Cytoplasmic Tail Domain Research Articles

Newcastle Disease Virus Expressing Cap Gene of Porcine Circovirus Type 2 Confers Protection in Mice and Induced Long-Lasting Neutralizing Antibodies in Pigs.

Background/Objectives: Porcine Circovirus 2 (PCV2) infection poses significant health and economic challenges to the global swine industry. The disease in pigs leads to lymphoid depletion, resulting in immunosuppression and increased susceptibility to co-infections with other bacterial and viral pathogens. This study evaluated the efficacy of two novel recombinant Newcastle disease virus (NDV) strain R2B vectored vaccines that express the cap gene of PCV2 alone and along with the transmembrane and cytoplasmic tail (TMCT) domains of the NDV F gene. The efficacy of the vaccine candidates was studied in mouse and pig models. Methods: Six-week-old BALB/c mice were divided into five groups and immunized intramuscularly three times at 14-day intervals with various vaccine candidates, namely rNDV-R2B-PCVcap-TMCT, rNDV-R2B-PCVcap, and CircoFLEX commercial vaccine, along with controls. Following immunization and PCV2d virus challenge, multiple assays assessed the immune responses in animal trials. In the pig animal trial, pigs were divided into four groups: a control group (PBS), NDV-vectored PCVcap-TMCT group, NDV-vectored-PCVcap group, and CircoFLEX vaccine group. Pigs were immunized intramuscularly twice at 28-day intervals. Blood samples were collected at regular intervals over 70 days to evaluate the humoral and cell-mediated immune responses. Results: Both mice and pigs' trials indicated that the NDV-vectored PCV2 cap-TMCT vaccine candidate elicited superior immune responses. In mice, the rNDV-R2B-PCVcap-TMCT group showed enhanced humoral and cellular immunity, increased PCV2-specific antibody levels, higher CD4+/CD8+ ratio, elevated IFN-γ and TNF-α levels, decreased IL-10 levels, reduced viral loads, and minimal histopathological changes. In pigs, the NDV-vectored PCVcap-TMCT group demonstrated better antibody responses, cytokine profiles (IFN-γ and IL-10), and higher levels of PCV2-specific neutralizing antibodies against the PCV2a, PCV2b and PCV2d genotypes when compared to other groups. Conclusions: These findings suggest NDV-vectored PCVcap-TMCT vaccine candidate, expressing the cap gene of PCV2 along with the TMCT domain, offers a promising alternative for protecting against PCV2 infection, potentially addressing the challenges posed by emerging PCV2 strains in the swine industry.

Role of protein domains in trafficking and localization of the voltage-gated sodium channel β2 subunit

The voltage-gated sodium (NaV) channel is critical for cardiomyocyte function since it is responsible for action potential initiation and its propagation throughout the cell. It consists of a protein complex made of a pore forming α subunit and associated β subunits, which regulate α subunit function and subcellular localization. We previously showed the implication of N-linked glycosylation and S-acylation of β2 in its polarized trafficking. Here, we present evidence of β2 dimerization. Moreover, we demonstrate the implication of the cytoplasmic tail, extracellular loop, and transmembrane domain on proper β2 folding and export to the cell surface of polarized Madin-Darby canine kidney cells. Substantial alteration, or lack of any of these domains, leads to accumulation of β2 in the endoplasmic reticulum, along with impaired complex N-glycosylation, which is needed for its efficient surface delivery. We also show that these alterations to β2 affected to certain extent NaV1.5 surface localization. Conversely, however, NaV1.5 had little or no influence on β2 trafficking, its localization to the surface, or homodimer formation. Altogether, our data link the architecture of the β2 domains to the establishment of its proper subcellular localization. These findings could provide valuable insights to gain a deeper comprehension of the elusive biology of β subunits in excitable cells, such as neurons and cardiomyocytes.

Coexistence of pulmonary arterial hypertension and straight back syndrome in a patient with a novel BMPR2 variant affecting cytoplasmic tail domain

BackgroundPathologic variants in the bone morphogenetic protein receptor-2 (BMPR2) gene cause a pulmonary arterial hypertension phenotype in an autosomal-dominant pattern with incomplete penetrance. Straight back syndrome is one of the causes of pseudo-heart diseases. To date, no cases of idiopathic or heritable pulmonary arterial hypertension with straight back syndrome have been reported.Case presentationA 30-year-old female was diagnosed with pulmonary arterial hypertension by right heart catheterization. Computed tomography revealed a decreased anteroposterior thoracic space with heart compression, indicating a straight back syndrome. Genetic analysis by whole exome sequencing identified a novel c.2423_2424delGT (p.G808Gfs*4) germline frameshift variant within BMPR2 affecting the cytoplasmic tail domain.ConclusionsThis is the first report of different straight back characteristics in heritable pulmonary arterial hypertension with a novel germline BMPR2 variant. This finding may provide a new perspective on the variable penetrance of the pulmonary arterial hypertension phenotype.

Characterization of target gene regulation by the two Epstein-Barr virus oncogene LMP1 domains essential for B-cell transformation.

Epstein-Barr virus (EBV) causes multiple human cancers, including B-cell lymphomas. In cell culture, EBV converts healthy human B-cells into immortalized ones that grow continuously, which model post-transplant lymphomas. Constitutive signaling from two cytoplasmic tail domains of the EBV oncogene latent membrane protein 1 (LMP1) is required for this transformation, yet there has not been systematic analysis of their host gene targets. We identified that only signaling from the membrane proximal domain is required for survival of these EBV-immortalized cells and that its loss triggers apoptosis. We identified key LMP1 target genes, whose abundance changed significantly with loss of LMP1 signals, or that were instead upregulated in response to switching on signaling by one or both LMP1 domains in an EBV-uninfected human B-cell model. These included major anti-apoptotic factors necessary for EBV-infected B-cell survival. Bioinformatics analyses identified clusters of B-cell genes that respond differently to signaling by either or both domains.

Connexin 37 sequestering of activated-ERK in the cytoplasm promotes p27-mediated endothelial cell cycle arrest.

Connexin37-mediated regulation of cell cycle modulators and, consequently, growth arrest lack mechanistic understanding. We previously showed that arterial shear stress up-regulates Cx37 in endothelial cells and activates a Notch/Cx37/p27 signaling axis to promote G1 cell cycle arrest, and this is required to enable arterial gene expression. However, how induced expression of a gap junction protein, Cx37, up-regulates cyclin-dependent kinase inhibitor p27 to enable endothelial growth suppression and arterial specification is unclear. Herein, we fill this knowledge gap by expressing wild-type and regulatory domain mutants of Cx37 in cultured endothelial cells expressing the Fucci cell cycle reporter. We determined that both the channel-forming and cytoplasmic tail domains of Cx37 are required for p27 up-regulation and late G1 arrest. Mechanistically, the cytoplasmic tail domain of Cx37 interacts with, and sequesters, activated ERK in the cytoplasm. This then stabilizes pERK nuclear target Foxo3a, which up-regulates p27 transcription. Consistent with previous studies, we found this Cx37/pERK/Foxo3a/p27 signaling axis functions downstream of arterial shear stress to promote endothelial late G1 state and enable up-regulation of arterial genes.

BST2, a Novel Inhibitory Receptor, Is Involved in NK Cell Cytotoxicity through Its Cytoplasmic Tail Domain.

Bone Marrow Stromal Cell Antigen 2 (BST2) is a type II transmembrane protein expressed on various cell types that tethers the release of viruses. Natural killer (NK) cells express low levels of BST2 under normal conditions but exhibit increased expression of BST2 upon activation. In this study, we show for the first time that murine BST2 can control the cytotoxicity of NK cells. The cytoplasmic tail of murine BST2 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM). The absence of BST2 on NK cells can enhance their cytotoxicity against tumor cells compared to wild type NK cells. NK cells isolated from NZW mice, which express ITIM-deficient BST2, also showed higher cytotoxicity than wild type NK cells. In addition, we found that galectin-8 and galectin-9 were ligands of BST2, since blocking galectin-8 or -9 with monoclonal antibodies enhanced the cytotoxicity of NK cells. These results suggested that BST2 might be a novel NK cell inhibitory receptor as it was involved in regulating NK cell cytotoxicity through its interaction with galectins.

P-selectin mobility undergoes a sol-gel transition as it diffuses from exocytosis sites into the cell membrane

In response to vascular damage, P-selectin molecules are secreted onto the surface of cells that line our blood vessels. They then serve as mechanical anchors to capture leucocytes from the blood stream. Here, we track individual P-selectin molecules released at the surface of live endothelial cells following stimulated secretion. We find P-selectin initially shows fast, unrestricted diffusion but within a few minutes, movement becomes increasingly restricted and ~50% of the molecules become completely immobile; a process similar to a sol-gel transition. We find removal of the extracellular C-type lectin domain (ΔCTLD) and/or intracellular cytoplasmic tail domain (ΔCT) has additive effects on diffusive motion while disruption of the adapter complex, AP2, or removal of cell-surface heparan sulphate restores mobility of full-length P-selectin close to that of ΔCT and ΔCTLD respectively. We have found P-selectin spreads rapidly from sites of exocytosis and evenly decorates the cell surface, but then becomes less mobile and better-suited to its mechanical anchoring function.

Protein Kinase A Catalytic subunit:Smoothened interface ‐ Role of a parallel holoenzyme in negative feedback in Hedgehog Signaling

During signal transduction in the Hedgehog pathway, the G protein‐coupled receptor (GPCR) Smoothened (SMO) signals intracellularly via a novel mechanism involving sequestration of the Protein Kinase A (PKA) catalytic (C)‐subunit at the membrane. Activation of Hedgehog signaling leads to negative feedback regulation mediated through inhibition of PKA‐C by SMO at the ciliary membrane. While it is known that PKA‐C interacts with SMO at its C‐terminal proximal cytoplasmic tail domain (pCT), the dynamics of PKA‐C:SMO interactions is poorly understood. Here, we report a map of the PKA‐C:SMO interface by Amide Hydrogen Deuterium Exchange Mass spectrometry (HDXMS) over 1‐10 min timescales. SMO binding on PKA‐C resulted in global decreases in deuterium exchange (Dex). Peptides spanning the kinase activation loop (188‐198), P+1 loop (199‐211) and pseudo‐substrate recognition site between β5 and αD (129‐145) showed significant protection (>0.5 Da) when bound to SMO, identifying these regions as the SMO binding interface on PKA‐C. Interestingly, the footprint of SMO binding on PKA‐C was identical to that of the RIa regulatory subunit in PKA‐I holoenzymes. Furthermore, increased Dex (0.4 Da) was observed at the region spanning αC and β4 (83‐100), indicating that SMO binding destabilizes the active kinase conformation to allosterically inhibit PKA‐C. Correspondingly on SMO, PKA‐C binding elicited only low magnitudes of Dex protection (0.4 Da) in regions flanking the pseudo substrate site (573‐590, 622‐631). Increased Dex (0.4 Da) was observed at the sterol binding pocket (96‐112) in the extracellular Cysteine rich domain, indicating that PKA‐C binding may allosterically impact SMO activation by modulating its ability to bind sterols. A significant increase in Dex (0.9 Da) was observed at SMO helix 8 (537‐546) which is positioned parallel to the membrane, indicating that C binding promoted a disordered helix 8 conformation. We speculate that the pCT exists in a membrane‐bound or folded conformation in free SMO, but PKA‐C interaction switches the binding mode of pCT leading to a significantly Dex‐protected state. Thus, PKA‐C:SMO interactions reveal an unexpected intersection between cyclic nucleotide signaling and the Hedgehog pathway, defining a novel mechanism of PKA‐C regulation beyond the canonical PKA signalosome.

The primary structure of the cytoplasmic tail domain of influenza hemagglutinin regulates its co-clustering with phosphatidylinositol 4,5-bisphosphate

The influenza glycoprotein hemagglutinin (HA) is highly involved in the attachment, entry, and assembly stages of the viral life cycle. Previous work has observed co-localization and evidence for an interaction between HA and the host cell lipid phosphatidylinositol 4,5-bisphosphate (PIP2) (Curthoys et al. 2019 Biophysical J. 116:893), although the details of this interaction are not yet fully understood. While mutations in HA occur frequently, in general there are 3 cysteines and 1-2 basic residues in the cytoplasmic tail domain (CTD) which remain highly conserved.

Improved SARS-CoV-2 Spike Glycoproteins for Pseudotyping Lentiviral Vectors

The spike (S) glycoprotein of SARS-Cov-2 facilitates viral entry into target cells via the cell surface receptor angiotensin-converting enzyme 2 (ACE2). Third generation HIV-1 lentiviral vectors can be pseudotyped to replace the native CD4 tropic envelope protein of the virus and thereby either limit or expand the target cell population. We generated a modified S glycoprotein of SARS-Cov-2 to pseudotype lentiviral vectors which efficiently transduced ACE2-expressing cells with high specificity and contain minimal off-target transduction of ACE2 negative cells. By utilizing optimized codons, modifying the S cytoplasmic tail domain, and including a mutant form of the spike protein, we generated an expression plasmid encoding an optimized protein that produces S-pseudotyped lentiviral vectors at an infectious titer (TU/mL) 1000-fold higher than the unmodified S protein and 4 to 10-fold more specific than the widely used delta-19 S-pseudotyped lentiviral vectors. S-pseudotyped replication-defective lentiviral vectors eliminate the need for biosafety-level-3 laboratories required when developing therapeutics against SARS-CoV-2 with live infectious virus. Furthermore, S-pseudotyped vectors with high activity and specificity may be used as tools to understand the development of immunity against SARS-CoV-2, to develop assays of neutralizing antibodies and other agents that block viral binding, and to allow in vivo imaging studies of ACE2-expressing cells.

Anti-Cancer Potential of Afzelin towards AGS Gastric Cancer Cells.

Afzelin demonstrates anti-inflammatory and anti-cancer properties. Our purpose was to assess its influence on apoptosis, Bax, caspases, MUC1, cancer-related carbohydrate antigens, enzymes participating in their formation, and galectin-3 in AGS gastric cancer cells. A total of 60 and 120 μM afzelin was used in all experiments. Flow cytometry was applied to determine apoptotic response. Western blotting and RT PCR were used to detect the expression of mentioned factors. Flavonoid at higher concentration revealed slight apoptotic respond. Bax, caspase-3, -8, -9 increased upon afzelin action. Stimulatory effect of the flavonoid on MUC1 cytoplasmic tail and extracellular domain in cell lysates and on MUC1 gene was revealed. MUC1 release into the culture medium was inhibited by the flavonoid. The 60 μM afzelin dose stimulated GalNAcTL5 protein expression and inhibited C1GalT1. ST6GalNAcT mRNA was inhibited by both flavonoid doses. ST3GalT was inhibited by 120 μM afzelin on protein and mRNA level. Lewisa/b protein was reduced by both afzelin concentrations. FUT3 and FUT4 mRNA was inhibited by 120 μM dose of afzelin. Galectin-3 protein increased in cell lysates and decreased in culture supernatant by 60 and 120 μM flavonoid. Galectin-3 gene expression was stimulated by two used concentrations of afzelin in comparison to control. We conclude that afzelin can be considered as the potential anti-cancer agent, supporting conventional cancer treatment.

Generation and Evaluation of Recombinant Thermostable Newcastle Disease Virus Expressing the HA of H9N2 Avian Influenza Virus.

H9N2 avian influenza virus (AIV) has become endemic in many countries, causing great economic losses when co-infected with other pathogens. So far, several live vaccines based on Newcastle disease virus (NDV) vectors expressing influenza hemagglutinin (HA) have been developed. However, the thermostable recombinant NDV is rarely reported. In this study, using a thermostable NDV rAHR09 strain as the vector, three recombinant NDVs expressing native HA, chimeric HA ectodomain with transmembrane domain/C-terminal cytoplasmic tail domain from fusion protein of NDV, and HA ectodomain were generated, designated rAHR09-HA, rAHR09-HAF, and rAHR09-HAE. The MDT value of three recombinant NDVs was above 120 h, their ICPI value was about 0.03, and the recombinant NDVs were still infectious when treated for 100 min under 56 °C, which demonstrated that the recombinant NDVs kept the lentogenic and thermostable nature of rAHR09. The immunization data showed that rAHR09-HA and rAHR09-HAF induced a higher HI antibody titer against H9N2 AIV and NDV. After being challenged with H9N2 AIV, the rAHR09-HA and rAHR09-HAF could significantly reduce the virus shedding in cloacal and tracheal swab samples. Our results suggest that rAHR09-HA and rAHR09-HAF might be vaccine candidates against H9N2 AIV.

Structural and Functional Characterization of a Single Chain β3 Integrin in Solution and on the Cell Surface and the Implications on Integrin Activation and Pathogenesis of Anti-HPA-1a Alloantibodies

The β3 integrins, including αIIbβ3 and αVβ3, are among the most well studied integrins. αIIbβ3 is specific to platelets and essential for hemostasis and thrombosis. αVβ3 is ubiquitously expressed in many cell types, including platelets and endothelial cells, and is important for cell growth and survival. The β3 subunit contains the headpiece formed by PSI, hybrid, βI, and I-EGF1 domains, the leg piece formed by the I-EGF 2-4 and β-tail domains, a transmembrane (TM) domain and a short cytoplasmic tail (CT). Structural studies revealed both αIIbβ3 and αVβ3 complexes as a bent conformation with the headpiece folded onto the leg piece, representing a resting state. Upon activating by ligand binding to the headpiece or talin/kindlin binding to the CT, β3 integrin undergoes a large conformational rearrangement, showing the headpiece extension from the leg piece and the separation of the leg from the α subunit. Only the interface at the headpiece between the β3 βI and the α β-propeller domains is maintained during the bent to extended transition. The interactions at the α/β TM, CT and leg domains are broken in the active conformation. It remains unknown how the extended state of β3 is triggered and maintained, especially whether the α subunit contributes to regulating the β3 conformation.A Leu33Pro polymorphism at the β3 PSI domain, forming human platelet antigen-1a/1b (HPA-1a/1b), can induce alloantibodies against incompatible HPA-1a during pregnancy or after blood transfusion, leading to two severe bleeding disorders, fetal/neonatal alloimmune thrombocytopenia (FNAIT) and post-transfusion purpura (PTP). The severe risk of FNAIT is intracranial hemorrhage (ICH), often leading to death or life-long neurological disability. The direct cause of ICH remains unknown, but recent studies suggest a strong connection between the ICH and the presence of a subtype of anti-HPA-1a that is specific to the αVβ3 complex on endothelial cells. The other anti-HPA-1a subtypes are αIIbβ3 specific and complex independent. The αVβ3-specific anti-HPA-1a can interfere with endothelial cell function by inhibiting the αVβ3-mediated cell adhesion and angiogenesis, which may be responsible for the development of ICH. Certain anti-HPA-1a antibodies were also found to affect αIIbβ3-mediated platelet function. It is unknown how the anti-HPA-1a antibodies, which bind to the β3 PSI domain, can distinguish the β3 between αVβ3 and αIIbβ3, and how they block β3 integrin function.To address the above questions, we engineered a chimeric β3 integrin (cβ3) by replacing the βI domain with the αI domain of αL integrin, enabling to express the full-length β3 as a monomer in the absence of α subunit. With this strategy, the conformation of β3 can be studied without α subunit. On the cell surface, the cβ3 constitutively adopts an extended active conformation, evidenced by the binding of the αL ligand ICAM-1 in a metal ion-dependent manner, and the binding of active conformation-dependent anti-β3 mAbs. Very surprisingly, the cβ3 ectodomain exists in a compact conformation in solution as indicated by size exclusion chromatography and dynamic light scattering. This was confirmed by a high-resolution crystal structure of cβ3, which shows a bent shape that is comparable with the β3 conformation in the bent structures of αVβ3 and αIIbβ3. Our cβ3 structure revealed a rotational movement at the ankle region connecting the I-EGF4 and the β-tail domains. Mutagenesis study demonstrated that the plasticity of the ankle region regulates the activation of β3 integrin. These data demonstrate that the β3 integrin can adopt either a bent or extended conformation even without α subunit. Our data suggest that the TM, CT, cell membrane, and/or other interacting molecules are responsible for the active form of β3 on the cell surface. We next used our monomeric cβ3 to detect the anti-HPA-1a antibodies in FNAIT and PTP, aiming to distinguish the complex-dependent and independent subtypes. Remarkably, we found that anti-αIIbβ3 alloantibodies in FNAIT only recognize the bent conformation of either αIIbβ3 or cβ3 but not the extended form. Structural analysis of the PSI domains of the available β3 crystal structures revealed conformational diversity and flexibility, which may account for the heterogeneity of the anti-HPA-1a antibodies. More importantly, the bent conformation specific anti-HPA-1a antibodies may stabilize the resting integrin and block its cellular function. DisclosuresCurtis:Ionis Pharmaceuticals: Consultancy.

Potential Benefit of Soluble Thrombomodulin in Coping with COVID-19/SARS-CoV-2-Induced Coagulopathy

Researchers should investigate the coagulopathy that accompanies Coronavirus Disease 2019 (COVID-19), as the incidence, as well as the mortality associated with coagulopathy, is higher than expected in COVID-19 patients. We read with interest the article published by Tang N et al. suggesting that elevated plasma levels of the fibrin degradation product D-dimer might be a predictive marker for mortality of patients with COVID-19 [1]. The same authors also reported that the use of heparin improved the prognosis of COVID-19 patients with severe coagulopathy and D-dimer levels >6-fold of the normal upper limit [2]. The high plasma levels of D-dimer suggest that the fibrinolytic system is not inhibited; nonetheless, thrombotic events are frequently noted in COVID-19 patients transferred to intensive care units [3]. Curiously, severe cases of COVID-19 are complicated by cytokine release syndrome in conjunction with Hemophagocytic Lymphohistiocytosis (HLH) [4], suggesting that extremely exaggerated hypercoagulability is induced in COVID-19. The pathophysiology of COVID-19-induced coagulopathy has not been fully elucidated. However, recently, postmortem examination of patients with COVID-19 who died of respiratory failure found thrombi in parallel with the deposition of the C5b-9 complex in the microvasculature, indicating terminal complement activation [5]. These pathological findings are reminiscent of the pathogenesis of Transplant-Associated Thrombotic Microangiopathy (TA-TMA) and atypical Hemolytic Uremic Syndrome (aHUS), in which activation of the complement system plays a role. Other possible factors involved in the pathogenesis of COVID-19-induced coagulopathy may be related to the tropism of Severe Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the pathogen that causes COVID-19, to vascular endothelial cells [6]. SARS-CoV-2 infects vascular endothelial cells via the Angiotensin-Converting Enzyme 2 (ACE2) receptor, leading to vascular derangement, so-called endotheliitis, which may result in coagulopathy and TMA. Thrombomodulin (TM) is a cell-surface expressed glycoprotein comprising six distinct domains, including an NH2-terminal lectinlike region, six tandem Epidermal Growth Factor (EGF)-like structures, an O-glycosylation site-rich domain, a transmembrane domain, and a cytoplasmic tail domain [7]. TM binds to thrombin via its EGF-like region and Converts Protein C (PC) to Activated PC (APC), which inhibits coagulant factors FVIIIa and FVa in the presence of protein S, thereby inhibiting further thrombin generation. Interestingly, TM has an anti-inflammatory effect; the lectin-like domain of TM binds and degrades High Mobility Group Box-1 (HMGB1) and attenuates inflammation. Additionally, after interacting with thrombin, TM converts carboxypeptidase Thrombin-Activatable Fibrinolysis Inhibitor (TAFI) to activated TAFI (TAFIa), which inactivates C3 and C5. Furthermore, the fifth domain of EGF-like region of TM (TME5) interacts with the cell-surface chemokine receptor G protein-coupled receptor protein 15 (GPR15) and then exerts cytoprotective effects on vascular endothelial cells in association with upregulation of antiapoptotic MCl-1 proteins, which is mediated by the activation of extracellular signal-regulated kinase [7]. Recombinant human soluble TM (rhTM) comprises extracellular domains of TM. A clinical trial comparing the efficacy and safety of rhTM with Unfractionated Heparin (UFH) in patients with Intravascular Coagulation (DIC) caused by hematological malignancies or infection found that a greater number of patients treated with rhTM recovered from DIC with acceptable toxicity profiles than those who received UFH. Based on these results, the use of rhTM has been approved for the treatment of DIC in Japan since 2008. The use of rhTM effectively treated many patients with DIC with various types of underlying diseases, including transplant-associated complications such as TA-TMA, hepatic sinusoidal obstruction syndrome/veno-occlusive disease and engraftment syndrome [8]. Importantly, endothliitis plays a role in the development of all of these conditions, suggesting the possible cytoprotective roles of rhTM in vascular endothelial cells. Moreover, prompt improvement of DIC caused by HLH developed in association with immune reconstitution syndrome after initiation of highly active antiretroviral therapy for acquired immunodeficiency syndrome was noted in the individual who received rhTM [9]. Thus, the use of anticoagulant rhTM with additional activities for immune systems and vascular endothelial cells could counteract severe COVID-19 with coagulopathy, although a systematic review and meta-analysis of rhTM therapy for sepsis-induced coagulopathy failed to prove the significant improvement of clinical outcomes of patients with sepsis-induced coagulopathy [10].

Immunogenicity of HIV-1-Based Virus-Like Particles with Increased Incorporation and Stability of Membrane-Bound Env.

An optimal prophylactic vaccine to prevent human immunodeficiency virus (HIV-1) transmission should elicit protective antibody responses against the HIV-1 envelope glycoprotein (Env). Replication-incompetent HIV-1 virus-like particles (VLPs) offer the opportunity to present virion-associated Env with a native-like structure during vaccination that closely resembles that encountered on infectious virus. Here, we optimized the incorporation of Env into previously designed mature-form VLPs (mVLPs) and assessed their immunogenicity in mice. The incorporation of Env into mVLPs was increased by replacing the Env transmembrane and cytoplasmic tail domains with those of influenza haemagglutinin (HA-TMCT). Furthermore, Env was stabilized on the VLP surface by introducing an interchain disulfide and proline substitution (SOSIP) mutations typically employed to stabilize soluble Env trimers. The resulting mVLPs efficiently presented neutralizing antibody epitopes while minimizing exposure of non-neutralizing antibody sites. Vaccination of mice with mVLPs elicited a broader range of Env-specific antibody isotypes than Env presented on immature VLPs or extracellular vesicles. The mVLPs bearing HA-TMCT-modified Env consistently induced anti-Env antibody responses that mediated modest neutralization activity. These mVLPs are potentially useful immunogens for eliciting neutralizing antibody responses that target native Env epitopes on infectious HIV-1 virions.

Improved SARS-CoV-2 Spike Glycoproteins for Pseudotyping Lentiviral Vectors

The spike (S) glycoprotein of SARS-Cov-2 facilitates viral entry into target cells via the cell surface receptor angiotensin-converting enzyme 2 (ACE2). Third generation HIV-1 lentiviral vectors can be pseudotyped to replace the native CD4 tropic envelope protein of the virus and thereby either limit or expand the target cell population. We generated a modified S glycoprotein of SARS-Cov-2 to pseudotype lentiviral vectors which efficiently transduced ACE2-expressing cells with high specificity and contain minimal off-target transduction of ACE-2 negative cells. By utilizing optimized codons, modifying the S cytoplasmic tail domain, and including a mutant form of the spike protein (D614G), we generated an expression plasmid encoding the optimized protein that produces S pseudotyped lentiviral vectors at an infectious titer (TU/mL) 1000-fold higher than the unmodified S protein. S pseudotyped replication-defective lentiviral vectors eliminate the need for biosafety-level-3 laboratories required when developing therapeutics against SARS-CoV-2 with live infectious virus. Furthermore, S pseudotyped vectors with high activity and specificity may be used as tools to understand the development of immunity against SARS-CoV-2, to develop assays of neutralizing antibodies and other agents that block viral binding, and to allow in vivo imaging studies of ACE-2 expressing cells.Funding: This work was supported by a UCLA David Geffen School of Medicine (DGSOM) and Broad Stem Cell Research Center (BSCRC) COVID 19 Research Award. Dr. Lili Yang (UCLA) and Dr. Pin Wang (USC) provided the ACE2 expressing 293T cell line (ACE-293T). Dr. Jocelyn Kim (UCLA) provided the VeroE6 cell lines. The Flow Cytometry Core of the UCLA Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, and the Virology Core of the UCLA Center for AIDS Research (CFAR) (5P30 AI028697) were used to support studies.

Abstract 4911: Characterizing the role of podocalyxin's cytoplasmic tail domain in collective tumor invasion

Abstract High expression of the single-pass transmembrane sialomucin, podocalyxin, has been shown by many groups to correlate with poor disease outcome in a number of solid tumor types, including colorectal, ovarian, pancreatic and breast cancers. We had previously identified that high podocalyxin expression in invasive ductal breast carcinoma was an independent indicator of poor survival (Somasiri et al., 2004, Cancer Res. 64:15). Further analysis of these tumor samples revealed that this decrease in survival occurred without the loss of membranous, junctional E-cadherin, suggesting that these tumors may have invaded collectively without necessitating an overt epithelial to mesenchymal transition (EMT). Experimentally, forced over-expression of podocalyxin in polarity-disrupted human MCF7 breast cancer cells drives the formation of invasive orthotopic xenograft tumors and elongated, cohesive, and E-Cadherin-expressing spheroids in three-dimensional (3D) culture as compared to control (Graves et al., 2016, Breast Canc. Res. 18:11). Further, treatment of these podocalyxin-overexpressing MCF7 cells with the myosin inhibitor, blebbistatin, and the small molecule inhibitor of ezrin-actin binding, NSC668394, resulted in decreased collective invasion and migration, respectively. Together these data suggest that podocalyxin, through interactions with the actin cytoskeleton via its cytoplasmic tail binding partners, can facilitate increased collective epithelial tumor cell motility, at least in some contexts. To test this hypothesis, we generated podocalyxin null MCF7 clones and cell populations using CRISPR-Cas9 genome editing and reconstituted these cells with mutant forms of podocalyxin that are unable to interact with the scaffolding proteins NHERF and/or ezrin and hence with the actin cytoskeleton. Preliminary results from 3D culture and live imaging of these mutant podocalyxin-expressing cells suggests that loss of podocalyxin's cytoplasmic tail results in decreased spheroid invasion that may be a result of deficiencies in actomyosin contractility. Hence, increased expression and mislocalization of podocalyxin may facilitate aberrant interactions with the actin cytoskeleton and contractile machinery, driving enhanced cell motility and, in certain tumor microenvironments, promote collective tumor invasion. Citation Format: Erin M. Bell, Marcia L. Graves, Pamela Dean, Kelly M. McNagny, Calvin D. Roskelley. Characterizing the role of podocalyxin's cytoplasmic tail domain in collective tumor invasion [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4911.

TREM2 suppresses the proinflammatory response to facilitate PRRSV infection via PI3K/NF-κB signaling.

Triggering receptor expressed on myeloid cells 2 (TREM2) serves as an anti-inflammatory receptor, negatively regulating the innate immune response. TREM2 is mainly expressed on dendritic cells and macrophages, the target cells of porcine reproductive and respiratory syndrome virus (PRRSV). Thus, we investigated the potential role of TREM2 in PRRSV infection in porcine alveolar macrophages (PAMs). We found that there was an increased expression of TREM2 upon PRRSV infection in vitro. TREM2 silencing restrained the replication of PRRSV, whereas TREM2 overexpression facilitated viral replication. The cytoplasmic tail domain of TREM2 interacted with PRRSV Nsp2 to promote infection. TREM2 downregulation led to early activation of PI3K/NF-κB signaling, thus reinforcing the expression of proinflammatory cytokines and type I interferons. Due to the enhanced cytokine expression, a disintegrin and metalloproteinase 17 was activated to promote the cleavage of membrane CD163, which resulted in suppression of infection. Furthermore, exogenous soluble TREM2 (sTREM2)-mediated inhibition of PRRSV attachment might be attributed to its competitive binding to viral envelope proteins. In pigs, following PRRSV challenge in vivo, the expression of TREM2 in lungs and lymph nodes as well as the production of sTREM2 were significantly increased. These novel findings indicate that TREM2 plays a role in regulating PRRSV replication via the inflammatory response. Therefore, our work describes a novel antiviral mechanism against PRRSV infection and suggests that targeting TREM2 could be a new approach in the control of the PRRSV infection.

An Essential Role for Perforin-2 in Type I IFN Signaling.

Type I IFNs play a complex role in determining the fate of microbial pathogens and may also be deleterious to the host during bacterial and viral infections. Upon ligand binding, a receptor proximal complex consisting of IFN-α and -β receptors 1 and 2 (IFNAR1, IFNAR2, respectively), tyrosine kinase 2 (Tyk2), Jak1, and STAT2 are assembled and promote the phosphorylation of STAT1 and STAT2. However, how the IFNARs proximal complex is assembled upon binding to IFN is poorly understood. In this study, we show that the membrane-associated pore-forming protein Perforin-2 (P2) is critical for LPS-induced endotoxic shock in wild-type mice. Type I IFN-mediated JAK-STAT signaling is severely impaired, and activation of MAPKs and PI3K signaling pathways are delayed in P2-deficient mouse bone marrow-derived macrophages, mouse embryonic fibroblasts (MEFs), and human HeLa cells upon IFN stimulation. The P2 N-glycosylated extracellular membrane attack complex/perforin domain and the P2 domain independently associate with the extracellular regions of IFNAR1 and IFNAR2, respectively, in resting MEFs. In addition, the P2 cytoplasmic tail domain mediated the constitutive interaction between STAT2 and IFNAR2 in resting MEFs, an interaction that is dependent on the association of the extracellular regions of P2 and IFNAR2. Finally, the constitutive association of P2 with both receptors and STAT2 is critical for the receptor proximal complex assembly and reciprocal transphosphorylation of Jak1 and Tyk2 as well as the phosphorylation and activation of STAT1 and STAT2 upon IFN-β stimulation.

Role of Mucin‐1 in Carcinomas

Mucin 1 (MUC1) is a large, heavy O‐glycosylated mucin glycoprotein with a molecular weight of 250 to 500 kDa, depending on the level of glycosylation, and extends 200–500 nm from the cell surface. MUC1 spans the cell membrane at the apical surface of normal simple epithelia and the surfaces of some hematopoietic cells. MUC1 is comprised of three major domains: the cytoplasmic tail, transmembrane, and extracellular domains. The cytoplasmic tail (CT) domain is highly conserved across species and interacts with various cell signaling networks. The transmembrane domain is composed of hydrophobic amino acids to anchor MUC1 in the membrane. The extracellular domain is not strictly conserved, but always consists of a series of 20–100 tandem repeats enriched in serine, threonine and proline residues providing sites for the heavy O‐linked glycosylation characteristic of mucin‐type glycoproteins. The extracellular domain contributes MUC1’s function as an inhibitor of cell‐cell and cell‐extracellular matrix adhesion. The heavy glycosylation of MUC1 normally serves to protect cells from infection and also lubricates the cell surface. In normal biology, MUC1 serves as an inhibitor of embryo attachment to the uterine cell surface and controls early aspects of the reproductive process. In cancer, carcinomas often overexpress MUC1 due to a combination of gene duplication events as well as stimulation of MUC gene expression by cytokines produced by cells of the immune system in their efforts to destroy the cancer cells. Cancer cells also lose their normally polarized distribution of MUC1 and instead express the glycoprotein over their entire surface, MUC1 protects tumor cells from the immune system in two ways. First, by inhibiting the cell contact required for cell‐mediated immunity, e.g. by NK cells. In addition MUC1 expressed at tumor cell surfaces or shed by the action of cell surface proteases is immunosuppressive, presumably by interacting with immune checkpoint inhibitors. Various mouse models have been used to conduct research about MUC1, showing the connection between MUC1 overexpression in mouse mammary glands and cancer development and other processes. MUC1 serves as a commonly used serum marker for various cancers and is being explored as a target for certain therapeutic approaches. The Walton SMART team has designed a 3D model of MUC1 to investigate the relationship between its structure and function.Support or Funding InformationMSOE Center for Biomolecular Modeling