Susceptible Target Cells Research Articles

Abstract 4699: Evaluating extracellular matrix invasion and cytotoxicity of natural killer cells in a novel co-culture assay

Abstract Background: The complex tumor microenvironment (TME) in solid tumors presents unique challenges for lymphocytes used in cancer immunotherapy. The extracellular matrix (ECM) in TME can limit the recruitment of lymphocytes and restrict access to cancer cells. This study utilized Matrigel to study the kinetics of natural killer cell invasion and cytotoxicity. We hypothesized that the kinetics of tumor cell killing by the NKs would depend on the distance for invasion and rely on matrix-metalloproteinases (MMPs) for remodeling the ECM. Methods: Increasing volumes (50-100 µL/well) of Matrigel (6 mg/mL) representing increasing distance for invasion was layered over MCF7-red target tumor cells expressing nuclear-localized mKate2 (red fluorescent protein). NK92 (Creative Bioarray) or eGFP-NK92 (BPS Bioscience) were seeded over the solidified Matrigel layer at E:T of 3:1. The broad spectrum MMP inhibitor, Ilomostat (GM6001, 2 µM and 10 µM), was added to the wells to investigate to role of MMPs. Label-free impedance and live cell imaging data was collected in real time on xCELLigence RTCA eSight to evaluate NK cell invasion and cytotoxicity. Percent cytolysis was calculated on the RTCA software from normalized cell impedance readings and imaging data using the formula [(1-(treated/untreated)*100]. Results: Impedance reading increases as the MCF7-red cells settle, attach, and proliferate. The readings plateau as the cells reach confluence. The addition of Matrigel modulates the impedance signature of MCF7 cells. Importantly, Matrigel delays cytolysis by NK92 cells added at 24h. The delay increases with increasing volume of Matrigel representing increasing invasion distance. The time to achieve 60% killing with respect to controls (KT60) was 67, 76 and 89 hours for 50, 75 and 100 µL/well, respectively. Consistent with the role of MMPs in invasion, percent cytolysis calculated independently from normalized impedance readings and live cell imaging data confirmed both delayed and reduced target cell killing by GFP-NK92 cells in the presence of Ilomostat. Interestingly, only a few GFP-NK92 were detected by live cell imaging when there was increased target cell death. These included highly active NK cells that made multiple contacts with different MCF7 red targets in clusters suggesting serial killing activity. Conclusions: The observations that only a few NK92 cells are detected during the killing of susceptible target cells and that cytolysis is not abrogated by the inhibition of MMPs suggest a potential role for distal effectors like cytokines, independent of invasion. This is a novel 2.5D assay that demonstrates the potential of RTCA eSight platform for studying the invasion and cytotoxicity function of various immune cells through both real-time impedance recording and live-cell time-lapse imaging simultaneously. (For Research Use Only. Not for use in diagnostic procedures. RA45236.3472453704) Citation Format: Jyothi Thyagabhavan Mony, Xiaoyu Zhang. Evaluating extracellular matrix invasion and cytotoxicity of natural killer cells in a novel co-culture assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4699.

Evaluation of Food Homogenates on Cell Survival In Vitro

A critical review on the approaches to assess the infectivity of the Hepatitis E virus (HEV) in food recommended that a cell culture-based method should be developed. Due to the observations that viral loads in food may be low, it is important to maximise the potential for detection of HEV in a food source in order to fully assess infectivity. To do so, would require minimal processing of any target material. In order to proceed with the development of an infectivity culture method that is simple, robust and reproducible, there are a number of points to address; one being to assess if food homogenates are cytotoxic to HEV susceptible target cells. Food matrices previously shown to have detectable HEV nucleic acid were selected for analysis and assessed for their effect on the percentage survival of three cell lines commonly used for infectivity assays. Target cells used were A549, PLC/PRF/5 and HepG2 cells. The results showed that, as expected, various food homogenates have differing effects on cells in vitro. In this study, the most robust cell line over a time period was the A549 cell line in comparison to HepG2, with PLC/PRF/5 cells being the most sensitive. Overall, this data would suggest that FH can be left in contact with A549 cells for a period of up to 72 h to maximise the potential for testing infection. Using food homogenates directly would negate any concerns over losing virus as a result of any additional processing steps.

Carbohydrate PL-M binds galectin-3 to inhibit SARS-CoV-2 viral entry into cells

Galectin-3 (Gal-3) binds to glycans on the spike protein S1 domain of the SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) virus, thereby facilitating viral entry into cells. Because little is known about how to antagonize Gal-3 to block viral activity, we developed a pectin-derived polysaccharide of a(1-6)-D-mannopyranose (termed “ProLectin M” or PL-M) and studied its effect on Vero cells being infected with SARS-CoV-2 virus. Using this novel glycovirology approach, we demonstrated a significant reduction in viral load with no demonstrable cytotoxicity. We also evaluated the efficacy of PL-M in a randomized, double-blinded, placebo-controlled clinical study in 34 patients with mild to moderately severe COVID-19. Overall, treatment with PL-M significantly (p = 0.001) increased RT-PCR cycle counts for N and ORF genes on days 3 (Ct values 32.09 ± 2.39 and 30.69 ± 3.38, respectively) and 7 (Ct values 34.91 ± 0.39 and 34.85 ± 0.61, respectively) compared to placebo. From day 3 post-treatment, all subjects were RT-PCR negative for both genes in the PL-M treatment group, whereas placebo subjects remained positive. On the molecular level, our NMR studies show that PL-M binds relatively strongly to Gal-3, supporting the idea of an antagonist effect on the lectin. Gal-3 also binds strongly to sugar binding sites on the SARS-CoV2 virus spike protein S1 domain as evidenced by competitive lactose binding. In this regard, PL-M competes with the spike protein for binding to Gal-3 and thereby compromises viral entry into susceptible target cells, thus helping to explain our positive clinical effect from PL-M on the course of viral infection. This report provides a brief review of what is already known about PL-M and its effects on SARS-CoV2 virus, as well as new results on an expanded clinical trial with PL-M and NMR studies on the molecular mechanism of action of PL-M, Gal-3, and the SARS-CoV2 viral spike protein S1 from the Omicron variant.

The reproduction number and its probability distribution for stochastic viral dynamics.

We consider stochastic models of individual infected cells. The reproduction number, R, is understood as a random variable representing the number of new cells infected by one initial infected cell in an otherwise susceptible (target cell) population. Variability in R results partly from heterogeneity in the viral burst size (the number of viral progeny generated from an infected cell during its lifetime), which depends on the distribution of cellular lifetimes and on the mechanism of virion release. We analyse viral dynamics models with an eclipse phase: the period of time after a cell is infected but before it is capable of releasing virions. The duration of the eclipse, or the subsequent infectious, phase is non-exponential, but composed of stages. We derive the probability distribution of the reproduction number for these viral dynamics models, and show it is a negative binomial distribution in the case of constant viral release from infectious cells, and under the assumption of an excess of target cells. In a deterministic model, the ultimate in-host establishment or extinction of the viral infection depends entirely on whether the mean reproduction number is greater than, or less than, one, respectively. Here, the probability of extinction is determined by the probability distribution of R, not simply its mean value. In particular, we show that in some cases the probability of infection is not an increasing function of the mean reproduction number.

Initial productive and latent HIV infections originate in vivo by infection of resting T cells.

Productively infected cells are generally thought to arise from HIV infection of activated CD4+ T cells, and these infected activated cells are thought to be a recurring source of latently infected cells when a portion of the population transitions to a resting state. We discovered and report here that productively and latently infected cells can instead originate from direct infection of resting CD4+ T cell populations in lymphoid tissues in Fiebig I, the earliest stage of detectable HIV infection. We found that direct infection of resting CD4+ T cells was correlated with the availability of susceptible target cells in lymphoid tissues largely restricted to resting CD4+ T cells in which expression of pTEFb enabled productive infection, and we documented persistence of HIV-producing resting T cells during antiretroviral therapy (ART). Thus, we provide evidence of a mechanism by which direct infection of resting T cells in lymphoid tissues to generate productively and latently infected cells creates a mechanism by which the productively infected cells can replenish both populations and maintain two sources of virus from which HIV infection can rebound, even if ART is instituted at the earliest stage of detectable infection.

Cellular vimentin regulates the infectivity of Newcastle disease virus through targeting of the HN protein

The haemagglutinin-neuraminidase (HN) protein plays a crucial role in the infectivity and virulence of Newcastle disease virus (NDV). In a previous study, the mutant HN protein was identified as a crucial virulence factor for the velogenic variant NDV strain JS/7/05/Ch, which evolved from the prototypic vaccine strain Mukteswar. Furthermore, macrophages are the main susceptible target cells of NDV. However, the possible involvement of cellular molecules in viral infectivity remains unclear. Herein, we elucidate the crucial role of vimentin, an intermediate filament protein, in regulating NDV infectivity through targeting of the HN protein. Using LC‒MS/MS mass spectrometry and coimmunoprecipitation assays, we identified vimentin as a host protein that differentially interacted with prototypic and mutant HN proteins. Further analysis revealed that the variant NDV strain induced more significant rearrangement of vimentin fibres compared to the prototypic NDV strain and showed an interdependence between vimentin rearrangement and virus replication. Notably, these mutual influences were pronounced in HD11 chicken macrophages. Moreover, vimentin was required for multiple infection processes of the variant NDV strain in HD11 cells, including viral internalization, fusion, and release, while it was not necessary for those of the prototypic NDV strain. Collectively, these findings underscore the pivotal role of vimentin in NDV infection through targeting of the HN protein, providing novel targets for antiviral treatment strategies for NDV.

ACE-2, TMPRSS2, and Neuropilin-1 Receptor Expression on Human Brain Astrocytes and Pericytes and SARS-CoV-2 Infection Kinetics.

Angiotensin Converting Enzyme 2 (ACE-2), Transmembrane Serine Protease 2 (TMPRSS-2) and Neuropilin-1 cellular receptors support the entry of SARS-CoV-2 into susceptible human target cells and are characterized at the molecular level. Some evidence on the expression of entry receptors at mRNA and protein levels in brain cells is available, but co-expression of these receptors and confirmatory evidence on brain cells is lacking. SARS-CoV-2 infects some brain cell types, but infection susceptibility, multiple entry receptor density, and infection kinetics are rarely reported in specific brain cell types. Highly sensitive Taqman ddPCR, flow-cytometry and immunocytochemistry assays were used to quantitate the expression of ACE-2, TMPRSS-2 and Neuropilin-1 at mRNA and protein levels on human brain-extracted pericytes and astrocytes, which are an integral part of the Blood-Brain-Barrier (BBB). Astrocytes showed moderate ACE-2 (15.9 ± 1.3%, Mean ± SD, n = 2) and TMPRSS-2 (17.6%) positive cells, and in contrast show high Neuropilin-1 (56.4 ± 39.8%, n = 4) protein expression. Whereas pericytes showed variable ACE-2 (23.1 ± 20.7%, n = 2), Neuropilin-1 (30.3 ± 7.5%, n = 4) protein expression and higher TMPRSS-2 mRNA (667.2 ± 232.3, n = 3) expression. Co-expression of multiple entry receptors on astrocytes and pericytes allows entry of SARS-CoV-2 and progression of infection. Astrocytes showed roughly four-fold more virus in culture supernatants than pericytes. SARS-CoV-2 cellular entry receptor expression and "in vitro" viral kinetics in astrocytes and pericytes may improve our understanding of viral infection "in vivo". In addition, this study may facilitate the development of novel strategies to counter the effects of SARS-CoV-2 and inhibit viral infection in brain tissues to prevent the spread and interference in neuronal functions.

MicroRNAs affecting the susceptibility of melanoma cells to CD8+ T cell-mediated cytolysis.

The regulatory functions of microRNAs (miRNAs) in anti-tumour immunity have been mainly described in immune effector cells. Since little is known about miRNA effects on the susceptibility of target cells during T cell-target cell interaction, this study focused on the identification of miRNAs expressed in tumour cells controlling their susceptibility to CD8+ T cell-mediated cytotoxicity. Luciferase expressing B16F10 melanoma (B16F10 Luci+ ) cells transfected with individual miRNAs covering a comprehensive murine miRNA library were screened for their susceptibility to lysis by an established cytotoxic T lymphocyte (CTL) line (5a, clone Nβ) specific for the melanoma-associated antigen tyrosinase-related protein 2. miRNAs with the most pronounced effects on T cell-mediated lysis were validated and stably expressed in B16F10 cells. In silico analyses identified common targets of miRNA sets determined by the screen, which were further confirmed by small interfering RNA (siRNA)-mediated silencing experiments modulating immune surveillance. The Ingenuity Pathway Analysis (IPA) software and RNA sequencing (RNA-seq) data from miRNA-overexpressing cell lines were applied to investigate the underlying mechanisms. The Cancer Genome Atlas (TCGA)-derived miRNA sequencing data were used to assess the correlation of miRNA expression with melanoma patients' survival. The miRNA screen resulted in the selection of seven miRNAs enhancing CTL-mediated melanoma cell killing in vitro. Upon stable overexpression of selected miRNAs, hsa-miR-320a-3p, mmu-miR-7037-5p and mmu-miR-666-3p were determined as most effective in enhancing susceptibility to CTL lysis. In silico analyses and subsequent siRNA-mediated silencing experiments identified Psmc3 and Ndufa1 as common miRNA targets possibly involved in the functional effects observed. The analyses of RNA-seq data with IPA showed pathways, networks, biological functions and key molecules potentially involved in the miRNA-mediated functional effects. Finally, based on TCGA data analysis, a positive correlation of the conserved miRNAs among the panel of the seven identified miRNAs with overall survival of melanoma patients was determined. For the first time, this study uncovered miRNA species that affect the susceptibility of melanoma cells to T cell-mediated killing. These miRNAs might represent attractive candidates for novel therapy approaches against melanoma and other tumour entities.

Identification of a novel mechanism for LFA-1 organization during NK cytolytic response.

The elimination of transformed and viral infected cells by natural killer (NK) cells requires a specialized junction between NK and target cells, denominated immunological synapse (IS). After initial recognition, the IS enables the directed secretion of lytic granules content into the susceptible target cell. The lymphocyte function-associated antigen (LFA)-1 regulates NK effector function by enabling NK-IS assembly and maturation. The pathways underlying LFA-1 accumulation at the IS in NK cells remained uncharacterized. A kinase anchoring protein 350 (AKAP350) is a centrosome/Golgi-associated protein, which, in T cells, participates in LFA-1 activation by mechanisms that have not been elucidated. We first evaluated AKAP350 participation in NK cytolytic activity. Our results showed that the decrease in AKAP350 levels by RNA interference (AKAP350KD) inhibited NK-YTS cytolytic activity, without affecting conjugate formation. The impairment of NK effector function in AKAP350KD cells correlated with decreased LFA-1 clustering and defective IS maturation. AKAP350KD cells that were exclusively activated via LFA-1 showed impaired LFA-1 organization and deficient lytic granule translocation as well. In NK AKAP350KD cells, activation signaling through Vav1 was preserved up to 10 min of interaction with target cells, but significantly decreased afterwards. Experiments in YTS and in ex vivo NK cells identified an intracellular pool of LFA-1, which partially associated with the Golgi apparatus and, upon NK activation, redistributed to the IS in an AKAP350-dependent manner. The analysis of Golgi organization indicated that the decrease in AKAP350 expression led to the disruption of the Golgi integrity in NK cells. Alteration of Golgi function by BFA treatment or AKAP350 delocalization from this organelle also led to impaired LFA-1 localization at the IS. Therefore, this study characterizes AKAP350 participation in the modulation of NK effector function, revealing the existence of a Golgi-dependent trafficking pathway for LFA-1, which is relevant for LFA-1 organization at NK-lytic IS.

Recent advances in inhibition of porcine reproductive and respiratory syndrome virus through targeting CD163.

Porcine reproductive and respiratory syndrome virus (PRRSV) has plagued the pig industry for more than 30 years and causes great economic losses. At present different commercial vaccines are available but limited tools. Until now at least six potential host factors are identified as the key receptors for PRRSV infection. Among them, CD163 molecule is the most important and critical in PRRSV life cycle responsible for mediating virus uncoating and genome release. It determines the susceptibility of target cells to the virus. Several PRRSV non-permissive cells (such as PK-15, 3D4/21, and BHK-21) are demonstrated to become completely susceptible to PRRSV infection in the presence of expression of porcine CD163 protein. Therefore, CD163 has become the target for the design of novel antiviral molecules disrupting the interaction between CD163 and viral glycoproteins, or the breeding of gene-modified animals against PRRSV infection. In this review, we comprehensively summarize the recent progress in inhibition of PRRSV replication via targeting CD163 receptor. In addition, whether there are other potential molecules interacting with CD163 in the process of uncoating of virus life cycle is also discussed.

Carbohydrate prolectin-M, a galectin-3 antagonist, blocks SARS-CoV-2 activity

The SARS-COV-2 (severe acute respiratory syndrome coronavirus 2) virus binds to human lectins to gain entry into cells to replicate. Blocking the virus’s entry using a complex polysaccharide component of [a (1-6)- D-mannopyranose termed “ProLectin M” has an effect on viral replication as a therapeutic tool and a safe alternative to existing antiviral therapies. Little is known about how galectin-3 inhibits viral entry into cells and its impact on the course of viral infection. Here, we investigated the effect of these non-cytotoxic polysaccharides on Vero cells infected with SARS-CoV-2 and demonstrated a dose-dependent reduction in viral load over a 48-hour viral incubation period. A pilot clinical study in five patients with laboratory-confirmed COVID-19 disease was treated with an oral formulation of ProLectin M, and all patients achieved complete disease remission with zero hospitalization or need for oxygen support. Moreover, the viral load was significantly lowered within 2 days of drug administration. On the viral envelope, glycans often play a crucial role in enabling pathogen transmission and/or entry into susceptible target cells. On the molecular level, our NMR spectroscopic studies show that ProLectin M binds relatively strongly to galectin-3, supporting the idea of an antagonist effect on the lectin.

Escaping stochastic extinction of mutant virus: Temporal pattern of emergence of drug resistance within a host

After infecting a host, a viral strain may increase rapidly within the body and produce mutants with a faster proliferation rate than the virus itself. However, most of the mutants become extinct because of the stochasticity caused by the small number of infected cells. In addition, the mean growth rate of a mutant strain decreases with time because the number of susceptible target cells is reduced by the original strain. In this study, we calculated the fraction of mutants that can escape stochastic extinction, based on a continuous-time branching process with a time-dependent growth rate. We analyzed two cases differing in the mode of viral transmission: (1) an infected cell transmits the virus through cell-to-cell contact with a susceptible target cell; (2) an infected cell releases numerous free viral particles that subsequently infect susceptible target cells. The chance for a mutant strain to be established decreases with time after infection of the original type, and it may oscillate before convergence at the stationary value. We then calculated the probability of escaping stochastic extinction for a drug-resistant mutant when a patient received an antiviral drug that suppressed the original strain. Combining the rate of mutant production from the original strain and the chance of escaping stochastic extinction, the number of emerging drug-resistant mutations may have two peaks: one soon after the infection of the original type and the second at the start of antiviral drug administration.

T cell-derived tumor necrosis factor induces cytotoxicity by activating RIPK1-dependent target cell death.

TNF ligation of TNF receptor 1 (TNFR1) promotes either inflammation and cell survival by (a) inhibiting RIPK1’s death-signaling function and activating NF-κB or (b) causing RIPK1 to associate with the death-inducing signaling complex to initiate apoptosis or necroptosis. The cellular source of TNF that results in RIPK1-dependent cell death remains unclear. To address this, we employed in vitro systems and murine models of T cell–dependent transplant or tumor rejection in which target cell susceptibility to RIPK1-dependent cell death could be genetically altered. We show that TNF released by T cells is necessary and sufficient to activate RIPK1-dependent cell death in target cells and thereby mediate target cell cytolysis independently of T cell frequency. Activation of the RIPK1-dependent cell death program in target cells by T cell–derived TNF accelerates murine cardiac allograft rejection and synergizes with anti-PD1 administration to destroy checkpoint blockade–resistant murine melanoma. Together, the findings uncover a distinct immunological role for TNF released by cytotoxic effector T cells following cognate interactions with their antigenic targets. Manipulating T cell TNF and/or target cell susceptibility to RIPK1-dependent cell death can be exploited to either mitigate or augment T cell–dependent destruction of allografts and malignancies to improve outcomes.

Functional organization of the endoplasmic reticulum dictates the susceptibility of target cells to arsenite-induced mitochondrial superoxide formation, mitochondrial dysfunction and apoptosis

Arsenite induces many critical effects associated with the formation of reactive oxygen species (ROS) through different mechanisms. We focused on Ca2+-dependent mitochondrial superoxide (mitoO2-.) formation and addressed questions on the effects of low concentrations of arsenite on the mobilization of the cation from the endoplasmic reticulum and the resulting mitochondrial accumulation. Using various differentiated and undifferentiated cell types uniquely expressing the inositol-1, 4, 5-triphosphate receptor (IP3R), or both the IP3R and the ryanodine receptor (RyR), we determined that expression of this second Ca2+ channel is an absolute requirement for mitoO2-. formation and for the ensuing mitochondrial dysfunction and downstream apoptosis. In arsenite-treated cells, RyR was recruited after IP3R stimulation and agonist studies provided an indirect indication for a close apposition between RyR and mitochondria. It was also interesting to observe that arsenite fails to promote mitochondrial Ca2+ accumulation, mitoO2-. formation and mitochondrial toxicity in RyR-devoid cells, in which the IP3R is in close contact with the mitochondria. We therefore conclude that low dose arsenite-induced mitoO2- formation, and the resulting mitochondrial dysfunction and toxicity, are prerequisite of cell types expressing the RyR in close apposition with mitochondria.

Microglia: The Real Foe in HIV-1-Associated Neurocognitive Disorders?

The current use of combined antiretroviral therapy (cART) is leading to a significant decrease in deaths and comorbidities associated with human immunodeficiency virus type 1 (HIV-1) infection. Nonetheless, none of these therapies can extinguish the virus from the long-lived cellular reservoir, including microglia, thereby representing an important obstacle to curing HIV. Microglia are the foremost cells infected by HIV-1 in the central nervous system (CNS) and are believed to be involved in the development of HIV-1-associated neurocognitive disorder (HAND). At present, the pathological mechanisms contributing to HAND remain unclear, but evidence suggests that removing these infected cells from the brain, as well as obtaining a better understanding of the specific molecular mechanisms of HIV-1 latency in these cells, should help in the design of new strategies to prevent HAND and achieve a cure for these diseases. The goal of this review was to study the current state of knowledge of the neuropathology and research models of HAND containing virus susceptible target cells (microglial cells) and potential pharmacological treatment approaches under investigation.

β-Catenin Restricts Zika Virus Internalization by Downregulating Axl.

The latest outbreak of Zika virus (ZIKV) in the Americas was associated with significant neurologic complications, including microcephaly of newborns. We evaluated mechanisms that regulate ZIKV entry into human fetal astrocytes (HFAs). Astrocytes are key players in maintaining brain homeostasis. We show that the central mediator of canonical Wnt signaling, β-catenin, regulates Axl, a receptor for ZIKV infection of HFAs, at the transcriptional level. In turn, ZIKV inhibited β-catenin, potentially as a mechanism to overcome its restriction of ZIKV internalization through regulation of Axl. This was evident with three ZIKV strains tested but not with a laboratory-adapted strain which has a large deletion in its envelope gene. Finally, we show that β-catenin-mediated Axl-dependent internalization of ZIKV may be of increased importance for brain cells, as it regulated ZIKV infection of astrocytes and human brain microvascular cells but not kidney epithelial (Vero) cells. Collectively, our studies reveal a role for β-catenin in ZIKV infection and highlight a dynamic interplay between ZIKV and β-catenin to modulate ZIKV entry into susceptible target cells. IMPORTANCE ZIKV is an emerging pathogen with sporadic outbreaks throughout the world. The most recent outbreak in North America was associated with small brains (microcephaly) in newborns. We studied the mechanism(s) that may regulate ZIKV entry into astrocytes. Astrocytes are a critical resident brain cell population with diverse functions that maintain brain homeostasis, including neurogenesis and neuronal survival. We show that three ZIKV strains (and not a heavily laboratory-adapted strain with a large deletion in its envelope gene) require Axl for internalization. Most importantly, we show that β-catenin, the central mediator of canonical Wnt signaling, negatively regulates Axl at the transcriptional level to prevent ZIKV internalization into human fetal astrocytes. To overcome this restriction, ZIKV downregulates β-catenin to facilitate Axl expression. This highlights a dynamic host-virus interaction whereby ZIKV inhibits β-catenin to promote its internalization into human fetal astrocytes through the induction of Axl.

Influence of Different Glycoproteins and of the Virion Core on SERINC5 Antiviral Activity.

Host plasma membrane protein SERINC5 is incorporated into budding retrovirus particles where it blocks subsequent entry into susceptible target cells. Three structurally unrelated proteins encoded by diverse retroviruses, human immunodeficiency virus type 1 (HIV-1) Nef, equine infectious anemia virus (EIAV) S2, and ecotropic murine leukemia virus (MLV) GlycoGag, disrupt SERINC5 antiviral activity by redirecting SERINC5 from the site of virion assembly on the plasma membrane to an internal RAB7+ endosomal compartment. Pseudotyping retroviruses with particular glycoproteins, e.g., vesicular stomatitis virus glycoprotein (VSV G), renders the infectivity of particles resistant to inhibition by virion-associated SERINC5. To better understand viral determinants for SERINC5-sensitivity, the effect of SERINC5 was assessed using HIV-1, MLV, and Mason-Pfizer monkey virus (M-PMV) virion cores, pseudotyped with glycoproteins from Arenavirus, Coronavirus, Filovirus, Rhabdovirus, Paramyxovirus, and Orthomyxovirus genera. SERINC5 restricted virions pseudotyped with glycoproteins from several retroviruses, an orthomyxovirus, a rhabdovirus, a paramyxovirus, and an arenavirus. Infectivity of particles pseudotyped with HIV-1, amphotropic-MLV (A-MLV), or influenza A virus (IAV) glycoproteins, was decreased by SERINC5, whether the core was provided by HIV-1, MLV, or M-PMV. In contrast, particles pseudotyped with glycoproteins from M-PMV, parainfluenza virus 5 (PIV5), or rabies virus (RABV) were sensitive to SERINC5, but only with particular retroviral cores. Resistance to SERINC5 did not correlate with reduced SERINC5 incorporation into particles, route of viral entry, or absolute infectivity of the pseudotyped virions. These findings indicate that some non-retroviruses may be sensitive to SERINC5 and that, in addition to the viral glycoprotein, the retroviral core influences sensitivity to SERINC5.

Modeling the Relationship Between Antibody-Dependent Enhancement and Disease Severity in Secondary Dengue Infection

Sequential infections with different dengue serotypes (DENV-1, 4) significantly increase the risk of a severe disease outcome (fever, shock, and hemorrhagic disorders). Two hypotheses have been proposed to explain the severity of the disease: (1) antibody-dependent enhancement (ADE) and (2) original T cell antigenic sin. In this work, we explored the first hypothesis through mathematical modeling. The proposed model reproduces the dynamic of susceptible and infected target cells and dengue virus in scenarios of infection-neutralizing and infection-enhancing antibody competition induced by two distinct serotypes of the dengue virus during secondary infection. The enhancement and neutralization functions are derived from basic concepts of chemical reactions and used to mimic binding to the virus by two distinct populations of antibodies. The analytic study of the model showed the existence of two equilibriums: a disease-free equilibrium and an endemic one. Using the concept of the basic reproduction number [Formula: see text], we performed the asymptotic stability analysis for the two equilibriums. To measure the severity of the disease, we considered the maximum value of infected cells as well as the time when this maximum is reached. We observed that it corresponds to the time when the maximum enhancing activity for the infection occurs. This critical time was calculated from the model to be a few days after the occurrence of the infection, which corresponds to what is observed in the literature. Finally, using as output [Formula: see text], we were able to rank the contribution of each parameter of the model. In particular, we highlighted that the cross-reactive antibody responses may be responsible for the disease enhancement during secondary heterologous dengue infection.

Regulatory functions of NK cells during infections and cancer.

After recognition, NK cells can kill susceptible target cells through perforin-dependent mechanisms or by inducing death receptor-mediated apoptosis, and they can also secrete cytokines that are pivotal for immunomodulation. Despite the critical role as effector cells against tumors and virus-infected cells, NK cells have been implicated in the regulation of T cell-mediated responses in different models of autoimmunity, transplantation, and viral infections. Here, we review the mechanisms described for NK cell-mediated inhibition of adaptive immune responses, with spotlight on the emerging evidence of their regulatory role that shapes antitumor immune responses.

Real-Time Analysis of Individual Ebola Virus Glycoproteins Reveals Pre-Fusion, Entry-Relevant Conformational Dynamics.

The Ebola virus (EBOV) envelope glycoprotein (GP) mediates the fusion of the virion membrane with the membrane of susceptible target cells during infection. While proteolytic cleavage of GP by endosomal cathepsins and binding of the cellular receptor Niemann-Pick C1 protein (NPC1) are essential steps for virus entry, the detailed mechanisms by which these events promote membrane fusion remain unknown. Here, we applied single-molecule Förster resonance energy transfer (smFRET) imaging to investigate the structural dynamics of the EBOV GP trimeric ectodomain, and the functional transmembrane protein on the surface of pseudovirions. We show that in both contexts, pre-fusion GP is dynamic and samples multiple conformations. Removal of the glycan cap and NPC1 binding shift the conformational equilibrium, suggesting stabilization of conformations relevant to viral fusion. Furthermore, several neutralizing antibodies enrich alternative conformational states. This suggests that these antibodies neutralize EBOV by restricting access to GP conformations relevant to fusion. This work demonstrates previously unobserved dynamics of pre-fusion EBOV GP and presents a platform with heightened sensitivity to conformational changes for the study of GP function and antibody-mediated neutralization.