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Abstract Human coronaviruses (hCoVs) infect millions of people every year. Among these, MERS, SARS-CoV-1, and SARS-CoV-2 caused significant morbidity and mortality and their emergence highlights the risk of possible future coronavirus outbreaks. Therefore, broadly-active anti-coronavirus drugs are needed. Pharmacological inhibition of the hCoV protease Nsp5 (3CLpro) is clinically beneficial as shown by the wide and effective use of Paxlovid (nirmatrelvir, ritonavir). However, further treatment options are required due to the risk of drug resistance. To facilitate the assessment of coronavirus protease function and its pharmacological inhibition, we developed an assay allowing rapid and reliable quantification of Nsp5 activity under biosafety level 1 conditions. It is based on an ACE2-Gal4 transcription factor fusion protein separated by a Nsp5 recognition site. Cleavage by Nsp5 releases the Gal4 transcription factor, which then induces the expression of Gaussia luciferase. Our assay is compatible with Nsp5 proteases from all hCoVs and allows simultaneous measurement of inhibitory and cytotoxic effects of the tested compounds. Proof-of-concept measurements confirmed that nirmatrelvir, GC376 and lopinavir inhibit SARS-CoV-2 Nsp5 function. Furthermore, the assay accurately predicted the impact of Nsp5 mutations on catalytic activity and inhibitor sensitivity. Overall, the reporter assay is suitable for evaluating viral protease activity.

Abstract Molecular pathways mediating systemic inflammation entering the brain parenchyma to induce sepsis-associated encephalopathy (SAE) remain elusive. Here, we report that in mice during the first 6 hours of peripheral lipopolysaccharide (LPS)-evoked systemic inflammation (6 hpi), the plasma level of adenosine quickly increased and enhanced the tone of central extracellular adenosine which then provoked neuroinflammation by triggering early astrocyte reactivity. Specific ablation of astrocytic Gi protein-coupled A1 adenosine receptors (A1ARs) prevented this early reactivity and reduced the levels of inflammatory factors (e.g., CCL2, CCL5, and CXCL1) in astrocytes, thereby alleviating microglial reaction, ameliorating blood-brain barrier disruption, peripheral immune cell infiltration, neuronal dysfunction, and depression-like behaviour in the mice. Chemogenetic stimulation of Gi signaling in A1AR-deficent astrocytes at 2 and 4 hpi of LPS injection could restore neuroinflammation and depression-like behaviour, highlighting astrocytes rather than microglia as early drivers of neuroinflammation. Our results identify early astrocyte reactivity towards peripheral and central levels of adenosine as an important pathway driving SAE and highlight the potential of targeting A1ARs for therapeutic intervention.

Abstract Long-term non-progressors (LTNPs) of HIV-1 infection may provide important insights into mechanisms involved in viral control and pathogenesis. Here, our results suggest that the ribosomal protein lateral stalk subunit P1 (RPLP1) is expressed at higher levels in LTNPs compared to regular progressors (RPs). Functionally, RPLP1 inhibits transcription of clade B HIV-1 strains by occupying the C/EBPβ binding sites in the viral long terminal repeat (LTR). This interaction requires the α-helixes 2 and 4 domains of RPLP1 and is evaded by HIV-1 group M subtype C and group N, O and P strains that do not require C/EBPβ for transcription. We further demonstrate that HIV-1-induced translocation of RPLP1 from the cytoplasm to the nucleus is essential for antiviral activity. Finally, knock-down of RPLP1 promotes reactivation of latent HIV-1 proviruses. Thus, RPLP1 may play a role in the maintenance of HIV-1 latency and resistance to RPLP1 restriction may contribute to the effective spread of clade C HIV-1 strains.

Abstract Innate antiviral factors are essential for effective defense against viral pathogens. However, the identity of major restriction mechanisms remains elusive. Current approaches to discover antiviral factors usually focus on the initial steps of viral replication and are limited to a single round of infection. Here, we engineered libraries of >1500 replication-competent HIV-1 constructs each expressing a single gRNAs to target >500 cellular genes for virus-driven discovery of antiviral factors. Passaging in CD4+ T cells robustly enriched HIV-1 encoding sgRNAs against GRN, CIITA, EHMT2, CEACAM3, CC2D1B and RHOA by >50-fold. Using an HIV-1 library lacking the accessory nef gene, we identified IFI16 as a Nef target. Functional analyses in cell lines and primary CD4+ T cells support that the HIV-driven CRISPR screen identified restriction factors targeting virus entry, transcription, release and infectivity. Our HIV-guided CRISPR technique enables sensitive discovery of physiologically relevant cellular defense factors throughout the entire viral replication cycle.

Abstract Traumatic brain injury leads to a highly orchestrated immune- and glial cell response partially responsible for long-lasting disability and the development of secondary neurodegenerative diseases. A holistic understanding of the mechanisms controlling the responses of specific cell types and their crosstalk is required to develop an efficient strategy for better regeneration. Here, we combine spatial and single-cell transcriptomics to chart the transcriptomic signature of the injured male murine cerebral cortex, and identify specific states of different glial cells contributing to this signature. Interestingly, distinct glial cells share a large fraction of injury-regulated genes, including inflammatory programs downstream of the innate immune-associated pathways Cxcr3 and Tlr1/2. Systemic manipulation of these pathways decreases the reactivity state of glial cells associated with poor regeneration. The functional relevance of the discovered shared signature of glial cells highlights the importance of our resource enabling comprehensive analysis of early events after brain injury.

Abstract Background: Spinal ventral root injuries generate significant motoneuron degeneration, which hinders full functional recovery. The poor prognosis of functional recovery can be attributed to the use or combination of different therapeutic approaches. Several molecules have been screened as potential treatments in combination with surgical reimplantation of the avulsed roots, the gold standard approach for such injuries. Among the studied molecules, human natural killer-1 (HNK-1) stands out as it is related to the stimulation of motor axon outgrowth. Therefore, we aimed to comparatively investigate the effects of local administration of an HNK-1 mimetic peptide (mp-HNK-1) and systemic treatment with ursolic acid (UA), another HNK-1 mimetic, after ventral root avulsion and reimplantation with heterologous fibrin biopolymer (HFB). Methods: Female mice of the isogenic strain C57BL/6JUnib were divided into five experimental groups: Avulsion, Reimplantation, mp-HNK-1 (in situ), and UA (systemic treatment). Mice were evaluated 2 and 12 weeks after surgery. Functional assessment was performed every four days using the Catwalk platform. Neuronal survival was analyzed by cytochemistry, and glial reactions and synaptic coverage were evaluated by immunofluorescence. Results: Treatment with UA elicited long-term neuroprotection, accompanied by a decrease in microglial reactions, and reactive astrogliosis. The neuroprotective effects of UA were preceded by increased glutamatergic and GABAergic inputs in the ventral spinal cord two weeks after injury. However, a single application of mp-HNK-1 had no significant effects. Functional analysis showed that UA treatment led to an improvement in motor and sensory recovery. Conclusion: Overall, the results indicate that UA is neuroprotective, acting on glial cells and synaptic maintenance, and the combination of these findings led to a better functional recovery.

Abstract Besides the many advantages of oral drug administration, challenges like premature drug degradation and limited bioavailability in the gastro-intestinal tract (GIT) remain. A prolonged residence time in the GIT is beneficial for enhancing the therapeutic outcome when treating diseases associated with an increased intestinal clearance rate, like inflammatory bowel disease (IBD). In this study, we synthesized rod-shaped mesoporous silica nanoparticles (MSNs) functionalized with polyethylene glycol (PEG) or hyaluronic acid (HA) and investigated their bio-distribution upon oral administration in vivo. The negatively charged, non-toxic particles showed different accumulation behavior over time in healthy mice and in mice with dextran sulfate sodium (DSS)-induced intestinal inflammation. PEGylated particles were shown to accumulate in the lower intestinal tract of healthy animals, whereas inflammation promoted retention of HA-functionalized particles in this area. Overall systemic absorption was low. However, some particles were detected in organs of mice with DSS-induced colitis, especially in the case of MSN-PEG. The in vivo findings were connected to surface chemistry-related differences in particle adhesion on Caco-2/Raji and mucus-producing Caco-2/Raji/HT29 cell co-culture epithelial models in vitro. While the particle adhesion behavior in vivo was mirrored in the in vitro results, this was not the case for the resorption results, suggesting that the in vitro model does not fully reflect the erosion of the inflamed epithelial tissue. Overall, our study demonstrates the possibility to modulate accumulation and retention of MSNs in the GIT of mice with and without inflammation through surface functionalization, which has important implications for the formulation of nanoparticle-based delivery systems for oral delivery applications.

Abstract Type I interferon (IFN) signalling is tightly controlled. Upon recognition of DNA by cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING) translocates along the endoplasmic reticulum (ER)-Golgi axis to induce IFN signalling. Termination is achieved through autophagic degradation or recycling of STING by retrograde Golgi-to-ER transport. Here, we identify the GTPase ADP-ribosylation factor 1 (ARF1) as a crucial negative regulator of cGAS-STING signalling. Heterozygous ARF1 missense mutations cause a previously unrecognized type I interferonopathy associated with enhanced IFN-stimulated gene expression. Disease-associated, GTPase-defective ARF1 increases cGAS-STING dependent type I IFN signalling in cell lines and primary patient cells. Mechanistically, mutated ARF1 perturbs mitochondrial morphology, causing cGAS activation by aberrant mitochondrial DNA release, and leads to accumulation of active STING at the Golgi/ERGIC due to defective retrograde transport. Our data show an unexpected dual role of ARF1 in maintaining cGAS-STING homeostasis, through promotion of mitochondrial integrity and STING recycling.

Abstract Utilization of human ACE2 allowed several bat coronaviruses (CoVs), including the causative agent of COVID-19, to infect humans directly or via intermediate hosts. However, the determinants of species-specific differences in ACE2 usage and the frequency of the ability of animal CoVs to use human ACE2 are poorly understood. Here we applied VSV pseudoviruses to analyze the ability of Spike proteins from 26 human or animal CoVs to use ACE2 receptors across nine reservoir, potential intermediate and human hosts. We show that SARS-CoV-2 Omicron variants evolved towards more efficient ACE2 usage but mutation of R493Q in BA.4/5 and XBB Spike proteins disrupts utilization of ACE2 from Greater horseshoe bats. Variations in ACE2 residues 31, 41 and 354 govern species-specific differences in usage by coronaviral Spike proteins. Mutation of T403R allows the RaTG13 bat CoV Spike to efficiently use all ACE2 orthologs for viral entry. Sera from COVID-19 vaccinated individuals neutralize the Spike proteins of various bat Sarbecoviruses. Our results define determinants of ACE2 receptor usage of diverse CoVs and suggest that COVID-19 vaccination may protect against future zoonoses of bat coronaviruses.

Summary: The accessory viral protein R (Vpr) is encoded by all primate lentiviruses. Vpr counteracts DNA repair pathways, modulates viral immune sensing, and induces cell-cycle arrest in cell culture. However, its impact in vivo is controversial. Here, we show that deletion of vpr is associated with delayed viral replication kinetics, rapid innate immune activation, development and maintenance of strong B and T cell responses, and increased neutralizing activity against SIVmac239 in rhesus macaques. All wild-type SIVmac239-infected animals maintained high viral loads, and five of six developed fatal immunodeficiency during ∼80 weeks of follow-up. Lack of Vpr was associated with better preservation of CD4+ T cells, lower viral loads, and an attenuated clinical course of infection in most animals. Our results show that Vpr contributes to efficient viral immune evasion and the full pathogenic potential of SIVmac in vivo. Inhibition of Vpr may improve humoral immune control of viral replication.

PYHIN proteins are only found in mammals and play key roles in the defense against bacterial and viral pathogens. The corresponding gene locus shows variable deletion and expansion ranging from 0 genes in bats, over 1 in cows, and 4 in humans to a maximum of 13 in mice. While initially thought to act as cytosolic immune sensors that recognize foreign DNA, increasing evidence suggests that PYHIN proteins also inhibit viral pathogens by more direct mechanisms. Here, we examined the ability of all 13 murine PYHIN proteins to inhibit HIV-1 and murine leukemia virus (MLV). We show that overexpression of p203, p204, p205, p208, p209, p210, p211, and p212 strongly inhibits production of infectious HIV-1; p202, p207, and p213 had no significant effects, while p206 and p214 showed intermediate phenotypes. The inhibitory effects on infectious HIV-1 production correlated significantly with the suppression of reporter gene expression by a proviral Moloney MLV-eGFP construct and HIV-1 and Friend MLV LTR luciferase reporter constructs. Altogether, our data show that the antiretroviral activity of PYHIN proteins is conserved between men and mice and further support the key role of nuclear PYHIN proteins in innate antiviral immunity.

ABSTRACT The type 1 interferon-regulated E3 ubiquitin ligase MARCH1 reduces surface expression of HIV-1 envelope glycoproteins (Env) and their packaging into nascent virions, a condition that restricts viral infectivity. However, how HIV-1 counters this restriction, notably during infection of macrophages, remains unclear. Here, we show that the HIV-1 accessory protein Vpu increases the levels of microRNAs-25 and -93 to target MARCH1 mRNA. By recruiting β-TRCP, a component of the SCFβ-TRCP E3 ligase complex that targets phosphorylated β-catenin for degradation, Vpu increases β-catenin levels, which, in concert with TCF4/LEF, drives transcription of the MARCH1-targeting microRNAs. This potentiates HIV-1 infectivity as a result of increased Env incorporation into nascent virions. Pharmacological targeting of the β-catenin pathway inhibits Vpu-mediated upregulation of microRNAs-25 and -93 and restores MARCH1 restriction on HIV-1 infectivity. Overall, our findings highlight a novel mechanism by which HIV-1 counteracts MARCH1 by downregulating its expression via Vpu-mediated induction of microRNAs-25 and -93. IMPORTANCE In order to efficiently produce infectious viral particles, HIV must counter several restrictions exerted by host cell antiviral proteins. MARCH1 is a member of the MARCH protein family that restricts HIV infection by limiting the incorporation of viral envelope glycoproteins into nascent virions. Here, we identified two regulatory RNAs, microRNAs-25 and -93, induced by the HIV-1 accessory protein Vpu, that downregulate MARCH1 mRNA. We also show that Vpu induces these cellular microRNAs in macrophages by hijacking the cellular β-catenin pathway. The notion that HIV-1 has evolved a mechanism to counteract MARCH1 restriction on viral infectivity underlines the importance of MARCH1 in the host antiviral response.

Severity of SARS-CoV-2 infection is different in adults and children which involves the immune response. Here using a parent and children cohort with 4 month and 12 month sampling times, the authors show enhanced levels and increased breadth of anti-spike antibody level over time but reduced specific T cell and B cell numbers in children.

ABSTRACT HIV-1 evades antibody-dependent cellular cytotoxicity (ADCC) responses not only by controlling Env conformation and quantity at the cell surface but also by altering NK cell activation via the downmodulation of several ligands of activating and co-activating NK cell receptors. The signaling lymphocyte activation molecule (SLAM) family of receptors, which includes NTB-A and 2B4, act as co-activating receptors to sustain NK cell activation and cytotoxic responses. These receptors cooperate with CD16 (FcγRIII) and other activating receptors to trigger NK cell effector functions. In that context, Vpu-mediated downregulation of NTB-A on HIV-1-infected CD4 T cells was shown to prevent NK cell degranulation via an homophilic interaction, thus contributing to ADCC evasion. However, less is known on the capacity of HIV-1 to evade 2B4-mediated NK cell activation and ADCC. Here, we show that HIV-1 downregulates the ligand of 2B4, CD48, from the surface of infected cells in a Vpu-dependent manner. This activity is conserved among Vpu proteins from the HIV-1/SIVcpz lineage and depends on conserved residues located in its transmembrane domain and dual phosphoserine motif. We show that NTB-A and 2B4 stimulate CD16-mediated NK cell degranulation and contribute to ADCC responses directed to HIV-1-infected cells to the same extent. Our results suggest that HIV-1 has evolved to downmodulate the ligands of both SLAM receptors to evade ADCC. IMPORTANCE Antibody-dependent cellular cytotoxicity (ADCC) can contribute to the elimination of HIV-1-infected cells and HIV-1 reservoirs. An in-depth understanding of the mechanisms used by HIV-1 to evade ADCC might help develop novel approaches to reduce the viral reservoirs. Members of the signaling lymphocyte activation molecule (SLAM) family of receptors, such as NTB-A and 2B4, play a key role in stimulating NK cell effector functions, including ADCC. Here, we show that Vpu downmodulates CD48, the ligand of 2B4, and this contributes to protect HIV-1-infected cells from ADCC. Our results highlight the importance of the virus to prevent the triggering of the SLAM receptors to evade ADCC.

Coronaviruses are a large family of enveloped RNA viruses found in numerous animal species. They are well known for their ability to cross species barriers and have been transmitted from bats or intermediate hosts to humans on several occasions. Four of the seven human coronaviruses (hCoVs) are responsible for approximately 20% of common colds (hCoV-229E, -NL63, -OC43, -HKU1). Two others (SARS-CoV-1 and MERS-CoV) cause severe and frequently lethal respiratory syndromes but have only spread to very limited extents in the human population. In contrast the most recent human hCoV, SARS-CoV-2, while exhibiting intermediate pathogenicity, has a profound impact on public health due to its enormous spread. In this review, we discuss which initial features of the SARS-CoV-2 Spike protein and subsequent adaptations to the new human host may have helped this pathogen to cause the COVID-19 pandemic. Our focus is on host forces driving changes in the Spike protein and their consequences for virus infectivity, pathogenicity, immune evasion and resistance to preventive or therapeutic agents. In addition, we briefly address the significance and perspectives of broad-spectrum therapeutics and vaccines.

BackgroundRecent data on immune evasion of new SARS-CoV-2 variants raise concerns about the efficacy of antibody-based COVID-19 therapies. Therefore, in this study the in-vitro neutralization capacity against SARS-CoV-2 variant B.1 and the Omicron subvariants BA.1, BA.2 and BA.5 of sera from convalescent individuals with and without boost by vaccination was assessed.Methods and findingsThe study included 313 serum samples from 155 individuals with a history of SARS-CoV-2 infection, divided into subgroups without (n=25) and with SARS-CoV-2 vaccination (n=130). We measured anti-SARS-CoV-2 antibody concentrations by serological assays (anti-SARS-CoV-2-QuantiVac-ELISA (IgG) and Elecsys Anti-SARS-CoV-2 S) and neutralizing titers against B.1, BA.1, BA.2 and BA.5 in a pseudovirus neutralization assay. Sera of the majority of unvaccinated convalescents did not effectively neutralize Omicron sublineages BA.1, BA.2 and BA.5 (51.7%, 24.1% and 51.7%, resp.). In contrast, 99.3% of the sera of superimmunized individuals (vaccinated convalescents) neutralized the Omicron subvariants BA.1 and BA.5 and 99.6% neutralized BA.2. Neutralizing titers against B.1, BA.1, BA.2 and BA.5 were significantly higher in vaccinated compared to unvaccinated convalescents (p

ABSTRACT Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns – conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we – a broad group of working virologists – seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology.

Summary: Opposing effects of interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) on SARS-CoV-2 infection have been reported. The reasons for this are unclear and the role of IFITMs in infection of other human coronaviruses (hCoVs) remains poorly understood. Here, we demonstrate that endogenous expression of IFITM2 and/or IFITM3 is critical for efficient replication of SARS-CoV-1, SARS-CoV-2 and hCoV-OC43 but has little effect on MERS-, NL63-and 229E-hCoVs. In contrast, overexpression of IFITMs inhibits all these hCoVs, and the corresponding spike-containing pseudo-particles, except OC43, which is enhanced by IFITM3. We further demonstrate that overexpression of IFITMs impairs cell surface expression of ACE2 representing the entry receptor of SARS-CoVs and hCoV-NL63 but not hCoV-OC43. Our results explain the inhibitory effects of artificial IFITM overexpression on ACE2-tropic SARS-CoVs and show that three hCoVs, including major causative agents of severe respiratory disease, hijack IFITMs for efficient infection of human cells.

IntroductionDurability of immune protection against reinfection with SARS-CoV-2 remains enigmatic, especially in the pediatric population and in the context of immune-evading variants of concern. Obviously, this knowledge is required for measures to contain the spread of infection and in selecting rational preventive measures.MethodsHere, we investigated the serum neutralization capacity of 36 seropositive adults and 34 children approximately one year after infection with the ancestral Wuhan strain of SARS-CoV-2 by using a pseudovirus neutralization assay.ResultsWe found that 88.9% of seropositive adult (32/36) and 94.1% of seropositive children (32/34) convalescents retained the neutralizing activity against the SARS-CoV-2 Wuhan strain (WT). Although, the neutralization effect against Omicron BA.1 (B.1.1.529.1) was significantly lower, 70.6% (24/34) of children and 41.7% (15/36) of adults possessed BA.1 cross-neutralizing antibodies. The spike 1 (S1)-specific T cell recall capacity using an activation-induced marker assay was analyzed in 18 adults and 16 children. All participants had detectable S1-specific CD4 T cells against WT, and 72.2% (13/18) adults and 81,3% (13/16) children had detectable S1 WT-specific CD8 T cells. CD4 cross-reactivity against BA.1 was demonstrated in all investigated adults (18/18), and 66.7% (12/18) adult participants had also detectable specific CD8 BA.1 T cells while we detected BA.1 S1 reactive CD4 and CD8 T cells in 81.3% (13/16) children.DiscussionTogether, our findings demonstrate that infection with the ancestral strain of SARS-CoV-2 in children as well as in adults induces robust serological as well as T cell memory responses that persist over at least 12 months. This suggests persistent immunological memory and partial cross-reactivity against Omicron BA.1.

Early postnatal interruption of the bidirectional GABA/TNFSF12 signaling between parvalbumin-positive interneurons and oligodendrocyte precursor cells impairs correct prefrontal cortical network activity and social cognitive behavior later in life.

Coronavirus infection induces interferon-stimulated genes, one of which encodes Tetherin, a transmembrane protein inhibiting the release of various enveloped viruses from infected cells. Previous studies revealed that SARS-CoV encodes two Tetherin antagonists: the Spike protein (S), inducing lysosomal degradation of Tetherin, and ORF7a, altering its glycosylation. Similarly, SARS-CoV-2 has also been shown to use ORF7a and Spike to enhance virion release in the presence of Tetherin. Here, we directly compare the abilities and mechanisms of these two viral proteins to counteract Tetherin. Therefore, cell surface and total Tetherin levels upon ORF7a or S expression were investigated using flow cytometry and Western blot analysis. SARS-CoV and SARS-CoV-2 S only marginally reduced Tetherin cell surface levels in a cell type-dependent manner. In HEK293T cells, under conditions of high exogenous Tetherin expression, SARS-CoV-2 S and ORF7a reduced total cellular Tetherin levels much more efficiently than the respective counterparts derived from SARS-CoV. Nevertheless, ORF7a from both species was able to alter Tetherin glycosylation. The ability to decrease total protein levels of Tetherin was conserved among S proteins from different SARS-CoV-2 variants (α, γ, δ, ο). While SARS-CoV-2 S and ORF7a both colocalized with Tetherin, only ORF7a directly interacted with the restriction factor in a two-hybrid assay. Despite the presence of multiple Tetherin antagonists, SARS-CoV-2 replication in Caco-2 cells was further enhanced upon Tetherin knockout. Altogether, our data show that endogenous Tetherin restricts SARS-CoV-2 replication and that the antiviral activity of Tetherin is only partially counteracted by viral antagonists with differential and complementary modes of action.

Summary: HIV-1 envelope (Env) conformation determines the susceptibility of infected CD4+ T cells to antibody-dependent cellular cytotoxicity (ADCC). Upon interaction with CD4, Env adopts more “open” conformations, exposing ADCC epitopes. HIV-1 limits Env-CD4 interaction and protects infected cells against ADCC by downregulating CD4 via Nef, Vpu, and Env. Limited data exist, however, of the role of these proteins in downmodulating CD4 on infected macrophages and how this impacts Env conformation. While Nef, Vpu, and Env are all required to efficiently downregulate CD4 on infected CD4+ T cells, we show here that any one of these proteins is sufficient to downmodulate most CD4 from the surface of infected macrophages. Consistent with this finding, Nef and Vpu have a lesser impact on Env conformation and ADCC sensitivity in infected macrophages compared with CD4+ T cells. However, treatment of infected macrophages with small CD4 mimetics exposes vulnerable CD4-induced Env epitopes and sensitizes them to ADCC.

The bat sarbecovirus RaTG13 is a close relative of SARS-CoV-2, but its spike protein doesn’t efficiently bind human ACE2. Here, the authors show that exchange of spike residue 403 between RaTG13 and SARS-CoV-2 spike proteins affects binding to human ACE2 and entry of pseudotyped viruses.

Aberrant CXCR4/CXCL12 signaling is involved in many pathophysiological processes such as cancer and inflammatory diseases. A natural fragment of serum albumin, named EPI-X4, has previously been identified as endogenous peptide antagonist and inverse agonist of CXCR4 and is a promising compound for the development of improved analogues for the therapy of CXCR4-associated diseases. To generate optimized EPI-X4 derivatives we here performed molecular docking analysis to identify key interaction motifs of EPI-X4/CXCR4. Subsequent rational drug design allowed to increase the anti-CXCR4 activity of EPI-X4. The EPI-X4 derivative JM#21 bound CXCR4 and suppressed CXCR4-tropic HIV-1 infection more efficiently than the clinically approved small molecule CXCR4 antagonist AMD3100. EPI-X4 JM#21 did not exert toxic effects in zebrafish embryos and suppressed allergen-induced infiltration of eosinophils and other immune cells into the airways of animals in an asthma mouse model. Moreover, topical administration of the optimized EPI-X4 derivative efficiently prevented inflammation of the skin in a mouse model of atopic dermatitis. Thus, rationally designed EPI-X4 JM#21 is a novel potent antagonist of CXCR4 and the first CXCR4 inhibitor with therapeutic efficacy in atopic dermatitis. Further clinical development of this new class of CXCR4 antagonists for the therapy of atopic dermatitis, asthma and other CXCR4-associated diseases is highly warranted.

Combining molecular modeling and in vitro bioassays, Sokkar et al. provide an understanding of how EPI-X4, an endogenous peptide antagonist, interacts with its receptor CXCR4. With computational model, validated by mutagenesis studies, they design a series of derivatives with optimized receptor antagonizing properties as new leads for the development of CXCR4 inhibitors.

Stroke is a major reason for persistent disability due to insufficient treatment strategies beyond reperfusion, leading to oligodendrocyte death and axon demyelination, persistent inflammation and astrogliosis in peri-infarct areas. After injury, oligodendroglial precursor cells (OPCs) have been shown to compensate for myelin loss and prevent axonal loss through the replacement of lost oligodendrocytes, an inefficient process leaving axons chronically demyelinated. Phenotypic screening approaches in demyelinating paradigms revealed substances that promote myelin repair. We established an ex vivo adult organotypic coronal slice culture (OCSC) system to study repair after stroke in a resource-efficient way. Post-photothrombotic OCSCs can be manipulated for 8 d by exposure to pharmacologically active substances testing remyelination activity. OCSCs were isolated from a NG2-CreERT2-td-Tomato knock-in transgenic mouse line to analyze oligodendroglial fate/differentiation and kinetics. Parbendazole boosted differentiation of NG2+ cells and stabilized oligodendroglial fate reflected by altered expression of associated markers PDGFR-α, CC1, BCAS1 and Sox10 and GFAP. In vitro scratch assay and chemical ischemia confirmed the observed effects upon parbendazole treatment. Adult OCSCs represent a fast, reproducible, and quantifiable model to study OPC differentiation competence after stroke. Pharmacological stimulation by means of parbendazole promoted OPC differentiation.

Abstract The COVID-19 pandemic poses a global threat to public health and economy. The continuously emerging SARS-CoV-2 variants present a major challenge to the development of antiviral agents and vaccines. In this study, we identified that EK1 and cholesterol-coupled derivative of EK1, EK1C4, as pan-CoV fusion inhibitors, exhibit potent antiviral activity against SARS-CoV-2 infection in both lung- and intestine-derived cell lines (Calu-3 and Caco2, respectively). They are also effective against infection of pseudotyped SARS-CoV-2 variants B.1.1.7 (Alpha) and B.1.1.248 (Gamma) as well as those with mutations in S protein, including N417T, E484K, N501Y, and D614G, which are common in South African and Brazilian variants. Crystal structure revealed that EK1 targets the HR1 domain in the SARS-CoV-2 S protein to block virus-cell fusion and provide mechanistic insights into its broad and effective antiviral activity. Nasal administration of EK1 peptides to hACE2 transgenic mice significantly reduced viral titers in lung and intestinal tissues. EK1 showed good safety profiles in various animal models, supporting further clinical development of EK1-based pan-CoV fusion inhibitors against SARS-CoV-2 and its variants.

IFITM proteins can inhibit several viruses, but effects on SARS-CoV-2 infection are not well understood. Here, the authors show that endogenous IFITMs support SARS-CoV-2 infection in different in vitro models by binding spike and enhancing virus entry.

In light of the decreasing immune protection against symptomatic SARS-CoV-2 infection after initial vaccinations and the now dominant immune-evasive Omicron variants, ‘booster’ vaccinations are regularly performed to restore immune responses. Many individuals have received a primary heterologous prime-boost vaccination with long intervals between vaccinations, but the resulting long-term immunity and the effects of a subsequent ‘booster’, particularly against Omicron BA.1, have not been defined. We followed a cohort of 23 young adults, who received a primary heterologous ChAdOx1 nCoV-19 BNT162b2 prime-boost vaccination, over a 7-month period and analysed how they responded to a BNT162b2 ‘booster’. We show that already after the primary heterologous vaccination, neutralization titers against Omicron BA.1 are recognizable but that humoral and cellular immunity wanes over the course of half a year. Residual responsive memory T cells recognized spike epitopes of the early SARS-CoV-2 B.1 strain as well as the Delta and BA.1 variants of concern (VOCs). However, the remaining antibody titers hardly neutralized these VOCs. The ‘booster’ vaccination was well tolerated and elicited both high antibody titers and increased memory T cell responses against SARS-CoV-2 including BA.1. Strikingly, in this young heterologously vaccinated cohort the neutralizing activity after the ‘booster’ was almost as potent against BA.1 as against the early B.1 strain. Our results suggest that a ‘booster’ after heterologous vaccination results in effective immune maturation and potent protection against the Omicron BA.1 variant in young adults.

In chemical synapses undergoing high frequency stimulation, vesicle components can be retrieved from the plasma membrane via a clathrin-independent process called activity-dependent bulk endocytosis (ADBE). Alix (ALG-2-interacting protein X/PDCD6IP) is an adaptor protein binding to ESCRT and endophilin-A proteins which is required for clathrin-independent endocytosis in fibroblasts. Alix is expressed in neurons and concentrates at synapses during epileptic seizures. Here, we used cultured neurons to show that Alix is recruited to presynapses where it interacts with and concentrates endophilin-A during conditions triggering ADBE. Using Alix knockout (ko) neurons, we showed that this recruitment, which requires interaction with the calcium-binding protein ALG-2, is necessary for ADBE. We also found that presynaptic compartments of Alix ko hippocampi display subtle morphological defects compatible with flawed synaptic activity and plasticity detected electrophysiologically. Furthermore, mice lacking Alix in the forebrain undergo less seizures during kainate-induced status epilepticus and reduced propagation of the epileptiform activity. These results thus show that impairment of ADBE due to the lack of neuronal Alix leads to abnormal synaptic recovery during physiological or pathological repeated stimulations.

Antiviral NK cell activity is regulated through the interaction of activating and inhibitory NK cell receptors with their ligands on infected cells. HLA class I molecules serve as ligands for most killer cell immunoglobulin-like receptors (KIRs), but no HLA class I ligands for the inhibitory NK cell receptor KIR2DL5 have been identified to date. Using a NK cell receptor/ligand screening approach, we observed no strong binding of KIR2DL5 to HLA class I or class II molecules, but confirmed that KIR2DL5 binds to the poliovirus receptor (PVR, CD155). Functional studies using primary human NK cells revealed a significantly decreased degranulation of KIR2DL5+ NK cells in response to CD155-expressing target cells. We subsequently investigated the role of KIR2DL5/CD155 interactions in HIV-1 infection, and showed that multiple HIV-1 strains significantly decreased CD155 expression levels on HIV-1-infected primary human CD4+ T cells via a Nef-dependent mechanism. Co-culture of NK cells with HIV-1-infected CD4+ T cells revealed enhanced anti-viral activity of KIR2DL5+ NK cells against wild-type versus Nef-deficient viruses, indicating that HIV-1-mediated downregulation of CD155 renders infected cells more susceptible to recognition by KIR2DL5+ NK cells. These data show that CD155 suppresses the antiviral activity of KIR2DL5+ NK cells and is downmodulated by HIV-1 Nef protein as potential trade-off counteracting activating NK cell ligands, demonstrating the ability of NK cells to counteract immune escape mechanisms employed by HIV-1.

Here, via screening of a polypeptide library from bronchoalveolar lavage, the authors identify and characterize α1-antitrypsin (α1AT) as SARS-CoV-2 inhibitor and show that α1AT binds and inactivates the serine protease TMPRSS2, which enzymatically primes the SARS-CoV-2 spike protein for membrane fusion.

The Neurovascular Unit (NVU), composed of glia (astrocytes, oligodendrocytes, microglia), neurons, pericytes and endothelial cells, is a dynamic interface ensuring the physiological functioning of the central nervous system (CNS), which gets affected and contributes to the pathology of several neurodegenerative diseases. Neuroinflammation is a common feature of neurodegenerative diseases and is primarily related to the activation state of perivascular microglia and astrocytes, which constitute two of its major cellular components. Our studies focus on monitoring in real time the morphological changes of perivascular astrocytes and microglia, as well as their dynamic interactions with the brain vasculature, under physiological conditions and following systemic neuroinflammation triggering both microgliosis and astrogliosis. To this end, we performed 2-photon laser scanning microscopy (2P-LSM) for intravital imaging of the cortex of transgenic mice visualizing the dynamics of microglia and astroglia following neuroinflammation induced by systemic administration of the endotoxin lipopolysaccharide (LPS). Our results indicate that following neuroinflammation the endfeet of activated perivascular astrocytes lose their close proximity and physiological cross-talk with vasculature, an event that most possibly contributes to a loss of blood–brain barrier (BBB) integrity. At the same time, microglial cells become activated and exhibit a higher extent of physical contact with the blood vessels. These dynamic responses of perivascular astrocytes and microglia are peaking at 4 days following LPS administration; however, they still persist at a lower level at 8 days after LPS injection, revealing incomplete reversal of inflammation affecting the glial properties and interactions within the NVU.

NK cells control SIV infection in secondary lymphoid tissues in the natural host that typically doesn’t progress toward disease. Here the authors show that this control is associated with terminal NK cell differentiation and improved MHC-E-dependent activity lacking in pathogenic SIV infection.

Self‐assembled peptide fibrils are abundant in in vivo systems and have been associated both with normal physiological functions and with different diseases. Herein, a proof‐of‐concept study is presented, where aggregates of synthetic, fibril‐forming peptides are studied as carriers for mesoporous silica nanoparticles (MSNs), giving a hydrogel@MSN hybrid structure. It is shown that when the peptide carries a cellular‐targeting motif, in this case arg‐gly‐asp (RGD), the fibrillar aggregates efficiently attach to cancer cells, and the attached MSNs can then be locally taken up by the cells and intracellularly release their cargo or release their cargo close to the outer membrane of the cells. If the targeting motif is not present on the fibrils, hardly any attachment of the fibrillar aggregates to the target cells occurs. The higher drug carrying capacity of the MSNs together with the efficient cellular attachment of the fibrillar aggregates represents a synergistic approach toward reaching a modular drug release system, bringing together the strengths of each component of the system. Finally, it is noted that although the experimental design is such that the fibrillar aggregates would not be capable of entering cells due to their large size, the approach would also be applicable to smaller fibrillar aggregates.

The spinal cord is the main pathway connecting brain and peripheral nervous system. Its functionality relies on the orchestrated activity of both neurons and glial cells. To date, most advancement in understanding the spinal cord inner mechanisms has been made either by in vivo exposure of its dorsal surface through laminectomy or by acute ex vivo slice preparation, likely affecting spinal cord physiology in virtue of the necessary extensive manipulation of the spinal cord tissue. This is especially true of cells immediately responding to alterations of the surrounding environment, such as microglia and astrocytes, reacting within seconds or minutes and for up to several days after the original insult. Ca2+ signaling is considered one of the most immediate, versatile, and yet elusive cellular responses of glia. Here, we induced the cell-specific expression of the genetically encoded Ca2+ indicator GCaMP3 to evaluate spontaneous intracellular Ca2+ signaling in astrocytes and microglia. Ca2+ signals were then characterized in acute ex vivo (both gray and white matter) as well as in chronic in vivo (white matter) preparations using MSparkles, a MATLAB-based software for automatic detection and analysis of fluorescence events. As a result, we were able to segregate distinct astroglial and microglial Ca2+ signaling patterns along with method-specific Ca2+ signaling alterations, which must be taken into consideration in the reliable evaluation of any result obtained in physiological as well as pathological conditions. Our study revealed a high degree of Ca2+ signaling diversity in glial cells of the murine spinal cord, thus adding to the current knowledge of the astonishing glial heterogeneity and cell-specific Ca2+ dynamics in non-neuronal networks.

Summary: Background: Heterologous COVID-19 vaccination regimens combining vector- and mRNA-based vaccines are already administered, but data on solicited adverse reactions, immunological responses and elicited protection are limited. Methods: To evaluate the reactogenicity and humoral as well as cellular immune responses towards most prevalent SARS-CoV-2 variants after a heterologous ChAdOx1 nCoV-19 BNT162b2 prime-boost vaccination, we analysed a cohort of 26 clinic employees aged 25-46 (median 30.5) years who received a ChAdOx1 nCoV-19 prime followed by a BNT162b2 boost after an 8-week interval. Serological data were compared to a cohort which received homologous BNT162b2 vaccination with a 3-week interval (14 individuals aged 25-65, median 42). Findings: Self-reported solicited symptoms after ChAdOx1 nCoV-19 prime were in line with previous reports and more severe than after the BNT162b2 boost. Antibody titres increased significantly over time resulting in strong neutralization titres two weeks after the BNT162b2 boost and subsequently slightly decreased over the course of 17 weeks. At the latest time point measured, all analysed sera retained neutralizing activity against the currently dominant Delta (B.1.617.2) variant. Two weeks post boost, neutralizing activity against the Alpha (B.1.1.7) and immune-evading Beta (B.1.351) variant was ∼4-fold higher than in individuals receiving homologous BNT162b2 vaccination. No difference was observed in neutralization of Kappa (B.1.617.1). In addition, heterologous vaccination induced CD4+ and CD8+ T cells reactive to SARS-CoV-2 spike peptides of all analysed variants; Wuhan-Hu-1, Alpha, Beta, Gamma (P.1), and Delta. Interpretation: In conclusion, heterologous ChAdOx1 nCoV-19 / BNT162b2 prime-boost vaccination is not associated with serious adverse events and induces potent humoral and cellular immune responses. The Alpha, Beta, Delta, and Kappa variants of spike are potently neutralized by sera from all participants and reactive T cells recognize spike peptides of all tested variants. These results suggest that this heterologous vaccination regimen is at least as immunogenic and protective as homologous vaccinations and also offers protection against current variants of concern. Funding: This project has received funding from the European Union's Horizon 2020 research and innovation programme, the German Research Foundation, the BMBF, the Robert Koch Institute (RKI), the Baden-Württemberg Stiftung, the county of Lower Saxony, the Ministry for Science, Research and the Arts of Baden-Württemberg, Germany, and the National Institutes of Health.

Abstract Fast, precise and sustained neurotransmission requires graded Ca2+ signals at the presynaptic terminal. Neurotransmitter release depends on a complex interplay of Ca2+ fluxes and Ca2+ buffering in the presynaptic terminal that is not fully understood. Here, we show that the angiotensin-receptor-associated protein (ATRAP) localizes to synaptic terminals throughout the central nervous system. In the retinal photoreceptor synapse and the cerebellar mossy fiber-granule cell synapse, we find that ATRAP is involved in the generation of depolarization-evoked synaptic Ca2+ transients. Compared to wild type, Ca2+ imaging in acutely isolated preparations of the retina and the cerebellum from ATRAP knockout mice reveals a significant reduction of the sarcoendoplasmic reticulum (SR) Ca2+-ATPase (SERCA) activity. Thus, in addition to its conventional role in angiotensin signaling, ATRAP also modulates presynaptic Ca2+ signaling within the central nervous system.

Summary: We present a protocol for analyzing the impact of SARS-CoV-2 proteins in interferon signaling using luciferase reporter assays. Here, the induction of defined promoters can be quantitatively assessed with high sensitivity and broad linear range. The results are similar to those obtained using qPCR to measure endogenous mRNA induction. The assay requires stringent normalization and confirmation of the results in more physiological settings. The protocol is adaptable for other viruses and other innate immune stimuli.For complete details on the use and execution of this protocol, please refer to Hayn et al. (2021).

Infection with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic, leads to profound remodeling of cellular membranes, promoting viral replication and virion assembly. A full understanding of this drastic remodeling and the process of virion morphogenesis remains lacking. In this study, we applied room temperature transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) tomography to visualize the SARS-CoV-2 replication factory in Vero cells, and present our results in comparison with published cryo-EM studies. We obtained cryo-EM-like clarity of the ultrastructure by employing high-pressure freezing, freeze substitution (HPF-FS) and embedding, allowing room temperature visualization of double-membrane vesicles (DMVs) in a near-native state. In addition, our data illustrate the consecutive stages of virion morphogenesis and reveal that SARS-CoV-2 ribonucleoprotein assembly and membrane curvature occur simultaneously. Finally, we show the tethering of virions to the plasma membrane in 3D, and that accumulations of virus particles lacking spike protein in large vesicles are most likely not a result of defective virion assembly at their membrane. In conclusion, this study puts forward a room-temperature EM technique providing near-native ultrastructural information about SARS-CoV-2 replication, adding to our understanding of the interaction of this pandemic virus with its host cell.

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. An archetypal contact between a negatively charged SH3 residue and a highly conserved arginine in Nef (Arg77) plays a key role here. Combining structural analyses with functional assays, we here show that Nef proteins have also developed a distinct structural strategy-termed the "R-clamp"-that favors the formation of this salt bridge via buttressing Arg77. Comparison of evolutionarily diverse Nef proteins revealed that several distinct R-clamps have evolved that are functionally equivalent but differ in the side chain compositions of Nef residues 83 and 120. Whereas a similar R-clamp design is shared by Nef proteins of HIV-1 groups M, O, and P, as well as SIVgor, the Nef proteins of SIV from the Eastern chimpanzee subspecies (SIVcpzP.t.s.) exclusively utilize another type of R-clamp. By contrast, SIV of Central chimpanzees (SIVcpzP.t.t.) and HIV-1 group N strains show more heterogenous R-clamp design principles, including a non-functional evolutionary intermediate of the aforementioned two classes. These data add to our understanding of the structural basis of SH3 binding and kinase deregulation by Nef, and provide an interesting example of primate lentiviral protein evolution.

Translating the CRISPR/Cas9 genome editing technology into clinics is still hampered by rather unspecific, unsafe and/or inconvenient approaches for the delivery of its main components - the Cas9 endonuclease and a guide RNA - into cells. Here, we describe the development of a novel transient and non-viral Cas9 delivery strategy based on the translocation machinery of the Bacillus anthracis anthrax toxin, PA (protective antigen). We show that Cas9 variants fused to the N-terminus of the lethal factor or to a hexahistidine tag are shuttled through channels formed by PA into the cytosol of human cells. As proof-of-principle, we applied our new approach, denoted as CRISPA, to knock out lipolysis-stimulated lipoprotein receptor (LSR) in the human colon cancer cell line HCT116 and green-fluorescent protein (GFP) in human embryonic kidney 293T cells stably expressing GFP. Notably, we confirmed that the transporter PA can be adapted to recognize specific host cell-surface receptor proteins and may be optimized for cell type-selective delivery of Cas9. Altogether, CRISPA provides a novel, transient and non-viral way to deliver Cas9 into specific cells. Thus, this system is an additional step towards safe translation of the CRISPR/Cas9 technology into clinics.

NG2 (nerve/glial antigen 2) glia are uniformly distributed in the gray and white matter of the central nervous system (CNS). They are the major proliferating cells in the brain and can differentiate into oligodendrocytes. NG2 glia do not only receive synaptic input from excitatory and inhibitory neurons, but also secrete growth factors and cytokines, modulating CNS homeostasis. They express several receptors and ion channels that play a role in rapidly responding upon synaptic signals and generating fast feedback, potentially regulating their own properties. Ca2+ influx via voltage-gated Ca2+ channels (VGCCs) induces an intracellular Ca2+ rise initiating a series of cellular activities. We confirmed that NG2 glia express L-type VGCCs in the white and gray matter during CNS development, particularly in the early postnatal stage. However, the function of L-type VGCCs in NG2 glia remains elusive. Therefore, we deleted L-type VGCC subtypes Cav1.2 and Cav1.3 genes conditionally in NG2 glia by crossbreeding NG2-CreERT2 knock-in mice to floxed Cav1.2 and flexed Cav1.3 transgenic mice. Our results showed that ablation of Cav1.2 and Cav1.3 strongly inhibited the proliferation of cortical NG2 glia, while differentiation in white and gray matter was not affected. As a consequence, no difference on myelination could be detected in various brain regions. In addition, we observed morphological alterations of the nodes of Ranvier induced by VGCC-deficient NG2 glia, i.e., shortened paired paranodes in the corpus callosum. Furthermore, deletion of Cav1.2 and Cav1.3 largely eliminated N-methyl-D-aspartate (NMDA)-dependent long-term depression (LTD) and potentiation in the hippocampus while the synaptic input to NG2 glia from axons remained unaltered. We conclude that L-type VGCCs of NG2 glia are essential for cell proliferation and proper structural organization of nodes of Ranvier, but not for differentiation and myelin compaction. In addition, L-type VGCCs of NG2 glia contribute to the regulation of long-term neuronal plasticity.