Deep mutational scanning reveals sequence to function constraints for SWEET family transporters.

Protein science is entering a transformative phase enabled by deep mutational scans that provide an unbiased view of the residue level interactions that mediate function. However, it has yet to be extensively used to characterize the mutational and evolutionary landscapes of plant proteins. Here, we apply the method to explore sequence-function relationships within the sugar transporter AtSWEET13.

Sequence basis for selectivity of ephrin-B2 ligand for Eph receptors and pathogenic henipavirus G glycoproteins.

Ephrin-B2 (EFNB2) is a ligand for six Eph receptors in humans and regulates multiple cell developmental and signaling processes. It also functions as the cell entry receptor for Nipah virus and Hendra virus, zoonotic viruses that can cause respiratory and/or neurological symptoms in humans with high mortality. Here, we investigate the sequence basis of EFNB2 specificity for binding the Nipah virus attachment G glycoprotein over Eph receptors.

Identification of a 1.4-kb-Long Sequence Located in the nsp12 and nsp13 Coding Regions of SARS-CoV-2 Genomic RNA That Mediates Efficient Viral RNA Packaging.

The specific packaging of the viral RNA genome into virus particles is an essential step in the replication cycle of coronaviruses (CoVs). Using a single-cycle, replicable severe acute respiratory syndrome CoV-2 (SARS-CoV-2) mutant, we demonstrated the preferential packaging of the SARS-CoV-2 genomic RNA into purified virus particles. Furthermore, based on the sequence of an efficiently packaged defective interfering RNA of SARS-CoV, a closely related CoV, that was generated after serial passages of SARS-CoV in cell culture, we designed a series of replication-competent SARS-CoV-2 minigenome RNAs to identify the specific viral RNA region that is important for SARS-CoV-2 RNA packaging into virus particles.

The Sequence Basis for Selectivity of Ephrin-B2 Ligand for Eph Receptors and Pathogenic Henipavirus G Glycoproteins: Selective Ephrin-B2 Decoys for Nipah and Hendra Virus.

Ephrin-B2 (EFNB2) is a ligand for six Eph receptors in humans and functions as a cell entry receptor for several henipaviruses including Nipah virus (NiV), a pathogenic zoonotic virus with pandemic potential. To understand the sequence basis of promiscuity for EFNB2 binding to the attachment glycoprotein of NiV (NiV-G) and Eph receptors, we performed deep mutagenesis on EFNB2 to identify mutations that enhance binding to NiV-G over EphB2, one of the highest affinity Eph receptors. The mutations highlight how different EFNB2 conformations are selected by NiV-G versus EphB2.

Mechanistic insight into the efficient packaging of antigenomic S RNA into Rift Valley fever virus particles.

Rift Valley fever virus (RVFV), a bunyavirus, has a single-stranded, negative-sense tri-segmented RNA genome, consisting of L, M and S RNAs. An infectious virion carries two envelope glycoproteins, Gn and Gc, along with ribonucleoprotein complexes composed of encapsidated viral RNA segments. The antigenomic S RNA, which serves as the template of the mRNA encoding a nonstructural protein, NSs, an interferon antagonist, is also efficiently packaged into RVFV particles.

Betulinic acid mitigates oxidative stress-mediated apoptosis and enhances longevity in the yeast model.

Betulinic acid (BA), a pentacyclic triterpenoid found in certain plant species, has been reported to have several health benefits including antioxidant and anti-apoptotic properties. However, the mechanism by which BA confers these properties is currently unknown. , a budding yeast with a short life cycle and conserved cellular mechanism with high homology to humans, was used as a model for determining the role of BA in aging and programmed cell death (PCD).

An ACE2 decoy can be administered by inhalation and potently targets omicron variants of SARS-CoV-2.

Monoclonal antibodies targeting the SARS-CoV-2 spike (S) neutralize infection and are efficacious for the treatment of COVID-19. However, SARS-CoV-2 variants, notably sublineages of B.1.

Parsing the role of NSP1 in SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to shutoff of protein synthesis, and nsp1, a central shutoff factor in coronaviruses, inhibits cellular mRNA translation. However, the diverse molecular mechanisms employed by nsp1 as well as its functional importance are unresolved. By overexpressing various nsp1 mutants and generating a SARS-CoV-2 mutant, we show that nsp1, through inhibition of translation and induction of mRNA degradation, targets translated cellular mRNA and is the main driver of host shutoff during infection.

An engineered ACE2 decoy receptor can be administered by inhalation and potently targets the BA.1 and BA.2 omicron variants of SARS-CoV-2.

Monoclonal antibodies targeting the SARS-CoV-2 spike (S) glycoprotein neutralize infection and are efficacious for the treatment of mild-to-moderate COVID-19. However, SARS-CoV-2 variants have emerged that partially or fully escape monoclonal antibodies in clinical use. Notably, the BA.

Parsing the role of NSP1 in SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 19 (COVID-19) pandemic. Despite its urgency, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis and its ability to antagonize innate immune responses. SARS-CoV-2 leads to shutoff of cellular protein synthesis and over-expression of nsp1, a central shutoff factor in coronaviruses, inhibits cellular gene translation.

An Enhanced Decoding Algorithm for Coded Compressed Sensing with Applications to Unsourced Random Access.

Unsourced random access (URA) has emerged as a pragmatic framework for next-generation distributed sensor networks. Within URA, concatenated coding structures are often employed to ensure that the central base station can accurately recover the set of sent codewords during a given transmission period. Many URA algorithms employ independent inner and outer decoders, which can help reduce computational complexity at the expense of a decay in performance.

Characterization of the Molecular Interactions That Govern the Packaging of Viral RNA Segments into Rift Valley Fever Phlebovirus Particles.

Rift Valley fever phlebovirus (RVFV) has a single-stranded, negative-sense RNA genome, consisting of L, M, and S segments. The virion carries two envelope glycoproteins, Gn and Gc, along with ribonucleoprotein complexes (RNPs), composed of encapsidated genomes carrying N protein and the viral polymerase, L protein. A quantitative analysis of the profile of viral RNA segments packaged into RVFV particles showed that all three genomic RNA segments had similar packaging abilities, whereas among antigenomic RNA segments, the antigenomic S RNA, which serves as the template for the transcription of mRNA expressing the RVFV virulence factor, NSs, displayed a significantly higher packaging ability.

Deep Mutational Scanning of Viral Glycoproteins and Their Host Receptors.

Deep mutational scanning or deep mutagenesis is a powerful tool for understanding the sequence diversity available to viruses for adaptation in a laboratory setting. It generally involves tracking an selection of protein sequence variants with deep sequencing to map mutational effects based on changes in sequence abundance. Coupled with any of a number of selection strategies, deep mutagenesis can explore the mutational diversity available to viral glycoproteins, which mediate critical roles in cell entry and are exposed to the humoral arm of the host immune response.

An engineered decoy receptor for SARS-CoV-2 broadly binds protein S sequence variants.

The spike S of SARS-CoV-2 recognizes ACE2 on the host cell membrane to initiate entry. Soluble decoy receptors, in which the ACE2 ectodomain is engineered to block S with high affinity, potently neutralize infection and, because of close similarity with the natural receptor, hold out the promise of being broadly active against virus variants without opportunity for escape. Here, we directly test this hypothesis.

An engineered decoy receptor for SARS-CoV-2 broadly binds protein S sequence variants.

The spike S of SARS-CoV-2 recognizes ACE2 on the host cell membrane to initiate entry. Soluble decoy receptors, in which the ACE2 ectodomain is engineered to block S with high affinity, potently neutralize infection and, due to close similarity with the natural receptor, hold out the promise of being broadly active against virus variants without opportunity for escape. Here, we directly test this hypothesis.

An Affordable Neurosurgical Training System for Neurosurgical Residents; The Indian Perspective.

Context: Neurosurgical training in India.

Aims: To establish a sustainable, functional, and relatively inexpensive neurosurgical training system.

Methods And Materials: The training system involved using a relatively inexpensive stereoscopic microscope and ophthalmological microinstruments , including two jewellers' forceps and a microscissors.

Isolation and characterization of SARS-CoV-2 from the first US COVID-19 patient.

The etiologic agent of the outbreak of pneumonia in Wuhan China was identified as severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) in January, 2020. The first US patient was diagnosed by the State of Washington and the US Centers for Disease Control and Prevention on January 20, 2020. We isolated virus from nasopharyngeal and oropharyngeal specimens, and characterized the viral sequence, replication properties, and cell culture tropism.

An Infectious cDNA Clone of SARS-CoV-2.

The ongoing pandemic of COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), underscores the urgency to develop experimental systems for studying this virus and identifying countermeasures. We report a reverse genetic system for SARS-CoV-2. Seven complimentary DNA (cDNA) fragments spanning the SARS-CoV-2 genome were assembled into a full-genome cDNA.

Severe Acute Respiratory Syndrome Coronavirus 2 from Patient with Coronavirus Disease, United States.

The etiologic agent of an outbreak of pneumonia in Wuhan, China, was identified as severe acute respiratory syndrome coronavirus 2 in January 2020. A patient in the United States was given a diagnosis of infection with this virus by the state of Washington and the US Centers for Disease Control and Prevention on January 20, 2020. We isolated virus from nasopharyngeal and oropharyngeal specimens from this patient and characterized the viral sequence, replication properties, and cell culture tropism.

A strand-specific real-time quantitative RT-PCR assay for distinguishing the genomic and antigenomic RNAs of Rift Valley fever phlebovirus.

Rift Valley Fever phlebovirus (RVFV), genus Phlebovirus, family Phenuiviridae, order Bunyavirales, has a single-stranded, negative-sense RNA genome, consisting of L, M and S segments. Here, we report the establishment of a strand-specific, quantitative reverse transcription (RT)-PCR assay system that can selectively distinguish between the genomic and antigenomic RNAs of each of the three viral RNA segments produced in RVFV-infected cells. To circumvent the obstacle of primer-independent cDNA synthesis during RT, we used a tagged, strand-specific RT primer, carrying a non-viral 'tag' sequence at the 5' end, which ensured the strand-specificity through the selective amplification of only the tagged cDNA in the real-time PCR assay.

Interplay between coronavirus, a cytoplasmic RNA virus, and nonsense-mediated mRNA decay pathway.

Coronaviruses (CoVs), including severe acute respiratory syndrome CoV and Middle East respiratory syndrome CoV, are enveloped RNA viruses that carry a large positive-sense single-stranded RNA genome and cause a variety of diseases in humans and domestic animals. Very little is known about the host pathways that regulate the stability of CoV mRNAs, which carry some unusual features. Nonsense-mediated decay (NMD) is a eukaryotic RNA surveillance pathway that detects mRNAs harboring aberrant features and targets them for degradation.

The Endonucleolytic RNA Cleavage Function of nsp1 of Middle East Respiratory Syndrome Coronavirus Promotes the Production of Infectious Virus Particles in Specific Human Cell Lines.

Middle East respiratory syndrome coronavirus (MERS-CoV) nsp1 suppresses host gene expression in expressed cells by inhibiting translation and inducing endonucleolytic cleavage of host mRNAs, the latter of which leads to mRNA decay. We examined the biological functions of nsp1 in infected cells and its role in virus replication by using wild-type MERS-CoV and two mutant viruses with specific mutations in the nsp1; one mutant lacked both biological functions, while the other lacked the RNA cleavage function but retained the translation inhibition function. In Vero cells, all three viruses replicated efficiently with similar replication kinetics, while wild-type virus induced stronger host translational suppression and host mRNA degradation than the mutants, demonstrating that nsp1 suppressed host gene expression in infected cells.

Inhibition of Stress Granule Formation by Middle East Respiratory Syndrome Coronavirus 4a Accessory Protein Facilitates Viral Translation, Leading to Efficient Virus Replication.

Stress granule (SG) formation is generally triggered as a result of stress-induced translation arrest. The impact of SG formation on virus replication varies among different viruses, and the significance of SGs in coronavirus (CoV) replication is largely unknown. The present study examined the biological role of SGs in Middle East respiratory syndrome (MERS)-CoV replication.

A Bayesian Network-Based Approach to Selection of Intervention Points in the Mitogen-Activated Protein Kinase Plant Defense Response Pathway.

An important problem in computational biology is the identification of potential points of intervention that can lead to modified network behavior in a genetic regulatory network. We consider the problem of deducing the effect of individual genes on the behavior of the network in a statistical framework. In this article, we make use of biological information from the literature to develop a Bayesian network and introduce a method to estimate parameters of this network using data that are relevant to the biological phenomena under study.

Immunization with inactivated Middle East Respiratory Syndrome coronavirus vaccine leads to lung immunopathology on challenge with live virus.

To determine if a hypersensitive-type lung pathology might occur when mice were given an inactivated MERS-CoV vaccine and challenged with infectious virus as was seen with SARS-CoV vaccines, we prepared and vaccinated mice with an inactivated MERS-CoV vaccine. Neutralizing antibody was induced by vaccine with and without adjuvant and lung virus was reduced in vaccinated mice after challenge. Lung mononuclear infiltrates occurred in all groups after virus challenge but with increased infiltrates that contained eosinophils and increases in the eosinophil promoting IL-5 and IL-13 cytokines only in the vaccine groups.