory duplex RNAs with broad-spectrum antiviral activity.

The current coronavirus disease 2019 (COVID-19) pandemic highlights the need for broad-spectrum antiviral therapeutics. Here we describe a new class of self-assembling immunostimulatory short duplex RNAs that potently induce production of type I and type III interferon (IFN-I and IFN-III). These RNAs require a minimum of 20 base pairs, lack any sequence or structural characteristics of known immunostimulatory RNAs, and instead require a unique sequence motif (sense strand, 5'-C; antisense strand, 3'-GGG) that mediates end-to-end dimer self-assembly. The presence of terminal hydroxyl or monophosphate groups, blunt or overhanging ends, or terminal RNA or DNA bases did not affect their ability to induce IFN. Unlike previously described immunostimulatory small interfering RNAs (siRNAs), their activity is independent of Toll-like receptor (TLR) 7/8, but requires the RIG-I/IRF3 pathway that induces a more restricted antiviral response with a lower proinflammatory signature compared with immunostimulant poly(I:C). Immune stimulation mediated by these duplex RNAs results in broad-spectrum inhibition of infections by many respiratory viruses with pandemic potential, including severe acute respiratory syndrome coronavirus (SARS-CoV)-2, SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus (HCoV)-NL63, and influenza A virus in cell lines, human lung chips that mimic organ-level lung pathophysiology, and a mouse SARS-CoV-2 infection model. These short double-stranded RNAs (dsRNAs) can be manufactured easily, and thus potentially could be harnessed to produce broad-spectrum antiviral therapeutics.

A human-airway-on-a-chip for the rapid identification of candidate antiviral therapeutics and prophylactics.

The rapid repurposing of antivirals is particularly pressing during pandemics. However, rapid assays for assessing candidate drugs typically involve in vitro screens and cell lines that do not recapitulate human physiology at the tissue and organ levels. Here we show that a microfluidic bronchial-airway-on-a-chip lined by highly differentiated human bronchial-airway epithelium and pulmonary endothelium can model viral infection, strain-dependent virulence, cytokine production and the recruitment of circulating immune cells. In airway chips infected with influenza A, the co-administration of nafamostat with oseltamivir doubled the treatment-time window for oseltamivir. In chips infected with pseudotyped severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), clinically relevant doses of the antimalarial drug amodiaquine inhibited infection but clinical doses of hydroxychloroquine and other antiviral drugs that inhibit the entry of pseudotyped SARS-CoV-2 in cell lines under static conditions did not. We also show that amodiaquine showed substantial prophylactic and therapeutic activities in hamsters challenged with native SARS-CoV-2. The human airway-on-a-chip may accelerate the identification of therapeutics and prophylactics with repurposing potential.

Temporal proteomic changes induced by nicotine in human cells: A quantitative proteomics approach.

Nicotine is a prominent active compound in tobacco and many smoking cessation products. Some of the biological effects of nicotine are well documented in in vitro and in vivo systems; however, data are scarce concerning the time-dependent changes on protein and phosphorylation events in response to nicotine. Here, we profiled the proteomes of SH-SY5Y and A549 cell lines subjected to acute (15 min, 1 h and 4 h) or chronic (24 h, 48 h) nicotine exposures. We used sample multiplexing (TMTpro16) and quantified more than 9000 proteins and over 7000 phosphorylation events per cell line. Among our findings, we determined a decrease in mitochondrial protein abundance for SH-SY5Y, while we detected alterations in several immune pathways, such as the complement system, for A549 following nicotine treatment. We also explored the proposed association between smoking (specifically nicotine) and SARS-CoV2. Here, we found several host proteins known to interact with viral proteins that were affected by nicotine in a time dependent manner. This dataset can be mined further to investigate the potential role of nicotine in different biological contexts. SIGNIFICANCE: Smoking is a major public health issue that is associated with several serious chronic, yet preventable diseases, including stroke, heart disease, type 2 diabetes, cancer, and susceptibility to infection. Tobacco smoke is a complex mixture of thousands of different compounds, among which nicotine is the main addictive compound. The biological effects of nicotine have been reported in several models, however very little data are available concerning the temporal proteomic and phosphoproteomic changes in response to nicotine. Here, we provide a dataset exploring the potential role of nicotine on different biological processes over time, including implications in the study of SARS-CoV2.

ORF10-Cullin-2-ZYG11B complex is not required for SARS-CoV-2 infection.

In order to understand the transmission and virulence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is necessary to understand the functions of each of the gene products encoded in the viral genome. One feature of the SARS-CoV-2 genome that is not present in related, common coronaviruses is ORF10, a putative 38-amino acid protein-coding gene. Proteomic studies found that ORF10 binds to an E3 ubiquitin ligase containing Cullin-2, Rbx1, Elongin B, Elongin C, and ZYG11B (CRL2ZYG11B). Since CRL2ZYG11B mediates protein degradation, one possible role for ORF10 is to "hijack" CRL2ZYG11B in order to target cellular, antiviral proteins for ubiquitylation and subsequent proteasomal degradation. Here, we investigated whether ORF10 hijacks CRL2ZYG11B or functions in other ways, for example, as an inhibitor or substrate of CRL2ZYG11B While we confirm the ORF10-ZYG11B interaction and show that the N terminus of ORF10 is critical for it, we find no evidence that ORF10 is functioning to inhibit or hijack CRL2ZYG11B Furthermore, ZYG11B and its paralog ZER1 are dispensable for SARS-CoV-2 infection in cultured cells. We conclude that the interaction between ORF10 and CRL2ZYG11B is not relevant for SARS-CoV-2 infection in vitro.