The conserved single-cleavage mechanism of animal DROSHA enzymes

RNase III enzymes typically cleave both strands of double-stranded RNAs (dsRNAs). We recently discovered that a human RNase III, DROSHA, exhibits a single cleavage on the one strand of primary microRNAs (pri-miRNAs). This study revealed that DROSHAs from the other animals, including worms and flies, also show the single cleavage on dsRNAs. Furthermore, we demonstrated that the mechanism of single cleavage is conserved in animal DROSHA enzymes. In addition, the dsRNA-binding domain (dsRBD) and a 3p-strand cleavage-supporting helix (3pCSH) of the DROSHA enzymes foster a weak single cleavage on one strand, which ensures their double cleavages. Disrupting the interaction of dsRBD-RNA and 3pCSH-RNA by an internal loop (IL) and a 3pCSH-loop in the lower stem of pri-miRNAs, respectively, inhibits one of the double cleavages of DROSHAs, and this results in the single cleavage. Our findings expand our understanding of the enzymatic mechanisms of animal DROSHAs. They also indicate that there are currently unknown cellular functions of DROSHA enzymes using their single cleavage activity.

Ribonuclease zymogen induces cytotoxicity upon HIV-1 infection

Background Targeting RNA is a promising yet underdeveloped modality for the selective killing of cells infected with HIV-1. The secretory ribonucleases (RNases) found in vertebrates have cytotoxic ribonucleolytic activity that is kept in check by a cytosolic ribonuclease inhibitor protein, RI. Methods We engineered amino acid substitutions that enable human RNase 1 to evade RI upon its cyclization into a zymogen that is activated by the HIV-1 protease. In effect, the zymogen has an HIV-1 protease cleavage site between the termini of the wild-type enzyme, thereby positioning a cleavable linker over the active site that blocks access to a substrate. Results The amino acid substitutions in RNase 1 diminish its affinity for RI by 106-fold and confer high toxicity for T-cell leukemia cells. Pretreating these cells with the zymogen leads to a substantial drop in their viability upon HIV-1 infection, indicating specific toxicity toward infected cells. Conclusions These data demonstrate the utility of ribonuclease zymogens as biologic prodrugs.

Cordycepin and its nucleoside analogs for the treatment of systemic COVID-19 infection

: Coronavirus disease (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a new coronavirus isolated from Wuhan, China. It is a global health emergency, and there is no effective antiviral therapeutics available to date. Continuous structural genomic insights of SARS-CoV-2 proteins provide a warranty for the development of rational-based antivirals. Nevertheless, a structure-based drug candidate with multiple therapeutic actions would be a practical choice of medication in the treatment of severe COVID-19 patients. Cordycepin from medicinal fungi (Cordyceps spp.) and its nucleoside analogs targeting viral RNA-dependent RNA polymerase and human RNase L have potent antiviral activity against various human viruses with additional immunomodulatory and anti-inflammatory effects. Anti-inflammation treatment is of pivotal importance and should be timely tailored to the individual patient along with antivirals. Our perspective on the combined antiviral and anti-inflammatory effects of cordycepin and its analogs suggests them as new therapeutics in the treatment of systemic COVID-19 infection.

Processing Hundreds of SARS-CoV-2 Samples with an In-House PCR-Based Method without Robotics

The SARS-CoV-2 pandemic has required the development of multiple testing systems to monitor and control the viral infection. Here, we developed a PCR test to screen COVID-19 infections that can process up to ~180 samples per day without the requirement of robotics. For this purpose, we implemented the use of multichannel pipettes and plate magnetics for the RNA extraction step and combined the reverse transcription with the qPCR within one step. We tested the performance of two RT-qPCR kits as well as different sampling buffers and showed that samples taken in NaCl or PBS are stable and compatible with different COVID-19 testing systems. Finally, we designed a new internal control based on the human RNase P gene that does not require a DNA digestion step. Our protocol is easy to handle and reaches the sensitivity and accuracy of the standardized diagnostic protocols used in the clinic to detect COVID-19 infections.

Fomites and the environment did not have an important role in COVID-19 transmission in a Brazilian mid-sized city

It is not clear if COVID-19 can be indirectly transmitted. It is not possible to conclude the role of the environment in transmission of SARS-CoV-2 without studying areas in which people transit in great numbers. In this work we aimed to better understand the role of environment in the spread of COVID-19. We investigated the presence of SARS-CoV-2 in fomites as well as in the air and in the sewage using RT-qPCR. We studied both, a reference market area and a COVID-19 reference hospital at Barreiras city, Brazil. We collected and analyzed a total of 418 samples from mask fronts, cell phones, paper money, card machines, sewage, air and bedding during the ascendant phase of the epidemiological curve of COVID-19 in Barreiras. As a result, we detected the human RNAse P gene in most of samples, which indicates the presence of human cells or their fragments in specimens. However, we did not detect any trace of SARS-CoV-2 in all samples analyzed. We conclude that, so far, the environment and inanimate materials did not have an important role in COVID-19 transmission in Barreiras city. Therefore, similar results can probably be found in other cities, mainly those with COVID-19 epidemiological scenarios similar to that of Barreiras city. Our study is a small piece indicating the possibility that fomites and the environment do not have an important role in COVID-19 transmission. However, further studies are necessary to better understand the world scenario.

Catalytically inactive, purified RNase H1: A specific and sensitive probe for RNA–DNA hybrid imaging

R-loops are three-stranded nucleic acid structures with both physiological and pathological roles in cells. R-loop imaging generally relies on detection of the RNA–DNA hybrid component of these structures using the S9.6 antibody. We show that the use of this antibody for imaging can be problematic because it readily binds to double-stranded RNA (dsRNA) in vitro and in vivo, giving rise to nonspecific signal. In contrast, purified, catalytically inactive human RNase H1 tagged with GFP (GFP-dRNH1) is a more specific reagent for imaging RNA–DNA hybrids. GFP-dRNH1 binds strongly to RNA–DNA hybrids but not to dsRNA oligonucleotides in fixed human cells and is not susceptible to binding endogenous RNA. Furthermore, we demonstrate that purified GFP-dRNH1 can be applied to fixed cells to detect hybrids after their induction, thereby bypassing the need for cell line engineering. GFP-dRNH1 therefore promises to be a versatile tool for imaging and quantifying RNA–DNA hybrids under a wide range of conditions.

Buccal swabs as non-invasive specimens for detection of severe acute respiratory syndrome coronavirus-2

Introduction The current gold standard for detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RNA involves subjecting nasopharyngeal or oropharyngeal swabs to reverse transcription quantitative PCR (RT-qPCR). However, both sample types need to be collected by trained professionals. Using self-collected buccal swabs as an alternative could simplify and accelerate diagnosis of coronavirus disease 2019 (COVID-19). Objective To assess self-collected buccal swab samples as an alternative method for SARS-CoV-2 detection in patients with COVID-19. Methods Buccal swab samples were self-collected by 73 patients with COVID-19. Total RNA was extracted using Qiagen kits. RNA encoding the SARS-CoV-2 Env protein and human RNase P as an internal control was amplified using the TRUPCR® SARS-CoV-2 RT-qPCR kit version 2.1 and a Bio-Rad CFX96 Real-Time Detection System. Result The sensitivity of RT-qPCR from buccal swabs was 58.9% (43/73; 95% confidence interval [CI] 46.77%–70.27%) and that of RT-qPCR from saliva was 62.90% (39/62; 95% CI 49.69%–74.84%) taking positive SARS-CoV-2 RT-qPCR from nasopharyngeal swabs as the gold standard. Conclusion Self-collected buccal swabs are promising alternatives to nasopharyngeal or oropharyngeal swabs for SARS CoV-2 detection.

Inanimate Objects and the Environment Are Far From Contributing to COVID-19 Spread

It is not clear if COVID-19 can be indirectly transmitted. It is not possible to conclude the role of environment in transmission of SARS-CoV-2 without studying areas in which people transit in great amounts, such as market areas. In this work we aimed to better understand the role of environment in the spread of COVID-19. We investigated the presence of SARS-CoV-2 in inanimate objects as well as in the air and in the sewage using RT-qPCR. We studied both, a reference market area and a COVID-19 reference hospital at Barreiras city, Brazil. We collected and analyzed a total of 268 samples from mask fronts, cell phones, paper moneys, card machines, sewage, air and bedding during the ascendant phase of the epidemiological curve of COVID-19 in Barreiras. As a result, we detected the human RNAse P gene in most of samples, which indicates the presence of human cells in specimens. However, we did not detect any trace of SARS-CoV-2 in all samples analyzed. To rule out the possibility of problems in sampling method we tested detection of SARS-CoV-2 by RT-qPCR in laboratory conditions to reproduce environmental temperature and humidity. As a result, we showed detection of the virus in different conditions. We conclude that our sampling method reliable and that, strikingly, the environment and inanimate materials do not have an important role in COVID-19 transmission.

Expression of human ACE2 N-terminal domain, part of the receptor for SARS-CoV-2, in fusion with maltose binding protein, E. coli ribonuclease I and human RNase A

The SARS-CoV-2 viral genome contains a positive-strand single-stranded RNA of ~30 kb. Human ACE2 protein is the receptor for SARS-CoV-2 virus attachment and initiation of infection. We propose to use ribonucleases (RNases) as antiviral agents to destroy the viral genome in vitro. In the virions the RNA is protected by viral capsid proteins, membrane proteins and nucleocapsid proteins. To overcome this protection we set out to construct RNase fusion with human ACE2 receptor N-terminal domain (ACE2NTD). We constructed six proteins expressed in E. coli cells: 1) MBP-ACE2NTD, 2) ACE2NTD-GFP, 3) RNase I (6xHis), 4) RNase III (6xHis), 5) RNase I-ACE2NTD (6xHis), and 6) human RNase A-ACE2NTD150 (6xHis). We evaluated fusion expression in different E. coli strains, partially purified MBP-ACE2NTD protein from the soluble fraction of bacterial cell lysate, and refolded MBP-ACE2NTD protein from inclusion body. The engineered RNase I-ACE2NTD (6xHis) and hRNase A-ACE2NTD (6xHis) fusions are active in cleaving COVID-19 RNA in vitro. The recombinant RNase I (6xHis) and RNase III (6xHis) are active in cleaving RNA and dsRNA in test tube. This study provides a proof-of-concept for construction of fusion protein between human cell receptor and nuclease that may be used to degrade viral nucleic acids in our environment.Graphical AbstractCartoon illustration part of this work (Human ACE2 N-terminal domain tethered to RNase A and RNA degradation by the fusion enzyme).