The Role of SARS-CoV Non-structural protein 1 in Regulating RNA stability

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected more than 600 million people across 219 countries during the past three years. SARS-CoV-2 consists of a positive-strand RNA genome that encodes structural and nonstructural proteins and shares a 79% sequence homology with severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1). Nonstructural proteins are necessary for viral replication and suppression of the host cell immune response. Nonstructural protein 1 (nsp1), a small protein conserved among most beta-coronaviruses, inhibits host messenger RNA (mRNA) translation by binding to ribosomal mRNA channels. Nsp1 also triggers degradation of host mRNA while viral RNA remains intact. We have previously shown that nsp1 localizes within stress granules (SGs), non-membranous vesicles of stalled mRNA that form in response to viral infection. We also found that upon induction of stress, SGs disperses within 60- 120 minutes in the presence of nsp1. Since SGs are known to store and protect translationally stalled mRNAs that are target of nsp1, we sought to analyze the level of mRNAs accumulation in SGs in the presence of nsp1. The goal of this project is to identify the impact of nsp1 on stress granule formation during SARS-CoV infection. We used human embryonic kidney cells (HEK293) and transfected them with DNA expressing SARSCoV- 1 nsp1 or a control plasmid. Cells were then incubated at 37degreeC under 5% CO2 concentration for 16 hours. Following incubation, cells were subjected to 30 min of oxidative stress using sodium arenite. Cells were collected and lysed using lysis buffer, then centrifuge at 18 000xg to collect SG pellets used for RNA isolation. Isolated mRNAs were quantified using quantitative RT-PCR. We specifically targeted mRNAs that tend to show a preferential accumulation in SGs without any viral infection. When nsp1 was expressed, we found majority of mRNAs have shown a 2-fold decrease in accumulation in SGs. These results suggest there is a direct effect of nsp1 in dispersing of RNA from SGs. We are currently investigating the effect of viral leader sequence in their accumulation in SGs in the presence of nsp1. This project was supported by the DRP award from SC INBRE (NIGMS, P20GM103499).Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Efficacy Validation of SARS-CoV-2-Inactivation and Viral Genome Stability in Saliva by a Guanidine Hydrochloride and Surfactant-Based Virus Lysis/Transport Buffer.

To enhance biosafety and reliability in SARS-CoV-2 molecular diagnosis, virus lysis/transport buffers should inactivate the virus and preserve viral RNA under various conditions. Herein, we evaluated the SARS-CoV-2-inactivating activity of guanidine hydrochloride (GuHCl)- and surfactant (hexadecyltrimethylammonium chloride (Hexa-DTMC))-based buffer, Prep Buffer A, (Precision System Science Co., Ltd., Matsudo, Japan) and its efficacy in maintaining the stability of viral RNA at different temperatures using the traditional real-time one-step RT-PCR and geneLEAD VIII sample-to-result platform. Although Prep Buffer A successfully inactivated SARS-CoV-2 in solutions with high and low organic substance loading, there was considerable viral genome degradation at 35 °C compared with that at 4 °C. The individual roles of GuHCl and Hexa-DTMC in virus inactivation and virus genome stability at 35 °C were clarified. Hexa-DTMC alone (0.384%), but not 1.5 M GuHCl alone, exhibited considerable virucidal activity, suggesting that it was essential for potently inactivating SARS-CoV-2 using Prep Buffer A. GuHCl and Hexa-DTMC individually reduced the viral copy numbers to the same degree as Prep Buffer A. Although both components inhibited RNase activity, Hexa-DTMC, but not GuHCl, directly destroyed naked viral RNA. Our findings suggest that samples collected in Prep Buffer A should be stored at 4 °C when RT-PCR will not be performed for several days.

Positive SARS-CoV-2 RT-qPCR of a nasal swab spot after 30 days of conservation on filter paper at room temperature.

We tested the use of nasal swabs spotted onto filter paper (Whatman 3M) for the molecular diagnosis of SARS-CoV-2 infection. Spots of a positive nasal swab in conservation medium (B.1.177 strain, 21Ct) were still positive (duo E-gene/IP4) after 10, 20, and 30 days of conservation at room temperature, with Ct values of 28, 27, and 26, respectively. Direct spotting of the swab at bedside (omicron strain) still gave a positive result after 10 days in two RT-qPCR systems: 33.7 Ct using duo E-gene/IP4, and 34.8 using a specific Omicron system. Spotting of a dilution range of media spiked with the Delta (strain 2021/FR/0610, lineage B 1.617.2) and Omicron strains (strain UVE/SARS-CoV-2/2021/FR/1514) showed a threshold of 0.04 TCID50 after 10 days of conservation. We show, for the first time, that this simple and low-cost conservation method can be used to store samples for RT-qPCR against SARS-CoV-2 for up to at least 1 month.

Comparison of SARS-CoV-2 Detection from Saliva Sampling and Oropharyngeal Swab.

We examined the detection rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using reverse transcription-PCR (RT-PCR) of side-by-side saliva and oropharyngeal swab (OPS) samples from 639 symptomatic and asymptomatic subjects, of which 47 subjects were found to be positive for SARS-CoV-2 in the OPS or saliva sample or both. It was found that the detection rate (93.6% for both OPS and saliva) as well as the sensitivity and specificity were comparable between the two sampling methods in this cohort. The sensitivity was 0.932 (95% confidence interval [CI], 0.818 to 0.977) and the specificity was 0.995 (95% CI, 0.985 to 0.998), both for saliva when OPS sampling was used as the reference and for OPS when saliva was used as the reference. Furthermore, the Cohen's kappa value was 0.926 (95% CI, 0.868 to 0.985), indicating strong agreement between the two sampling methods. In addition, the viral RNA stability in pure saliva and saliva mixed with preservation buffers was examined following storage at room temperature and at 4°C. It was found that pure saliva kept the viral RNA stable for 9 days at both temperatures and that the type of preservation buffer can either enhance or reduce the stability of the RNA. We conclude that self-administered saliva sampling is an attractive alternative to oropharyngeal swabbing for SARS-CoV-2 detection, and it might be useful in large-scale testing. IMPORTANCE It is not inconceivable that we will witness recurring surges of COVID-19 before the pandemic finally recedes. It is therefore still relevant to look for feasible, simple, and flexible screening methods so that schools, workplaces, and communities in general can avoid lockdowns. In this work, we analyzed two different sampling methods: oropharyngeal swabs and saliva collection. Oropharyngeal swabs must be collected by trained health personnel at clinics, whereas self-assisted saliva collection can be performed at any given location. It was found that the two sampling methods were comparable. Saliva sampling is a simple method that allows easy mass testing using minimal resources from the existing health care system, and this method may therefore prove to be an effective tool for containing the COVID-19 pandemic.

Pre-analytical sample stabilization by different sampling devices for PCR-based COVID-19 diagnostics.

The outbreak of the SARS-CoV-2 pandemic created an unprecedented requirement for diagnostic testing, challenging not only healthcare workers and laboratories, but also providers. Quantitative RT-PCR of various specimen types is considered the diagnostic gold standard for the detection of SARS-CoV-2, both in terms of sensitivity and specificity. The pre-analytical handling of patient specimens is a critical factor to ensure reliable and valid test results. Therefore, the effect of storage duration and temperature on SARS-CoV-2 RNA copy number stability was examined in various commercially available specimen collection, transport and storage devices for naso/oropharyngeal swabs and saliva. The swab specimen transport and storage devices tested showed no significant alteration of viral RNA copy numbers when stored at room temperature, except for one system when stored for up to 96 h. However, at 37 °C a significant reduction of detectable RNA was found in 3 out of 4 of the swab solutions tested. It was also found that detectability of viral RNA remained unchanged in all 7 saliva devices as well as in unstabilized saliva when stored for 96 h at room temperature, but one device showed marked RNA copy number loss at 37 °C. All tested saliva collection devices inhibited SARS-CoV-2 infectivity immediately, whereas SARS-CoV-2 remained infectious in the swab transport systems examined, which are designed to be used for viral or bacterial growth in cell culture systems.

Direct Regulation of Estrogen Receptor Transcription Activity by NF1

This project centers on the NF1/neurofibromin tumor suppressor, which was best known as a GTPase Activating Protein (GAP)that repress Ras activity. The parent DoD award has successfully defined a new and GAP-independent activity that NF1 is also a transcriptional co-repressor for estrogen receptor (ER) in ER+ breast cancer. While the parent DoD award focused on endocrine therapy resistance caused by NF1 loss, in this Expansion Award, the focus instead is on metastasis, for which currently has no cure. An important feature of ER+ breast cancer metastasis is that greater than 70% of the metastasis is in the bone. We hypothesized that the transcriptional co-repressor role of NF1 is also responsible for driving bone metastasis in ER+ breast cancer. Therefore, the objective of this Expansion Award is to assess NF1s role in metastasis in order to establish a strategy to stop it. We have made progress in accomplish Task1/Aim 1 to fully define NF1-controlled genes that can impact bone metastasis. This was a key part of the data that was just published in the high impact journal Cancer Cell. This award has also supported the launching a Phase-II clinical trial to treat ER+ NF1-depleted breast cancer, and the awards of a SPORE and another DoD Level-2 project. However, in Aim 2 (Tasks 2 and 30) we are dependent on the use of animals to study how NF1-depleted cancer cells interact with the bone, but this line of study has been severely and negatively impacted by COVID-19. We discuss how we plan to overcome this problem in the future.

Detailed Dissection and Critical Evaluation of the Pfizer/BioNTech and Moderna mRNA Vaccines.

The design of Pfizer/BioNTech and Moderna mRNA vaccines involves many different types of optimizations. Proper optimization of vaccine mRNA can reduce dosage required for each injection leading to more efficient immunization programs. The mRNA components of the vaccine need to have a 5'-UTR to load ribosomes efficiently onto the mRNA for translation initiation, optimized codon usage for efficient translation elongation, and optimal stop codon for efficient translation termination. Both 5'-UTR and the downstream 3'-UTR should be optimized for mRNA stability. The replacement of uridine by N1-methylpseudourinine (Ψ) complicates some of these optimization processes because Ψ is more versatile in wobbling than U. Different optimizations can conflict with each other, and compromises would need to be made. I highlight the similarities and differences between Pfizer/BioNTech and Moderna mRNA vaccines and discuss the advantage and disadvantage of each to facilitate future vaccine improvement. In particular, I point out a few optimizations in the design of the two mRNA vaccines that have not been performed properly.

Gargle lavage & saliva: Feasible & cheaper alternatives to nasal & throat swabs for diagnosis of COVID-19.

Background & objectives: In the present scenario, the most common sample for diagnosis of COVID-19 by reverse transcription polymerase chain reaction (RT-PCR) is nasal and throat swab (NTS). Other sampling options such as gargle lavage have found limited application in clinical use mostly because of unavailability of an appropriate gargling liquid. This study was conducted to assess the stability of SARS-CoV-2 RNA in normal saline at 4°C that can serve as a gargling liquid as well as a transport medium. The study also looked at the agreement between NTS and gargle lavage/saliva for the detection of SARS-CoV-2. Methods: In 29 consecutive real-time RT-PCR (rRT-PCR) positive COVID-19 patients, paired NTS, gargle and saliva samples were taken. Samples were processed by rRT-PCR for the detection of SARS-CoV-2 RNA. To assess the SARS-CoV-2 RNA stability in normal saline, gargle lavage specimens were divided into two aliquots; one subset of the specimen was run within 4-6 h along with the routine samples (NTS and saliva) and the other subset was stored at 4°C and processed after 24-30 h. Agreement between cycle threshold (Ct) values from both the runs was compared using Bland-Altman (BA) analysis. Results: The positivity rates of rRT-PCR in NTS, saliva and gargle lavage samples were 82.7 (24/29), 79.3 (23/29) and 86.2 per cent (25/29), respectively. BA plot showed a good agreement between the Ct values of fresh and stored gargle samples, stipulating that there were no significant differences in the approximate viral load levels between the fresh and stored gargle lavage samples (bias: E gene -0.64, N gene -0.51, ORF gene -0.19). Interpretation & conclusions: Our study results show stability of SARS-CoV-2 RNA in the gargle samples collected using normal saline up to 24-30 h. Gargle lavage and saliva specimen collection are cost-effective and acceptable methods of sampling for the detection of SARS-CoV-2 RNA by rRT-PCR. These simplified, inexpensive and acceptable methods of specimen collection would reduce the cost and workload on healthcare workers for sample collection.

Evaluation of Transport Media and Specimen Transport Conditions for the Detection of SARS-CoV-2 by Use of Real-Time Reverse Transcription-PCR.

The global coronavirus (CoV) disease 2019 (COVID-19) pandemic has resulted in a worldwide shortage of viral transport media and raised questions about specimen stability. The objective of this study was to determine the stability of severe acute respiratory syndrome CoV 2 (SARS-CoV-2) RNA in specimen transport media under various storage conditions. Transport media tested included UTM, UTM-RT, ESwab, M4, and saline (0.9% NaCl). Specimen types tested included nasopharyngeal/oropharyngeal swabs in the above-named transport media, bronchoalveolar lavage (BAL) fluid, and sputum. A high-titer SARS-CoV-2 remnant patient specimen was spiked into pooled SARS-CoV-2 RNA-negative specimen remnants for the various medium types. Aliquots of samples were stored at 18°C to 26°C, 2°C to 8°C, and -10°C to -30°C and then tested at time points up to 14 days. Specimens consistently yielded amplifiable RNA with mean cycle threshold differences of <3 over the various conditions assayed, thus supporting the use and transport of alternative collection media and specimen types under a variety of temperature storage conditions.

Postmortem Stability of SARS-CoV-2 in Nasopharyngeal Mucosa.

Analyses of infection chains have demonstrated that severe acute respiratory syndrome coronavirus 2 is highly transmissive. However, data on postmortem stability and infectivity are lacking. Our finding of nasopharyngeal viral RNA stability in 79 corpses showed no time-dependent decrease. Maintained infectivity is supported by virus isolation up to 35 hours postmortem.

The interface between coronaviruses and host cell RNA biology: Novel potential insights for future therapeutic intervention.

Coronaviruses, including SARS-Cov-2, are RNA-based pathogens that interface with a large variety of RNA-related cellular processes during infection. These processes include capping, polyadenylation, localization, RNA stability, translation, and regulation by RNA binding proteins or noncoding RNA effectors. The goal of this article is to provide an in-depth perspective on the current state of knowledge of how various coronaviruses interact with, usurp, and/or avoid aspects of these cellular RNA biology machineries. A thorough understanding of how coronaviruses interact with RNA-related posttranscriptional processes in the cell should allow for new insights into aspects of viral pathogenesis as well as identify new potential avenues for the development of anti-coronaviral therapeutics. This article is categorized under: RNA in Disease and Development > RNA in Disease.

Stability of RNA sequences derived from the coronavirus genome in human cells.

Most viruses inhibit the innate immune system and/or the RNA degradation processes of host cells to construct an advantageous intracellular environment for their survival. Characteristic RNA sequences within RNA virus genomes or RNAs transcribed from DNA virus genomes contribute toward this inhibition. In this study, we developed a method called "Fate-seq" to comprehensively identify the RNA sequences derived from RNA and DNA viruses, contributing RNA stability in the cells. We examined the stabilization activity of 5,924 RNA fragments derived from 26 different viruses (16 RNA viruses and 10 DNA viruses) using next-generation sequencing of these RNAs fused 3' downstream of GFP reporter RNA. With the Fate-seq approach, we detected multiple virus-derived RNA sequences that stabilized GFP reporter RNA, including sequences derived from severe acute respiratory syndrome-related coronavirus (SARS-CoV). Comparative genomic analysis revealed that these RNA sequences and their predicted secondary structures are highly conserved between SARS-CoV and the novel coronavirus, SARS-CoV-2, which is responsible for the global outbreak of the coronavirus-associated disease that emerged in December 2019 (COVID-19). These sequences have the potential to enhance the stability of viral RNA genomes, thereby augmenting viral replication efficiency and virulence.