The Network of Interactions between the Porcine Epidemic Diarrhea Virus Nucleocapsid and Host Cellular Proteins.

Host-virus protein interactions are critical for intracellular viral propagation. Understanding the interactions between cellular and viral proteins may help us develop new antiviral strategies. Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that causes severe damage to the global swine industry. Here, we employed co-immunoprecipitation and liquid chromatography-mass spectrometry to characterize 426 unique PEDV nucleocapsid (N) protein-binding proteins in infected Vero cells. A protein-protein interaction network (PPI) was created, and gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) database analyses revealed that the PEDV N-bound proteins belong to different cellular pathways, such as nucleic acid binding, ribonucleoprotein complex binding, RNA methyltransferase, and polymerase activities. Interactions of the PEDV N protein with 11 putative proteins: tripartite motif containing 21, DEAD-box RNA helicase 24, G3BP stress granule assembly factor 1, heat shock protein family A member 8, heat shock protein 90 alpha family class B member 1, YTH domain containing 1, nucleolin, Y-box binding protein 1, vimentin, heterogeneous nuclear ribonucleoprotein A2/B1, and karyopherin subunit alpha 1, were further confirmed by in vitro co-immunoprecipitation assay. In summary, studying an interaction network can facilitate the identification of antiviral therapeutic strategies and novel targets for PEDV infection.

Immunomodulatory roles of red blood cells.

PURPOSE OF THE REVIEW: To discuss recent advances supporting the role of red blood cells (RBCs) in the host immune response. RECENT FINDINGS: Over the last century, research has demonstrated that red blood cells exhibit functions beyond oxygen transport, including immune function. Recent work indicates that the nucleic acid sensing receptor, toll-like receptor 9 (TLR9), is expressed on the RBC surface and implicated in innate immune activation and red cell clearance during inflammatory states. In addition to this DNA-sensing role of RBCs, there is growing evidence that RBCs may influence immune function by inducing vascular dysfunction. RBC proteomics and metabolomics have provided additional insight into RBC immune function, with several studies indicating changes to RBC membrane structure and metabolism in response to severe acute respiratory syndrome coronavirus 2 infection. These structural RBC changes may even provide insight into the pathophysiology of the 'long-coronavirus disease 2019' phenomenon. Finally, evidence suggests that RBCs may influence host immune responses via complement regulation. Taken together, these recent findings indicate RBCs possess immune function. Further studies will be required to elucidate better how RBC immune function contributes to the heterogeneous host response during inflammatory states. SUMMARY: The appreciation for nongas exchanging, red blood cell immune functions is rapidly growing. A better understanding of these RBC functions may provide insight into the heterogeneity observed in the host immune response to infection and inflammation.

Kinetic Characterization of SARS-CoV-2 nsp13 ATPase Activity and Discovery of Small-Molecule Inhibitors.

SARS-CoV-2 non-structural protein 13 (nsp13) is a highly conserved helicase and RNA 5'-triphosphatase. It uses the energy derived from the hydrolysis of nucleoside triphosphates for directional movement along the nucleic acids and promotes the unwinding of double-stranded nucleic acids. Nsp13 is essential for replication and propagation of all human and non-human coronaviruses. Combined with its defined nucleotide binding site and druggability, nsp13 is one of the most promising candidates for the development of pan-coronavirus therapeutics. Here, we report the development and optimization of bioluminescence assays for kinetic characterization of nsp13 ATPase activity in the presence and absence of single-stranded DNA. Screening of a library of 5000 small molecules in the presence of single-stranded DNA resulted in the discovery of six nsp13 small-molecule inhibitors with IC50 values ranging from 6 ± 0.5 to 50 ± 6 µM. In addition to providing validated methods for high-throughput screening of nsp13 in drug discovery campaigns, the reproducible screening hits we present here could potentially be chemistry starting points toward the development of more potent and selective nsp13 inhibitors, enabling the discovery of antiviral therapeutics.

Extracellular Vesicles: New Tools for Early Diagnosis of Breast and Genitourinary Cancers.

Breast cancers and cancers of the genitourinary tract are the most common malignancies among men and women and are still characterized by high mortality rates. In order to improve the outcomes, early diagnosis is crucial, ideally by applying non-invasive and specific biomarkers. A key role in this field is played by extracellular vesicles (EVs), lipid bilayer-delimited structures shed from the surface of almost all cell types, including cancer cells. Subcellular structures contained in EVs such as nucleic acids, proteins, and lipids can be isolated and exploited as biomarkers, since they directly stem from parental cells. Furthermore, it is becoming even more evident that different body fluids can also serve as sources of EVs for diagnostic purposes. In this review, EV isolation and characterization methods are described. Moreover, the potential contribution of EV cargo for diagnostic discovery purposes is described for each tumor.

Insights into the specificity for the interaction of the promiscuous SARS-CoV-2 nucleocapsid protein N-terminal domain with deoxyribonucleic acids.

The SARS-CoV-2 nucleocapsid protein (N) is a multifunctional promiscuous nucleic acid-binding protein, which plays a major role in nucleocapsid assembly and discontinuous RNA transcription, facilitating the template switch of transcriptional regulatory sequences (TRS). Here, we dissect the structural features of the N protein N-terminal domain (N-NTD) and N-NTD plus the SR-rich motif (N-NTD-SR) upon binding to single and double-stranded TRS DNA, as well as their activities for dsTRS melting and TRS-induced liquid-liquid phase separation (LLPS). Our study gives insights on the specificity for N-NTD(-SR) interaction with TRS. We observed an approximation of the triple-thymidine (TTT) motif of the TRS to ß-sheet II, giving rise to an orientation difference of ~25° between dsTRS and non-specific sequence (dsNS). It led to a local unfavorable energetic contribution that might trigger the melting activity. The thermodynamic parameters of binding of ssTRSs and dsTRS suggested that the duplex dissociation of the dsTRS in the binding cleft is entropically favorable. We showed a preference for TRS in the formation of liquid condensates when compared to NS. Moreover, our results on DNA binding may serve as a starting point for the design of inhibitors, including aptamers, against N, a possible therapeutic target essential for the virus infectivity.

Clinical predictive risk factors prolonged the duration of SARS-CoV-2 clearance in 279 moderate COVID-19 patients: A multicenter retrospective cohort study.

ABSTRACT: The results of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid as one of the criteria has been widely applied to assess whether the coronavirus disease 2019 (COVID-19) patients could discharge, however, the risk factors that affect the duration of the SARS-CoV-2 clearance remained to be an enigma. Our research was to identify risk factors correlated with prolonged duration of the SARS-CoV-2 clearance in moderate COVID-19 patients.We retrospectively analyzed 279 consecutive ordinary COVID-19 patients in 3 hospitals in Hubei province including Huangshi Hospital of Infectious Disease, Wuhan Thunder God Mountain Hospital, and Tongji Hospital. Eight clinical characters were contained as risk factors. We used a logistic regression model and nomogram to assess the possibility that the SARS-CoV-2 nucleic acid may turn negative in 14 days.Time from symptoms onset to diagnosis (odds ratio [OR] = 3.18; 95% confidence interval [CI] 1.56-6.46; P = .001), time from onset use of antiviral drugs to onset of symptoms (OR = 0.41; 95% CI 0.23-0.72; P = .02), and bacterial coinfection (OR = 0.07; 95% CI 0.01-0.86; P = .038) were independent risks factors for the duration of SARS-CoV-2 nucleic acid clearance. The regression model showed good accuracy and sensitivity (area under the curve  = 0.96). Nomogram was also provided to predict the negative conversion rate of SARS-CoV-2 nucleic acids within 14 days.Time from symptoms onset to diagnosi, time from onset use of antiviral drugs to onset of symptoms, and bacterial coinfection were independent risk factors for the time of SARS-CoV-2 nucleic acid turning negative in ordinary COVID-19 patients. However, the age, gender, underlying disease, fungal coinfection, and duration use of antiviral drugs were irrelevant factors.

The method utilized to purify the SARS-CoV-2 N protein can affect its molecular properties.

One of the main structural proteins of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the nucleocapsid protein (N). The basic function of this protein is to bind genomic RNA and to form a protective nucleocapsid in the mature virion. The intrinsic ability of the N protein to interact with nucleic acids makes its purification very challenging. Therefore, typically employed purification methods appear to be insufficient for removing nucleic acid contamination. In this study, we present a novel purification protocol that enables the N protein to be prepared without any bound nucleic acids. We also performed comparative structural analysis of the N protein contaminated with nucleic acids and free of contamination and showed significant differences in the structural and phase separation properties of the protein. These results indicate that nucleic-acid contamination may severely affect molecular properties of the purified N protein. In addition, the notable ability of the N protein to form condensates whose morphology and behaviour suggest more ordered forms resembling gel-like or solid structures is described.

CRISPR-based diagnostics.

The accurate and timely diagnosis of disease is a prerequisite for efficient therapeutic intervention and epidemiological surveillance. Diagnostics based on the detection of nucleic acids are among the most sensitive and specific, yet most such assays require costly equipment and trained personnel. Recent developments in diagnostic technologies, in particular those leveraging clustered regularly interspaced short palindromic repeats (CRISPR), aim to enable accurate testing at home, at the point of care and in the field. In this Review, we provide a rundown of the rapidly expanding toolbox for CRISPR-based diagnostics, in particular the various assays, preamplification strategies and readouts, and highlight their main applications in the sensing of a wide range of molecular targets relevant to human health.

HISNAPI: a bioinformatic tool for dynamic hot spot analysis in nucleic acid-protein interface with a case study.

Protein-nucleic acid interactions play essential roles in many biological processes, such as transcription, replication and translation. In protein-nucleic acid interfaces, hotspot residues contribute the majority of binding affinity toward molecular recognition. Hotspot residues are commonly regarded as potential binding sites for compound molecules in drug design projects. The dynamic property is a considerable factor that affects the binding of ligands. Computational approaches have been developed to expedite the prediction of hotspot residues on protein-nucleic acid interfaces. However, existing approaches overlook hotspot dynamics, despite their essential role in protein function. Here, we report a web server named Hotspots In silico Scanning on Nucleic Acid and Protein Interface (HISNAPI) to analyze hotspot residue dynamics by integrating molecular dynamics simulation and one-step free energy perturbation. HISNAPI is capable of not only predicting the hotspot residues in protein-nucleic acid interfaces but also providing insights into their intensity and correlation of dynamic motion. Protein dynamics have been recognized as a vital factor that has an effect on the interaction specificity and affinity of the binding partners. We applied HISNAPI to the case of SARS-CoV-2 RNA-dependent RNA polymerase, a vital target of the antiviral drug for the treatment of coronavirus disease 2019. We identified the hotspot residues and characterized their dynamic behaviors, which might provide insight into the target site for antiviral drug design. The web server is freely available via a user-friendly web interface at http://chemyang.ccnu.edu.cn/ccb/server/HISNAPI/ and http://agroda.gzu.edu.cn:9999/ccb/server/HISNAPI/.

1H, 13C, and 15N backbone chemical shift assignments of the nucleic acid-binding domain of SARS-CoV-2 non-structural protein 3e.

The ongoing pandemic caused by the Betacoronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2) demonstrates the urgent need of coordinated and rapid research towards inhibitors of the COVID-19 lung disease. The covid19-nmr consortium seeks to support drug development by providing publicly accessible NMR data on the viral RNA elements and proteins. The SARS-CoV-2 genome encodes for approximately 30 proteins, among them are the 16 so-called non-structural proteins (Nsps) of the replication/transcription complex. The 217-kDa large Nsp3 spans one polypeptide chain, but comprises multiple independent, yet functionally related domains including the viral papain-like protease. The Nsp3e sub-moiety contains a putative nucleic acid-binding domain (NAB) with so far unknown function and consensus target sequences, which are conceived to be both viral and host RNAs and DNAs, as well as protein-protein interactions. Its NMR-suitable size renders it an attractive object to study, both for understanding the SARS-CoV-2 architecture and drugability besides the classical virus' proteases. We here report the near-complete NMR backbone chemical shifts of the putative Nsp3e NAB that reveal the secondary structure and compactness of the domain, and provide a basis for NMR-based investigations towards understanding and interfering with RNA- and small-molecule-binding by Nsp3e.