Ring around the Ro-sie: RNA-mediated alterations of PNPase activity.

Chen et al. demonstrate a new way by which noncoding RNAs tailor the function of multicomponent complexes. They show that a noncoding RNA interacts with an exoribonuclease, altering its substrate specificity and enzymatic activity by serving as a ribonucleoprotein scaffold and, perhaps, a gate for entry of the RNA substrate.

Multiplexed Capillary-Flow Driven Immunoassay for Respiratory Illnesses.

Multiplexed analysis in medical diagnostics is widely accepted as a more thorough and complete method compared to single-analyte detection. While analytical methods like polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) exist for multiplexed detection of biomarkers, they remain time-consuming and expensive. Lateral flow assays (LFAs) are an attractive option for point-of-care testing, and examples of multiplexed LFAs exist. However, these devices are limited by spatial resolution of test lines, large sample volume requirements, cross-reactivity, and poor sensitivity. Recent work has developed capillary-flow microfluidic ELISA platforms as a more sensitive alternative to LFAs; however, multiplexed detection on these types of devices has yet to be demonstrated. In the aftermath of the initial SARS-CoV-2 pandemic, the need for rapid, sensitive point-of-care devices has become ever clearer. Moving forward, devices that can distinguish between diseases with similar presenting symptoms would be the ideal home diagnostic. Here, the first example of a multiplexed capillary-flow immunoassay device for the simultaneous detection of multiple biomarkers is reported. From a single sample addition step, the reagents and washing steps required for two simultaneous ELISAs are delivered to spatially separated test strips. Visual results can be obtained in <15 min, and images captured with a smartphone can be analyzed for quantitative data. This device was used to distinguish between and quantify H1N1 hemagglutinin (HA) and SARS-CoV-2 nucleocapsid protein (N-protein). Using this device, analytical detection limits of 840 and 133 pg/mL were obtained for hemagglutinin and nucleocapsid protein, respectively. The presence of one target in the device did not increase the signal on the other test line, indicating no cross-reactivity between the assays. Additionally, simultaneous detection of both N-protein and HA was performed as well as simultaneous detection of N-protein and human C-reactive protein (CRP). Elevated levels of CRP in a patient infected with SARS-CoV-2 have been shown to correlate with more severe outcomes and a greater risk of death as well. To further expand on the simultaneous detection of two biomarkers, CRP and N-protein were detected simultaneously, and the presence of SARS-CoV-2 N-protein did not interfere with the detection of CRP when both targets were present in the sample.

Progress toward a Simplified UTI Diagnostic: Pump-Free Magnetophoresis for E. coli Detection.

Urinary tract infections (UTIs) are one of the most common infections across the world and can lead to serious complications such as sepsis if not treated in a timely manner. Uropathogenic Escherichia coli account for 75% of all UTIs. Early diagnosis is crucial to help control UTIs, but current culturing methods are expensive and time-consuming and lack sensitivity. The existing point-of-care methods fall short because they rely on indirect detection from elevated nitrates in urine rather than detecting the actual bacteria causing the infection. Magnetophoresis is a powerful method used to separate and/or isolate cells of interest from complex matrices for analysis. However, magnetophoresis typically requires complex and expensive instrumentation to control flow in microfluidic devices. Coupling magnetophoresis with microfluidic paper-based analytical devices (µPADs) enables pump-free flow control and simple and low-cost operation. Early magnetophoresis µPADs showed detection limits competitive with traditional methods but higher than targets for clinical use. Here, we demonstrate magnetophoresis using hybrid µPADs that rely on capillary action in hydrophilic polyethylene terephthalate channels combined with paper pumps. We were able to detect E. coli with a calculated limit of detection of 2.40 × 102 colony-forming units per mL.

Electrochemical Immunoassay for the Detection of SARS-CoV-2 Nucleocapsid Protein in Nasopharyngeal Samples.

Point-of-care (POC) methods currently available for detecting SARS-CoV-2 infections still lack accuracy. Here, we report the development of a highly sensitive electrochemical immunoassay capable of quantitatively detecting the presence of the SARS-CoV-2 virus in patient nasopharyngeal samples using stencil-printed carbon electrodes (SPCEs) functionalized with capture antibodies targeting the SARS-CoV-2 nucleocapsid protein (N protein). Samples are added to the electrode surface, followed by horseradish peroxidase (HRP)-conjugated detection antibodies also targeting the SARS-CoV-2 N protein. The concentration of the virus in samples is quantified using chronoamperometry in the presence of 3,3'5,5'-tetramethylbenzidine. Limits of detection equivalent to less than 50 plaque forming units/mL (PFU/mL) were determined with virus sample volumes of 20 µL. No cross-reactivity was detected with the influenza virus and other coronavirus N proteins. Patient nasopharyngeal samples were tested as part of a proof-of-concept clinical study where samples were also tested using the gold-standard real-time quantitative polymerase chain reaction (RT-qPCR) method. Preliminary results from a data set of 22 samples demonstrated a clinical specificity of 100% (n = 9 negative samples according to RT-qPCR) and a clinical sensitivity of 70% for samples with RT-PCR cycle threshold (Ct) values under 30 (n = 10) and 100% for samples with Ct values under 25 (n = 5), which complies with the World Health Organization (WHO) criteria for POC COVID-19 diagnostic tests. Our functionalized SPCEs were also validated against standard plaque assays, and very good agreement was found between both methods (R2 = 0.9993, n = 6), suggesting that our assay could be used to assess patient infectivity. The assay currently takes 70 min from sampling to results.

Motif V regulates energy transduction between the flavivirus NS3 ATPase and RNA-binding cleft.

The unwinding of dsRNA intermediates is critical for the replication of flavivirus RNA genomes. This activity is provided by the C-terminal helicase domain of viral nonstructural protein 3 (NS3). As a member of the superfamily 2 (SF2) helicases, NS3 requires the binding and hydrolysis of ATP/NTP to translocate along and unwind double-stranded nucleic acids. However, the mechanism of energy transduction between the ATP- and RNA-binding pockets is not well-understood. Previous molecular dynamics simulations conducted by our group have identified Motif V as a potential "communication hub" for this energy transduction pathway. To investigate the role of Motif V in this process, here we combined molecular dynamics, biochemistry, and virology approaches. We tested Motif V mutations in both the replicon and recombinant protein systems to investigate viral genome replication, RNA-binding affinity, ATP hydrolysis activity, and helicase-mediated unwinding activity. We found that the T407A and S411A substitutions in NS3 reduce viral replication and increase the helicase-unwinding turnover rates by 1.7- and 3.5-fold, respectively, suggesting that flaviviruses may use suboptimal NS3 helicase activity for optimal genome replication. Additionally, we used simulations of each mutant to probe structural changes within NS3 caused by each mutation. These simulations indicate that Motif V controls communication between the ATP-binding pocket and the helical gate. These results help define the linkage between ATP hydrolysis and helicase activities within NS3 and provide insight into the biophysical mechanisms for ATPase-driven NS3 helicase function.

A Hyperactive Kunjin Virus NS3 Helicase Mutant Demonstrates Increased Dissemination and Mortality in Mosquitoes.

The unwinding of double-stranded RNA intermediates is critical for the replication and packaging of flavivirus RNA genomes. This unwinding activity is achieved by the ATP-dependent nonstructural protein 3 (NS3) helicase. In previous studies, we investigated the mechanism of energy transduction between the ATP and RNA binding pockets using molecular dynamics simulations and enzymatic characterization. Our data corroborated the hypothesis that motif V is a communication hub for this energy transduction. More specifically, mutations T407A and S411A in motif V exhibit a hyperactive helicase phenotype, leading to the regulation of translocation and unwinding during replication. However, the effect of these mutations on viral infection in cell culture and in vivo is not well understood. Here, we investigated the role of motif V in viral replication using West Nile virus (Kunjin subtype) T407A and S411A mutants (T407A and S411A Kunjin, respectively) in cell culture and in vivo We were able to recover S411A Kunjin but unable to recover T407A Kunjin. Our results indicated that S411A Kunjin decreased viral infection and increased cytopathogenicity in cell culture compared to wild-type (WT) Kunjin. Similarly, decreased infection rates in surviving S411A Kunjin-infected Culex quinquefasciatus mosquitoes were observed, but S411A Kunjin infection resulted in increased mortality compared to WT Kunjin infection. Additionally, S411A Kunjin infection increased viral dissemination and saliva positivity rates in surviving mosquitoes compared to WT Kunjin infection. These data suggest that S411A Kunjin increases viral pathogenesis in mosquitoes. Overall, these data indicate that NS3 motif V may play a role in the pathogenesis, dissemination, and transmission efficiency of Kunjin virus.IMPORTANCE Kunjin and West Nile viruses belong to the arthropod-borne flaviviruses, which can result in severe symptoms, including encephalitis, meningitis, and death. Flaviviruses have expanded into new populations and emerged as novel pathogens repeatedly in recent years, demonstrating that they remain a global threat. Currently, there are no approved antiviral therapeutics against either Kunjin or West Nile viruses. Thus, there is a pressing need for understanding the pathogenesis of these viruses in humans. In this study, we investigated the role of the Kunjin virus helicase on infection in cell culture and in vivo This work provides new insight into how flaviviruses control pathogenesis and mosquito transmission through the nonstructural protein 3 helicase.

Padlock probe-based rolling circle amplification lateral flow assay for point-of-need nucleic acid detection.

Sensitive, reliable and cost-effective detection of pathogens has wide ranging applications in clinical diagnostics and therapeutics, water and food safety, environmental monitoring, biosafety and epidemiology. Nucleic acid amplification tests (NAATs) such as PCR and isothermal amplification methods provide excellent analytical performance and significantly faster turnaround times than conventional culture-based methods. However, the inherent cost and complexity of NAATs limit their application in resource-limited settings and the developing world. To help address this urgent need, we have developed a sensitive method for nucleic acid analysis based on padlock probe rolling circle amplification (PLRCA), nuclease protection (NP) and lateral flow detection (LFA), referred to as PLAN-LFA, that can be used in resource-limited settings. The assay involves solution-phase hybridization of a padlock probe to target, sequence-specific ligation of the probe to form a circular template that undergoes isothermal rolling circle amplification in the presence of a polymerase and a labeled probe DNA. The RCA product is a long, linear concatenated single-stranded DNA that contains binding sites for the labeled probe. The sample is then exposed to a nuclease which selectively cleaves single-stranded DNA, the double-stranded labeled probe is protected from nuclease digestion and detected in a lateral flow immunoassay format to provide a visual, colorimetric readout of results. We have developed specific assays targeting beta-lactamase resistance gene for monitoring of antimicrobial resistance and Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2, the novel coronavirus discovered in 2019) using the PLAN-LFA platform. The assay provides a limit of detection of 1.1 pM target DNA (or 1.3 × 106 copies per reaction). We also demonstrate the versatility and robustness of the method by performing analysis on DNA and RNA targets, and perform analysis in complex sample matrices like saliva, plant tissue extract and bacterial culture without any sample pretreatment steps.

Flow control in a laminate capillary-driven microfluidic device.

Capillary-driven microfluidic devices are of significant interest for on-site analysis because they do not require external pumps and can be made from inexpensive materials. Among capillary-driven devices, those made from paper and polyester film are among the most common and have been used in a wide array of applications. However, since capillary forces are the only driving force, flow is difficult to control, and passive flow control methods such as changing the geometry must be used to accomplish various analytical applications. This study presents several new flow control methods that can be utilized in a laminate capillary-driven microfluidic device to increase available functionality. First, we introduce push and burst valve systems that can stop and start flow. These valves can stop flow for >30 min and be opened by either pressing the channel or inflowing other fluids to the valve region. Next, we propose flow control methods for Y-shaped channels that enable more functions. In one example, we demonstrate the ability to accurately control concentration to create laminar, gradient, and fully mixed flows. In a second example, flow velocity in the main channel is controlled by adjusting the length of the inlet channel. In addition, the flow velocity is constant as the inlet length increases. Finally, the flow velocity in the Y-shaped device as a function of channel height and fluid properties such as viscosity and surface tension was examined. As in previous studies on capillary-driven channels, the flow rate was affected by each parameter. The fluidic control tools presented here will enable new designs and functions for low cost point of need assays across a variety of fields.

Zika virus noncoding sfRNAs sequester multiple host-derived RNA-binding proteins and modulate mRNA decay and splicing during infection.

Insect-borne flaviviruses produce a 300-500-base long noncoding RNA, termed subgenomic flavivirus RNA (sfRNA), by stalling the cellular 5'-3'-exoribonuclease 1 (XRN1) via structures located in their 3' UTRs. In this study, we demonstrate that sfRNA production by Zika virus represses XRN1 analogous to what we have previously shown for other flaviviruses. Using protein-RNA reconstitution and a stringent RNA pulldown assay with human choriocarcinoma (JAR) cells, we demonstrate that the sfRNAs from both dengue type 2 and Zika viruses interact with a common set of 21 RNA-binding proteins that contribute to the regulation of post-transcriptional processes in the cell, including splicing, RNA stability, and translation. We found that four of these sfRNA-interacting host proteins, DEAD-box helicase 6 (DDX6) and enhancer of mRNA decapping 3 (EDC3) (two RNA decay factors), phosphorylated adaptor for RNA export (a regulator of the biogenesis of the splicing machinery), and apolipoprotein B mRNA-editing enzyme catalytic subunit 3C (APOBEC3C, a nucleic acid-editing deaminase), inherently restrict Zika virus infection. Furthermore, we demonstrate that the regulations of cellular mRNA decay and RNA splicing are compromised by Zika virus infection as well as by sfRNA alone. Collectively, these results reveal the large extent to which Zika virus-derived sfRNAs interact with cellular RNA-binding proteins and highlight the potential for widespread dysregulation of post-transcriptional control that likely limits the effective response of these cells to viral infection.

Paper-based nuclease protection assay with on-chip sample pretreatment for point-of-need nucleic acid detection.

Pathogen detection is crucial for human, animal, and environmental health; crop protection; and biosafety. Current culture-based methods have long turnaround times and lack sensitivity. Nucleic acid amplification tests offer high specificity and sensitivity. However, their cost and complexity remain a significant hurdle to their applications in resource-limited settings. Thus, point-of-need molecular diagnostic platforms that can be used by minimally trained personnel are needed. The nuclease protection assay (NPA) is a nucleic acid hybridization-based technique that does not rely on amplification, can be paired with other methods to improve specificity, and has the potential to be developed into a point-of-need device. In traditional NPAs, hybridization of an anti-sense probe to the target sequence is followed by single-strand nuclease digestion. The double-stranded target-probe hybrids are protected from nuclease digestion, precipitated, and visualized using autoradiography or other methods. We have developed a paper-based nuclease protection assay (PB-NPA) that can be implemented in field settings as the detection approach requires limited equipment and technical expertise. The PB-NPA uses a lateral flow format to capture the labeled target-probe hybrids onto a nitrocellulose membrane modified with an anti-label antibody. A colorimetric enzyme-substrate pair is used for signal visualization, producing a test line. The nuclease digestion of non-target and mismatched DNA provides high specificity while signal amplification with the reporter enzyme-substrate provides high sensitivity. We have also developed an on-chip sample pretreatment step utilizing chitosan-modified paper to eliminate possible interferents from the reaction and preconcentrate nucleic acids, thereby significantly reducing the need for auxiliary equipment. Graphical abstract.

Review-Chemical and Biological Sensors for Viral Detection.

Infectious diseases commonly occur in contaminated water, food, and bodily fluids and spread rapidly, resulting in death of humans and animals worldwide. Among infectious agents, viruses pose a serious threat to public health and global economy because they are often difficult to detect and their infections are hard to treat. Since it is crucial to develop rapid, accurate, cost-effective, and in-situ methods for early detection viruses, a variety of sensors have been reported so far. This review provides an overview of the recent developments in electrochemical sensors and biosensors for detecting viruses and use of these sensors on environmental, clinical and food monitoring. Electrochemical biosensors for determining viruses are divided into four main groups including nucleic acid-based, antibody-based, aptamer-based and antigen-based electrochemical biosensors. Finally, the drawbacks and advantages of each type of sensors are identified and discussed.

Allostery in the dengue virus NS3 helicase: Insights into the NTPase cycle from molecular simulations.

The C-terminus domain of non-structural 3 (NS3) protein of the Flaviviridae viruses (e.g. HCV, dengue, West Nile, Zika) is a nucleotide triphosphatase (NTPase) -dependent superfamily 2 (SF2) helicase that unwinds double-stranded RNA while translocating along the nucleic polymer. Due to these functions, NS3 is an important target for antiviral development yet the biophysics of this enzyme are poorly understood. Microsecond-long molecular dynamic simulations of the dengue NS3 helicase domain are reported from which allosteric effects of RNA and NTPase substrates are observed. The presence of a bound single-stranded RNA catalytically enhances the phosphate hydrolysis reaction by affecting the dynamics and positioning of waters within the hydrolysis active site. Coupled with results from the simulations, electronic structure calculations of the reaction are used to quantify this enhancement to be a 150-fold increase, in qualitative agreement with the experimental enhancement factor of 10-100. Additionally, protein-RNA interactions exhibit NTPase substrate-induced allostery, where the presence of a nucleotide (e.g. ATP or ADP) structurally perturbs residues in direct contact with the phosphodiester backbone of the RNA. Residue-residue network analyses highlight pathways of short ranged interactions that connect the two active sites. These analyses identify motif V as a highly connected region of protein structure through which energy released from either active site is hypothesized to move, thereby inducing the observed allosteric effects. These results lay the foundation for the design of novel allosteric inhibitors of NS3.

Highly Sensitive Detection of Salmonella typhimurium Using a Colorimetric Paper-Based Analytical Device Coupled with Immunomagnetic Separation.

Salmonella causes over a million foodborne illnesses per year in the United States resulting in more hospitalizations and deaths than any other foodborne bacterial pathogen. To help prevent outbreaks, a rapid, portable, sensitive, and reliable method for onsite detection of bacteria that can be used in different sample matrices would be beneficial. Herein, we present a colorimetric paper-based analytical device (PAD) combined with immunomagnetic separation (IMS) for detecting Salmonella typhimurium. IMS anti-Salmonella coated magnetic beads were applied to capture and separate bacteria from the sample matrix and preconcentrate it into small volumes before testing on paper. To directly detect S. typhimurium after IMS, a sandwich immunoassay was implemented into the procedure with ß-galactosidase (ß-gal) as the detection enzyme. Using the antibody/enzyme complex, we performed a colorimetric assay with chlorophenol red-ß-d-galactopyranoside (CPRG) for bacteria quantification. The method was confirmed to be highly specific to S. typhimurium without interference from other pathogenic bacteria like Escherichia coli. Using this system, the limit of detection of S. typhimurium was found to be 102 CFU mL-1 in culturing solution without any pre-enrichment. In addition, distance-based detection where the concentration is read as the length of colored band formed on the reaction was also demonstrated. This assay had a detection limit of 102 CFU mL-1 for S. typhimurium, providing an instrument-free quantitative analysis alternative to spot tests, which require image analysis. Finally, the proposed platform was applied for detection of S. typhimurium in inoculated Starling bird fecal samples and whole milk with detection limits of 105 CFU g-1 and 103 CFU mL-1, respectively, and this is the first published paper-based detection method for S. typhimurium in bird feces and whole milk.

Development of an Electrochemical Paper-Based Analytical Device for Trace Detection of Virus Particles.

Viral pathogens are a serious health threat around the world, particularly in resource limited settings, where current sensing approaches are often insufficient and slow, compounding the spread and burden of these pathogens. Here, we describe a label-free, point-of-care approach toward detection of virus particles, based on a microfluidic paper-based analytical device with integrated microwire Au electrodes. The device is initially characterized through capturing of streptavidin modified nanoparticles by biotin-modified microwires. An order of magnitude improvement in detection limits is achieved through use of a microfluidic device over a classical static paper-based device, due to enhanced mass transport and capturing of particles on the modified electrodes. Electrochemical impedance spectroscopy detection of West Nile virus particles was carried out using antibody functionalized Au microwires, achieving a detection limit of 10.2 particles in 50 µL of cell culture media. No increase in signal is found on addition of an excess of a nonspecific target (Sindbis). This detection motif is significantly cheaper (∼$1 per test) and faster (∼30 min) than current methods, while achieving the desired selectivity and sensitivity. This sensing motif represents a general platform for trace detection of a wide range of biological pathogens.

Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone.

Zika virus (ZIKV; family Flaviviridae, genus Flavivirus) is a rapidly expanding global pathogen that has been associated with severe clinical manifestations, including devastating neurological disease in infants. There are currently no molecular clones of a New World ZIKV available that lack significant attenuation, hindering progress toward understanding determinants of transmission and pathogenesis. Here we report the development and characterization of a novel ZIKV reverse genetics system based on a 2015 isolate from Puerto Rico (PRVABC59). We generated a two-plasmid infectious clone system from which infectious virus was rescued that replicates in human and mosquito cells with growth kinetics representative of wild-type ZIKV. Infectious clone-derived virus initiated infection and transmission rates in Aedes aegypti mosquitoes comparable to those of the primary isolate and displayed similar pathogenesis in AG129 mice. This infectious clone system provides a valuable resource to the research community to explore ZIKV molecular biology, vaccine development, antiviral development, diagnostics, vector competence, and disease pathogenesis. IMPORTANCE: ZIKV is a rapidly spreading mosquito-borne pathogen that has been linked to Guillain-Barré syndrome in adults and congenital microcephaly in developing fetuses and infants. ZIKV can also be sexually transmitted. The viral molecular determinants of any of these phenotypes are not well understood. There is no reverse genetics system available for the current epidemic virus that will allow researchers to study ZIKV immunity, develop novel vaccines, or develop antiviral drugs. Here we provide a novel infectious clone system generated from a recent ZIKV isolated from a patient infected in Puerto Rico. This infectious clone produces virus with in vitro and in vivo characteristics similar to those of the primary isolate, providing a critical tool to study ZIKV infection and disease.

Utilizing Paper-Based Devices for Antimicrobial-Resistant Bacteria Detection.

Antimicrobial resistance (AMR), the ability of a bacterial species to resist the action of an antimicrobial drug, has been on the rise due to the widespread use of antimicrobial agents. Per the World Health Organization, AMR has an estimated annual cost of USD 34 billion in the US and is predicted to be the number one cause of death worldwide by 2050. One way AMR bacteria can spread, and by which individuals can contract AMR infections, is through contaminated water. Monitoring AMR bacteria in the environment currently requires that samples be transported to a central laboratory for slow and labor intensive tests. We have developed an inexpensive assay using paper-based analytical devices (PADs) that can test for the presence of ß-lactamase-mediated resistance. To demonstrate viability, the PAD was used to detect ß-lactam resistance in wastewater and sewage and identified resistance in individual bacterial species isolated from environmental water sources.

PAIRpred: partner-specific prediction of interacting residues from sequence and structure.

We present a novel partner-specific protein-protein interaction site prediction method called PAIRpred. Unlike most existing machine learning binding site prediction methods, PAIRpred uses information from both proteins in a protein complex to predict pairs of interacting residues from the two proteins. PAIRpred captures sequence and structure information about residue pairs through pairwise kernels that are used for training a support vector machine classifier. As a result, PAIRpred presents a more detailed model of protein binding, and offers state of the art accuracy in predicting binding sites at the protein level as well as inter-protein residue contacts at the complex level. We demonstrate PAIRpred's performance on Docking Benchmark 4.0 and recent CAPRI targets. We present a detailed performance analysis outlining the contribution of different sequence and structure features, together with a comparison to a variety of existing interface prediction techniques. We have also studied the impact of binding-associated conformational change on prediction accuracy and found PAIRpred to be more robust to such structural changes than existing schemes. As an illustration of the potential applications of PAIRpred, we provide a case study in which PAIRpred is used to analyze the nature and specificity of the interface in the interaction of human ISG15 protein with NS1 protein from influenza A virus. Python code for PAIRpred is available at http://combi.cs.colostate.edu/supplements/pairpred/.

PAbFold: Linear Antibody Epitope Prediction using AlphaFold2.

Defining the binding epitopes of antibodies is essential for understanding how they bind to their antigens and perform their molecular functions. However, while determining linear epitopes of monoclonal antibodies can be accomplished utilizing well-established empirical procedures, these approaches are generally labor- and time-intensive and costly. To take advantage of the recent advances in protein structure prediction algorithms available to the scientific community, we developed a calculation pipeline based on the localColabFold implementation of AlphaFold2 that can predict linear antibody epitopes by predicting the structure of the complex between antibody heavy and light chains and target peptide sequences derived from antigens. We found that this AlphaFold2 pipeline, which we call PAbFold, was able to accurately flag known epitope sequences for several well-known antibody targets (HA / Myc) when the target sequence was broken into small overlapping linear peptides and antibody complementarity determining regions (CDRs) were grafted onto several different antibody framework regions in the single-chain antibody fragment (scFv) format. To determine if this pipeline was able to identify the epitope of a novel antibody with no structural information publicly available, we determined the epitope of a novel anti-SARS-CoV-2 nucleocapsid targeted antibody using our method and then experimentally validated our computational results using peptide competition ELISA assays. These results indicate that the AlphaFold2-based PAbFold pipeline we developed is capable of accurately identifying linear antibody epitopes in a short time using just antibody and target protein sequences. This emergent capability of the method is sensitive to methodological details such as peptide length, AlphaFold2 neural network versions, and multiple-sequence alignment database. PAbFold is available at https://github.com/jbderoo/PAbFold.

The leader RNA of SARS-CoV-2 sequesters polypyrimidine tract binding protein (PTBP1) and influences pre-mRNA splicing in infected cells.

The large amount of viral RNA produced during infections has the potential to interact with and effectively sequester cellular RNA binding proteins, thereby influencing aspects of post-transcriptional gene regulation in the infected cell. Here we demonstrate that the abundant 5' leader RNA region of SARS-CoV-2 viral RNAs can interact with the cellular polypyrimidine tract binding protein (PTBP1). Interestingly, the effect of a knockdown of PTBP1 protein on cellular gene expression is also mimicked during SARS-CoV-2 infection, suggesting that this protein may be functionally sequestered by viral RNAs. Consistent with this model, the alternative splicing of mRNAs that is normally controlled by PTBP1 is dysregulated during SARS-CoV-2 infection. Collectively, these data suggest that the SARS-CoV-2 leader RNA sequesters the cellular PTBP1 protein during infection, resulting in significant impacts on the RNA biology of the host cell. These alterations in post-transcriptional gene regulation may play a role in SARS-CoV-2 mediated molecular pathogenesis.

Identification of a novel antiviral inhibitor of the flavivirus guanylyltransferase enzyme.

Arthropod-borne flavivirus infection causes serious morbidity and mortality worldwide, but there are currently no effective antiflaviviral chemotherapeutics available for human use. Therefore, it is critical that new therapeutics against virus-specific targets be developed. To identify new compounds that may be used as broadly active flavivirus therapeutics, we have performed a high-throughput screening of 235,456 commercially available compounds for small-molecule inhibitors of the dengue virus NS5 RNA capping enzyme. We identified a family of compounds, the 2-thioxothiazolidin-4-ones, that show potent biochemical inhibition of capping enzyme GTP binding and guanylyltransferase function. During the course of structure-activity relationship analysis, a molecule within this family, (E)-{3-[5-(4-tert-butylbenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]propanoic acid} (BG-323), was found to possess significant antiviral activity in a dengue virus subgenomic replicon assay. Further testing of BG-323 demonstrated that this molecule is able to reduce the replication of infectious West Nile virus and yellow fever virus in cell culture with low toxicity. The results of this study describe the first inhibitor that targets the GTP-binding/guanylyltransferase activity of the flavivirus RNA capping enzyme.