Structural insight into rabies virus neutralization revealed by an engineered antibody scaffold.

Host-cell entry of the highly pathogenic rabies virus (RABV) is mediated by glycoprotein (G) spikes, which also comprise the primary target for the humoral immune response. RABV glycoprotein (RABV-G) displays several antigenic sites that are targeted by neutralizing monoclonal antibodies (mAbs). In this study, we determined the epitope of a potently neutralizing human mAb, CR57, which we engineered into a diabody format to facilitate crystallization.

Comparison of Automated and Traditional Western Blotting Methods.

Traditional Western blotting is one of the most used analytical techniques in biological research. However, it can be time-consuming and suffer from a lack of reproducibility. Consequently, devices with different degrees of automation have been developed.

Intranasal administration of adenoviral vaccines expressing SARS-CoV-2 spike protein improves vaccine immunity in mouse models.

The ongoing SARS-CoV-2 pandemic is controlled but not halted by public health measures and mass vaccination strategies which have exclusively relied on intramuscular vaccines. Intranasal vaccines can prime or recruit to the respiratory epithelium mucosal immune cells capable of preventing infection. Here we report a comprehensive series of studies on this concept using various mouse models, including HLA class II-humanized transgenic strains.

Intranasal trimeric sherpabody inhibits SARS-CoV-2 including recent immunoevasive Omicron subvariants.

The emergence of increasingly immunoevasive SARS-CoV-2 variants emphasizes the need for prophylactic strategies to complement vaccination in fighting the COVID-19 pandemic. Intranasal administration of neutralizing antibodies has shown encouraging protective potential but there remains a need for SARS-CoV-2 blocking agents that are less vulnerable to mutational viral variation and more economical to produce in large scale. Here we describe TriSb92, a highly manufacturable and stable trimeric antibody-mimetic sherpabody targeted against a conserved region of the viral spike glycoprotein.

SARS-CoV-2 and type 1 diabetes in children in Finland: an observational study.

Background: Some epidemiological studies have suggested an increase in incidence of type 1 diabetes during the COVID-19 pandemic, however the mechanism(s) behind such an increase have yet to be identified. In this study we aimed to evaluate the possible role of the SARS-CoV-2 virus in the reported increase in the rate of type 1 diabetes.

Methods: In this observational cohort study using data from the Finnish Pediatric Diabetes Register (FPDR), we assessed the incidence of type 1 diabetes (number of children with newly diagnosed type 1 diabetes per 100 000 person-years during the pandemic and the reference period) during the first 18 months of the COVID-19 pandemic in children in Finland younger than 15 years old compared with a reference period which included three corresponding pre-pandemic periods also obtained from the FPDR.

Blood and saliva SARS-CoV-2 antibody levels in self-collected dried spot samples.

We examined the usefulness of dried spot blood and saliva samples in SARS-CoV-2 antibody analyses. We analyzed 1231 self-collected dried spot blood and saliva samples from healthcare workers. Participants filled in a questionnaire on their COVID-19 exposures, infections, and vaccinations.

Structure of SNX9 SH3 in complex with a viral ligand reveals the molecular basis of its unique specificity for alanine-containing class I SH3 motifs.

Class I SH3 domain-binding motifs generally comply with the consensus sequence [R/K]xØPxxP, the hydrophobic residue Ø being proline or leucine. We have studied the unusual Ø = Ala-specificity of SNX9 SH3 by determining its complex structure with a peptide present in eastern equine encephalitis virus (EEEV) nsP3. The structure revealed the length and composition of the n-Src loop as important factors determining specificity.

Antidepressant and Antipsychotic Drugs Reduce Viral Infection by SARS-CoV-2 and Fluoxetine Shows Antiviral Activity Against the Novel Variants .

Repurposing of currently available drugs is a valuable strategy to tackle the consequences of COVID-19. Recently, several studies have investigated the effect of psychoactive drugs on SARS-CoV-2 in cell culture models as well as in clinical practice. Our aim was to expand these studies and test some of these compounds against newly emerged variants.

Common Laboratory Mice Are Susceptible to Infection with the SARS-CoV-2 Beta Variant.

Small animal models are of crucial importance for assessing COVID-19 countermeasures. Common laboratory mice would be well-suited for this purpose but are not susceptible to infection with wild-type SARS-CoV-2. However, the development of mouse-adapted virus strains has revealed key mutations in the SARS-CoV-2 spike protein that increase infectivity, and interestingly, many of these mutations are also present in naturally occurring SARS-CoV-2 variants of concern.

Evolutionary plasticity of SH3 domain binding by Nef proteins of the HIV-1/SIVcpz lentiviral lineage.

The accessory protein Nef of human and simian immunodeficiency viruses (HIV and SIV) is an important pathogenicity factor known to interact with cellular protein kinases and other signaling proteins. A canonical SH3 domain binding motif in Nef is required for most of these interactions. For example, HIV-1 Nef activates the tyrosine kinase Hck by tightly binding to its SH3 domain.

Plasma proteins, cognitive decline, and 20-year risk of dementia in the Whitehall II and Atherosclerosis Risk in Communities studies.

Introduction: Plasma proteins affect biological processes and are common drug targets but their role in the development of Alzheimer's disease and related dementias remains unclear. We examined associations between 4953 plasma proteins and cognitive decline and risk of dementia in two cohort studies with 20-year follow-ups.

Methods: In the Whitehall II prospective cohort study proteins were measured using SOMAscan technology.

A Generic, Scalable, and Rapid Time-Resolved Förster Resonance Energy Transfer-Based Assay for Antigen Detection-SARS-CoV-2 as a Proof of Concept.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has seen an unprecedented increase in the demand for rapid and reliable diagnostic tools, leaving many laboratories scrambling for resources. We present a fast and simple assay principle for antigen detection and demonstrate its functionality by detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens in nasopharyngeal swabs. The method is based on the detection of SARS-CoV-2 nucleoprotein (NP) and S protein (SP) via time-resolved Förster resonance energy transfer (TR-FRET) with donor- and acceptor-labeled polyclonal anti-NP and -SP antibodies.

Indoor Model Simulation for COVID-19 Transport and Exposure.

Transmission of respiratory viruses is a complex process involving emission, deposition in the airways, and infection. Inhalation is often the most relevant transmission mode in indoor environments. For severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the risk of inhalation transmission is not yet fully understood.

T cell circuits that sense antigen density with an ultrasensitive threshold.

Overexpressed tumor-associated antigens [for example, epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2)] are attractive targets for therapeutic T cells, but toxic "off-tumor" cross-reaction with normal tissues that express low levels of target antigen can occur with chimeric antigen receptor (CAR)-T cells. Inspired by natural ultrasensitive response circuits, we engineered a two-step positive-feedback circuit that allows human cytotoxic T cells to discriminate targets on the basis of a sigmoidal antigen-density threshold. In this circuit, a low-affinity synthetic Notch receptor for HER2 controls the expression of a high-affinity CAR for HER2.

HIV-1 Nef-Induced Secretion of the Proinflammatory Protease TACE into Extracellular Vesicles Is Mediated by Raf-1 and Can Be Suppressed by Clinical Protein Kinase Inhibitors.

Chronic immune activation is an important driver of human immunodeficiency virus type 1 (HIV-1) pathogenesis and has been associated with the presence of tumor necrosis factor-α converting enzyme (TACE) in extracellular vesicles (EVs) circulating in infected individuals. We have recently shown that activation of the Src-family tyrosine kinase hematopoietic cell kinase (Hck) by HIV-1 Nef can trigger the packaging of TACE into EVs via an unconventional protein secretion pathway. Using a panel of HIV-1 Nef mutants and natural HIV-2 and simian immunodeficiency virus (SIV) Nef alleles, we now show that the capacity to promote TACE secretion depends on the superior ability of HIV-1-like Nef alleles to induce Hck kinase activity, whereas other Nef effector functions are dispensable.

MC159 of Molluscum Contagiosum Virus Suppresses Autophagy by Recruiting Cellular SH3BP4 via an SH3 Domain-Mediated Interaction.

MC159 is a viral FLIP (FLICE inhibitory protein) encoded by the molluscum contagiosum virus (MCV) enabling MCV to evade antiviral immunity and to establish persistent infections in humans. Here, we show that MC159 contains a functional SH3 binding motif, which mediates avid and selective binding to SH3BP4, a signaling protein known to regulate endocytic trafficking and suppress cellular autophagy. The capacity to bind SH3BP4 was dispensable for regulation of NF-κB-mediated transcription and suppression of proapoptotic caspase activation but contributed to inhibition of amino acid starvation-induced autophagy by MC159.

Tyrosine phosphorylation directs TACE into extracellular vesicles via unconventional secretion.

When studying how HIV-1 Nef can promote packaging of the proinflammatory transmembrane protease TACE (tumor necrosis factor-α converting enzyme) into extracellular vesicles (EVs) we have revealed a novel tyrosine kinase-regulated unconventional protein secretion (UPS) pathway for TACE. When TACE was expressed without its trafficking cofactor iRhom allosteric Hck activation by Nef triggered translocation of TACE into EVs. This process was insensitive to blocking of classical secretion by inhibiting endoplasmic reticulum (ER) to Golgi transport, and involved a distinct form of TACE devoid of normal glycosylation and incompletely processed for prodomain removal.

ANKRD54 preferentially selects Bruton's Tyrosine Kinase (BTK) from a Human Src-Homology 3 (SH3) domain library.

Bruton's Tyrosine Kinase (BTK) is a cytoplasmic protein tyrosine kinase with a fundamental role in B-lymphocyte development and activation. The nucleocytoplasmic shuttling of BTK is specifically modulated by the Ankyrin Repeat Domain 54 (ANKRD54) protein and the interaction is known to be exclusively SH3-dependent. To identify the spectrum of the ANKRD54 SH3-interactome, we applied phage-display screening of a library containing all the 296 human SH3 domains.

Large-Scale Screening of Preferred Interactions of Human Src Homology-3 (SH3) Domains Using Native Target Proteins as Affinity Ligands.

The Src Homology-3 (SH3) domains are ubiquitous protein modules that mediate important intracellular protein interactions via binding to short proline-rich consensus motifs in their target proteins. The affinity and specificity of such core SH3 - ligand contacts are typically modest, but additional binding interfaces can give rise to stronger and more specific SH3-mediated interactions. To understand how commonly such robust SH3 interactions occur in the human protein interactome, and to identify these in an unbiased manner we have expressed 324 predicted human SH3 ligands as full-length proteins in mammalian cells, and screened for their preferred SH3 partners using a phage display-based approach.

Identification of specific residues in avian influenza A virus NS1 that enhance viral replication and pathogenicity in mammalian systems.

Reassortment of their segmented genomes allows influenza A viruses (IAV) to gain new characteristics, which potentially enable them to cross the species barrier and infect new hosts. Improved replication was observed for reassortants of the strictly avian IAV A/FPV/Rostock/34 (FPV, H7N1) containing the NS segment from A/Goose/Guangdong/1/1996 (GD, H5N1), but not for reassortants containing the NS segment of A/Mallard/NL/12/2000 (MA, H7N3). The NS1 of GD and MA differ only in 8 aa positions.

Structural Basis of the High Affinity Interaction between the Alphavirus Nonstructural Protein-3 (nsP3) and the SH3 Domain of Amphiphysin-2.

We show that a peptide from Chikungunya virus nsP3 protein spanning residues 1728-1744 binds the amphiphysin-2 (BIN1) Src homology-3 (SH3) domain with an unusually high affinity (Kd 24 nm). Our NMR solution complex structure together with isothermal titration calorimetry data on several related viral and cellular peptide ligands reveal that this exceptional affinity originates from interactions between multiple basic residues in the target peptide and the extensive negatively charged binding surface of amphiphysin-2 SH3. Remarkably, these arginines show no fixed conformation in the complex structure, indicating that a transient or fluctuating polyelectrostatic interaction accounts for this affinity.

HIV-Nef and ADAM17-Containing Plasma Extracellular Vesicles Induce and Correlate with Immune Pathogenesis in Chronic HIV Infection.

Antiretroviral therapy (ART) efficiently suppresses HIV replication but immune activation and low CD4 T cell counts often persist. The underlying mechanism of this ART-resistant pathogenesis is not clear. We observed that levels of plasma extracellular vesicles (pEV) are strongly elevated in HIV infection and do not decline during ART.

Nuclear Translocation of Crk Adaptor Proteins by the Influenza A Virus NS1 Protein.

The non-structural protein-1 (NS1) of many influenza A strains, especially those of avian origin, contains an SH3 ligand motif, which binds tightly to the cellular adaptor proteins Crk (Chicken tumor virus number 10 (CT10) regulator of kinase) and Crk-like adapter protein (CrkL). This interaction has been shown to potentiate NS1-induced activation of the phosphatidylinositol 3-kinase (PI3K), but additional effects on the host cell physiology may exist. Here we show that NS1 can induce an efficient translocation of Crk proteins from the cytoplasm into the nucleus, which results in an altered pattern of nuclear protein tyrosine phosphorylation.