PACS-1 Interacts with TRPC3 and ESyt1 to Mediate Protein Trafficking while Promoting SOCE and Cooperatively Regulating Hormone Secretion.

Corticotropic cells of the anterior pituitary gland release adrenocorticotropic hormone (ACTH) in a regulated manner to promote the production of cortisol and androgens. The process of ACTH secretion is partly mediated by the phosphofurin acidic cluster sorting protein 1 (PACS-1); however, the underlying mechanisms behind this regulation remain unclear. Herein, we demonstrated PACS-1 interactions with the short transient receptor potential channel 3 (TRPC3) calcium transporter and the extended synaptotagmin-1 (ESyt1) endoplasmic reticulum-plasma membrane tethering protein. Importantly, PACS-1 promoted interactions between TRPC3 and ESyt1 and regulated their plasma membrane localization. Lastly, we demonstrated that PACS-1 is required for a proper store-operated calcium entry (SOCE) response and that ESyt1 regulates ACTH secretion through an unknown mechanism regulated by PACS-1. Overall, our study provides new insights into the physiological role PACS-1 plays in modulating intracellular calcium levels and regulating ACTH secretion in corticotropic cells.

SARS-CoV-2 NSP5 antagonizes MHC II expression by subverting histone deacetylase 2.

SARS-CoV-2 interferes with antigen presentation by downregulating major histocompatibility complex (MHC) II on antigen-presenting cells, but the mechanism mediating this process is unelucidated. Herein, analysis of protein and gene expression in human antigen-presenting cells reveals that MHC II is downregulated by the SARS-CoV-2 main protease, NSP5. This suppression of MHC II expression occurs via decreased expression of the MHC II regulatory protein CIITA. CIITA downregulation is independent of the proteolytic activity of NSP5, and rather, NSP5 delivers HDAC2 to the transcription factor IRF3 at an IRF-binding site within the CIITA promoter. Here, HDAC2 deacetylates and inactivates the CIITA promoter. This loss of CIITA expression prevents further expression of MHC II, with this suppression alleviated by ectopic expression of CIITA or knockdown of HDAC2. These results identify a mechanism by which SARS-CoV-2 limits MHC II expression, thereby delaying or weakening the subsequent adaptive immune response.

Sensing Dying Cells in Health and Disease: The Importance of Kidney Injury Molecule-1.

Kidney injury molecule-1 (KIM-1), also known as T-cell Ig and mucin domain-1 (TIM-1), is a widely recognized biomarker for AKI, but its biological function is less appreciated. KIM-1/TIM-1 belongs to the T-cell Ig and mucin domain family of conserved transmembrane proteins, which bear the characteristic six-cysteine Ig-like variable domain. The latter enables binding of KIM-1/TIM-1 to its natural ligand, phosphatidylserine, expressed on the surface of apoptotic cells and necrotic cells. KIM-1/TIM-1 is expressed in a variety of tissues and plays fundamental roles in regulating sterile inflammation and adaptive immune responses. In the kidney, KIM-1 is upregulated on injured renal proximal tubule cells, which transforms them into phagocytes for clearance of dying cells and helps to dampen sterile inflammation. TIM-1, expressed in T cells, B cells, and natural killer T cells, is essential for cell activation and immune regulatory functions in the host. Functional polymorphisms in the gene for KIM-1/TIM-1, HAVCR1 , have been associated with susceptibility to immunoinflammatory conditions and hepatitis A virus-induced liver failure, which is thought to be due to a differential ability of KIM-1/TIM-1 variants to bind phosphatidylserine. This review will summarize the role of KIM-1/TIM-1 in health and disease and its potential clinical applications as a biomarker and therapeutic target in humans.

Acetylenic tricyclic bis-(cyano enone) interacts with Cys 374 of actin, a residue necessary for stress fiber formation and cell migration.

The migratory and invasive potential of tumour cells relies on the actin cytoskeleton. We previously demonstrated that the tricyclic compound, TBE-31, inhibits actin polymerization and here we further examine the precise interaction between TBE-31 and actin. We demonstrate that iodoacetamide, a cysteine (Cys) alkylating agent, interferes with the ability of TBE-31 to interact with actin. In addition, in silico analysis identified Cys 217, Cys 272, Cys 285 and Cys 374 as potential binding sites for TBE-31. Using mass spectrometry analysis, we determined that TBE-31 associates with actin with a stoichiometric ratio of 1:1. We mutated the identified cysteines of actin to alanine and performed a pull-down analysis with a biotin labeled TBE-31 and demonstrated that by mutating Cys 374 to alanine the association between TBE-31 and actin was significantly reduced, suggesting that TBE-31 binds to Cys 374. A characterization of the NIH3T3 cells overexpressing eGFP-actin-C374A showed reduced stress fiber formation, suggesting Cys 374 is necessary for efficient incorporation into filamentous actin. Furthermore, migration of eGFP-Actin-WT expressing cells were observed to be inhibited by TBE-31, however fewer eGFP-Actin-C374A expressing cells were observed to migrate compared to the cells expressing eGFP-Actin-WT in the presence or absence of TBE-31. Taken together, our results suggest that TBE-31 binds to Cys 374 of actin to inhibit actin stress fiber formation and may potentially be a mechanism through which TBE-31 inhibits cell migration.

In vivo tracking of adenoviral-transduced iron oxide-labeled bone marrow-derived dendritic cells using magnetic particle imaging.

BACKGROUND: Despite widespread study of dendritic cell (DC)-based cancer immunotherapies, the in vivo postinjection fate of DC remains largely unknown. Due in part to a lack of quantifiable imaging modalities, this is troubling as the amount of DC migration to secondary lymphoid organs correlates with therapeutic efficacy. Magnetic particle imaging (MPI) has emerged as a suitable modality to quantify in vivo migration of superparamagnetic iron oxide (SPIO)-labeled DC. Herein, we describe a popliteal lymph node (pLN)-focused MPI scan to quantify DC in vivo migration accurately and consistently. METHODS: Adenovirus (Ad)-transduced SPIO+ (Ad SPIO+) and SPIO+ C57BL/6 bone marrow-derived DC were generated and assessed for viability and phenotype, then fluorescently labeled and injected into mouse hind footpads (n = 6). Two days later, in vivo DC migration was quantified using whole animal, pLN-focused, and ex vivo pLN MPI scans. RESULTS: No significant differences in viability, phenotype and in vivo pLN migration were noted for Ad SPIO+ and SPIO+ DC. Day 2 pLN-focused MPI quantified DC migration in all instances while whole animal MPI only quantified pLN migration in 75% of cases. Ex vivo MPI and fluorescence microscopy confirmed that pLN MPI signal was due to originally injected Ad SPIO+ and SPIO+ DC. CONCLUSION: We overcame a reported limitation of MPI by using a pLN-focused MPI scan to quantify pLN-migrated Ad SPIO+ and SPIO+ DC in 100% of cases and detected as few as 1000 DC (4.4 ng Fe) in vivo. MPI is a suitable preclinical imaging modality to assess DC-based cancer immunotherapeutic efficacy. RELEVANCE STATEMENT: Tracking the in vivo fate of DC using noninvasive quantifiable magnetic particle imaging can potentially serve as a surrogate marker of therapeutic effectiveness. KEY POINTS: • Adenoviral-transduced and iron oxide-labeled dendritic cells are in vivo migration competent. • Magnetic particle imaging is a suitable modality to quantify in vivo dendritic cell migration. • Magnetic particle imaging focused field of view overcomes dynamic range limitation.

Phylogenetic Reconstruction and Functional Characterization of the Ancestral Nef Protein of Primate Lentiviruses.

Nef is an accessory protein unique to the primate HIV-1, HIV-2, and SIV lentiviruses. During infection, Nef functions by interacting with multiple host proteins within infected cells to evade the immune response and enhance virion infectivity. Notably, Nef can counter immune regulators such as CD4 and MHC-I, as well as the SERINC5 restriction factor in infected cells. In this study, we generated a posterior sample of time-scaled phylogenies relating SIV and HIV Nef sequences, followed by reconstruction of ancestral sequences at the root and internal nodes of the sampled trees up to the HIV-1 Group M ancestor. Upon expression of the ancestral primate lentivirus Nef protein within CD4+ HeLa cells, flow cytometry analysis revealed that the primate lentivirus Nef ancestor robustly downregulated cell-surface SERINC5, yet only partially downregulated CD4 from the cell surface. Further analysis revealed that the Nef-mediated CD4 downregulation ability evolved gradually, while Nef-mediated SERINC5 downregulation was recovered abruptly in the HIV-1/M ancestor. Overall, this study provides a framework to reconstruct ancestral viral proteins and enable the functional characterization of these proteins to delineate how functions could have changed throughout evolutionary history.

Optimal Expression, Function, and Immunogenicity of an HIV-1 Vaccine Derived from the Approved Ebola Vaccine, rVSV-ZEBOV.

Vesicular stomatitis virus (VSV) remains an attractive platform for a potential HIV-1 vaccine but hurdles remain, such as selection of a highly immunogenic HIV-1 Envelope (Env) with a maximal surface expression on recombinant rVSV particles. An HIV-1 Env chimera with the transmembrane domain (TM) and cytoplasmic tail (CT) of SIVMac239 results in high expression on the approved Ebola vaccine, rVSV-ZEBOV, also harboring the Ebola Virus (EBOV) glycoprotein (GP). Codon-optimized (CO) Env chimeras derived from a subtype A primary isolate (A74) are capable of entering a CD4+/CCR5+ cell line, inhibited by HIV-1 neutralizing antibodies PGT121, VRC01, and the drug, Maraviroc. The immunization of mice with the rVSV-ZEBOV carrying the CO A74 Env chimeras results in anti-Env antibody levels as well as neutralizing antibodies 200-fold higher than with the NL4-3 Env-based construct. The novel, functional, and immunogenic chimeras of CO A74 Env with the SIV_Env-TMCT within the rVSV-ZEBOV vaccine are now being tested in non-human primates.

The Staphylococcus aureus protein IsdA increases SARS CoV-2 replication by modulating JAK-STAT signaling.

The Severe Acute Respiratory Syndrome Coronavirus 2 (CoV-2) pandemic has affected millions globally. A significant complication of CoV-2 infection is secondary bacterial co-infection, as seen in approximately 25% of severe cases. The most common organism isolated during co-infection is Staphylococcus aureus. Here, we describe the development of an in vitro co-infection model where both viral and bacterial replication kinetics may be examined. We demonstrate CoV-2 infection does not alter bacterial interactions with host epithelial cells. In contrast, S. aureus enhances CoV-2 replication by 10- to 15-fold. We identify this pro-viral activity is due to the S. aureus iron-regulated surface determinant A (IsdA) protein and demonstrate IsdA modifies host transcription. We find that IsdA alters Janus Kinase - Signal Transducer and Activator of Transcription (JAK-STAT) signaling, by affecting JAK2-STAT3 levels, ultimately leading to increased viral replication. These findings provide key insight into the molecular interactions between host cells, CoV-2 and S. aureus during co-infection.

Virology under the Microscope-a Call for Rational Discourse.

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.

Virology under the Microscope-a Call for Rational Discourse.

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.

Virology under the Microscope-a Call for Rational Discourse.

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.

Correction: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2.

[This corrects the article DOI: 10.1371/journal.ppat.1010092.].

The Phosphofurin Acidic Cluster Sorting Protein 2 (PACS-2) E209K Mutation Responsible for PACS-2 Syndrome Increases Susceptibility to Apoptosis.

Phosphofurin acidic cluster sorting protein 2 (PACS-2) is a multifunctional cytosolic membrane trafficking protein with distinct roles in maintaining cellular homeostasis. Recent clinical reports have described 28 individuals possessing a de novo PACS-2 E209K mutation that present with epileptic seizures and cerebellar dysgenesis. As the PACS-2 E209K missense mutation has become a marker for neurodevelopmental disorders, we sought to characterize its biochemical properties. Accordingly, we observed that the PACS-2 E209K protein exhibited a slower turnover rate relative to PACS-2 wild type (WT) upon cycloheximide treatment in 293T cells. The longer half-life of PACS-2 E209K suggests a disruption in its proteostasis, with the potential for altered protein-protein interactions. Indeed, a regulatory protein in neurodevelopment known as 14-3-3ε was identified as having an increased association with PACS-2 E209K. Subsequently, when comparing the effect of PACS-2 WT and E209K expression on the staurosporine-induced apoptosis response, we found that PACS-2 E209K increased susceptibility to staurosporine-induced apoptosis in HCT 116 cells. Overall, our findings suggest PACS-2 E209K alters PACS-2 proteostasis and favors complex formation with 14-3-3ε, leading to increased cell death in the presence of environmental stressors.

Identification and differential usage of a host metalloproteinase entry pathway by SARS-CoV-2 Delta and Omicron.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike glycoprotein (S) binds to angiotensin-converting enzyme 2 (ACE2) to mediate membrane fusion via two distinct pathways: 1) a surface, serine protease-dependent or 2) an endosomal, cysteine protease-dependent pathway. In this study, we found that SARS-CoV-2 S has a wider protease usage and can also be activated by TMPRSS13 and matrix metalloproteinases (MMPs). We found that MMP-2 and MMP-9 played roles in SARS-CoV-2 S cell-cell fusion and TMPRSS2- and cathepsin-independent viral entry in cells expressing high MMP levels. MMP-dependent viral entry required cleavage at the S1/S2 junction in viral producer cells, and differential processing of variants of concern S dictated its usage; the efficiently processed Delta S preferred metalloproteinase-dependent entry when available, and less processed Omicron S was unable to us metalloproteinases for entry. As MMP-2/9 are released during inflammation, they may play roles in S-mediated cytopathic effects, tropism, and disease outcome.

Magnetic Particle Imaging Is a Sensitive In Vivo Imaging Modality for the Detection of Dendritic Cell Migration.

PURPOSE: The purpose of this study was to evaluate magnetic particle imaging (MPI) as a method for the in vivo tracking of dendritic cells (DC). DC are used in cancer immunotherapy and must migrate from the site of implantation to lymph nodes to be effective. The magnitude of the ensuing T cell response is proportional to the number of lymph node-migrated DC. With current protocols, less than 10% of DC are expected to reach target nodes. Therefore, imaging techniques for studying DC migration must be sensitive and quantitative. Here, we describe the first study using MPI to detect and track DC injected into the footpads of C57BL/6 mice migrating to the popliteal lymph nodes (pLNs). PROCEDURES: DC were labelled with Synomag-D™ and injected into each hind footpad of C57BL/6 mice (n = 6). In vivo MPI was conducted immediately and repeated 48 h later. The MPI signal was measured from images and related to the signal from a known number of cells to calculate iron content. DC numbers were estimated by dividing iron content in the image by the iron per cell measured from a separate cell sample. The presence of SPIO-labeled DC in nodes was validated by ex vivo MPI, histology, and fluorescence microscopy. RESULTS: Day 2 imaging showed a decrease in MPI signal in the footpads and an increase in signal at the pLNs, indicating DC migration. MPI signal was detected in the left pLN in four of the six mice and two of the six mice showed MPI signal in the right pLN. Ex vivo imaging detected signal in 11/12 nodes. We report a sensitivity of approximately 4000 cells (0.015 µg Fe) in vivo and 2000 cells (0.007 µg Fe) ex vivo. CONCLUSIONS: Here, we describe the first study to use MPI to detect and track DC in a migration model with immunotherapeutic applications. We also bring attention to the issue of resolving unequal signals within close proximity, a challenge for any pre-clinical study using a highly concentrated tracer bolus that shadows nearby lower signals.

Inhibiteurs de Nef du VIH-1 : applications et développements pour une guérison efficace.

Résumé La thérapie anti-rétrovirale peut contrôler la réplication du virus de l'immunodéficience humaine de type 1 (VIH-1) chez les individus vivant avec le VIH. Par contre, ces traitements ne constituent pas une guérison et aucune approche pour une guérison du VIH-1 n'a encore montré de succès lors des études cliniques. Les approches de guérison sont souvent contrées in vivo par des barrières développées par le VIH-1. L'inhibition pharmacologique de la protéine accessoire Nef du VIH-1 représente une approche ambitieuse et prometteuse pour développer une nouvelle stratégie de guérison. Des petites molécules inhibitrices de Nef peuvent inverser les défauts reliés à l'infection par le VIH dans la signalisation des récepteurs des cellules T et les kinases, l'apoptose, l'autophagie et surtout, la présentation d'antigène. Ensemble, ces activités démontrent la grande capacité des inhibiteurs de Nef à être appliqués comme agents thérapeutiques dans un traitement contre le VIH-1. Dans cette revue, nous présentons les motifs pour lesquels Nef constitue une cible thérapeutique et nous soulignons les progrès effectués dans l'identification et le développement d'inhibiteurs de Nef.

Inhibitors of HIV-1 Nef: applications and developments for a practical cure.

Antiretroviral therapy can control human immunodeficiency virus type 1 (HIV-1) replication in people living with HIV; however, these treatments are not curative and no practical approach for an HIV-1 cure has yet shown success in clinical trials. Counteracting the multiple barriers HIV-1 presents against a practical cure is a direct means to functionalize these curative approaches in vivo. Pharmacological inhibition of the HIV-1 accessory protein, Nef, represents a particularly promising and ambitious approach, with Nef inhibitors holding the potential to reverse HIV-1-related defects in T cell receptor and kinase signaling, apoptosis, autophagy and most importantly, antigen presentation. Together, the capacity for Nef inhibitors to restore these activities underscores their potential as supportive agents in a practical HIV-1 cure. In this review, we outline a rationale for pharmacologically targeting Nef and review the progress made in the identification and development of Nef inhibitors.

Bimolecular Fluorescence Complementation to Visualize Protein-Protein Interactions in Cells.

Examining protein-protein interactions provides critical insight into numerous human diseases and infections. Here we describe a protocol for bimolecular fluorescence complementation, which can be used to directly visualize and characterize intracellular protein-protein interactions and ascertain their localization using fluorescence microscopy.

PACS-1 contains distinct motifs for nuclear-cytoplasmic transport and interacts with the RNA-binding protein PTBP1 in the nucleus and cytosol.

Phosphofurin acidic cluster sorting protein 1 (PACS-1) is canonically a cytosolic trafficking protein, yet recent reports have described nuclear roles for PACS-1. Herein, we sought to define the nuclear transport mechanism of PACS-1. We demonstrate that PACS-1 nucleocytoplasmic trafficking is dependent on its interaction with the nuclear transport receptors importin alpha 5 and exportin 1. PACS-1 nuclear entry and exit are defined by a nuclear localization signal (NLS, residues 311-318) and nuclear export signal (NES3, residues 366-375). Mutation of the PACS-1 NLS and NES3 altered the localization of a complex formed between PACS-1 and an RNA-binding protein, polypyrimidine tract-binding protein 1. Overall, we identify the nuclear localization mechanism of PACS-1 and highlight a potential role for PACS-1 in RNA-binding protein trafficking.

KIM-1/TIM-1 is a Receptor for SARS-CoV-2 in Lung and Kidney.

SARS-CoV-2 precipitates respiratory distress by infection of airway epithelial cells and is often accompanied by acute kidney injury. We report that Kidney Injury Molecule-1/T cell immunoglobulin mucin domain 1 (KIM-1/TIM-1) is expressed in lung and kidney epithelial cells in COVID-19 patients and is a receptor for SARS-CoV-2. Human and mouse lung and kidney epithelial cells express KIM-1 and endocytose nanoparticles displaying the SARS-CoV-2 spike protein (virosomes). Uptake was inhibited by anti-KIM-1 antibodies and TW-37, a newly discovered inhibitor of KIM-1-mediated endocytosis. Enhanced KIM-1 expression by human kidney tubuloids increased uptake of virosomes. KIM-1 binds to the SARS-CoV-2 Spike protein in vitro . KIM-1 expressing cells, not expressing angiotensin-converting enzyme 2 (ACE2), are permissive to SARS-CoV-2 infection. Thus, KIM-1 is an alternative receptor to ACE2 for SARS-CoV-2. KIM-1 targeted therapeutics may prevent and/or treat COVID-19.