140 research outputs found
TNF-alpha induces macroautophagy and regulates MHC class II expression in human skeletal myocytes
No full textTNF-{alpha} induces macroautophagy and regulates MHC class II expression in human skeletal muscle cells
No full textMacroautophagy, a homeostatic process that shuttles cytoplasmic constituents into endosomal and lysosomal compartments, has recently been shown to deliver antigens for presentation on major histocompatibility complex (MHC) class II molecules. Skeletal muscle fibers show a high level of constitutive macroautophagy and express MHC class II molecules upon immune activation. We found that tumor necrosis factor-α (TNF-α), a monokine overexpressed in inflammatory myopathies, led to a marked up-regulation of macroautophagy in skeletal myocytes. Furthermore, TNF-α augmented surface expression of MHC class II molecules in interferon-γ (IFN-γ)-treated myoblasts. The synergistic effect of TNF-α and IFN-γ on the induction of MHC class II surface expression was not reflected by higher intracellular human leukocyte antigen (HLA)-DR levels and was reversed by macroautophagy inhibition, suggesting that TNF-α facilitates antigen processing via macroautophagy for more efficient MHC class II loading. Muscle biopsies from patients with sporadic inclusion body myositis, a well defined myopathy with chronic inflammation, showed that over 20% of fibers that contained autophagosomes costained for MHC class II molecules and that more than 40% of double-positive muscle fibers had contact with CD4(+) and CD8(+) immune cells. These findings establish a mechanism through which TNF-α regulates both macroautophagy and MHC class II expression and suggest that macroautophagy-mediated antigen presentation contributes to the immunological environment of the inflamed human skeletal muscle
TNF-alpha induces macroautophagy and regulates MHC class II expression in human skeletal myocytes
No full textSARS-CoV-2 neutralizing antibodies: Longevity, breadth, and evasion by emerging viral variants
No full textThe Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) antibody neutralization response and its evasion by emerging viral variants and variant of concern (VOC) are unknown, but critical to understand reinfection risk and breakthrough infection following vaccination. Antibody immunoreactivity against SARS-CoV-2 antigens and Spike variants, inhibition of Spike-driven virus–cell fusion, and infectious SARS-CoV-2 neutralization were characterized in 807 serial samples from 233 reverse transcription polymerase chain reaction (RT-PCR)–confirmed Coronavirus Disease 2019 (COVID-19) individuals with detailed demographics and followed up to 7 months. A broad and sustained polyantigenic immunoreactivity against SARS-CoV-2 Spike, Membrane, and Nucleocapsid proteins, along with high viral neutralization, was associated with COVID-19 severity. A subgroup of “high responders” maintained high neutralizing responses over time, representing ideal convalescent plasma donors. Antibodies generated against SARS-CoV-2 during the first COVID-19 wave had reduced immunoreactivity and neutralization potency to emerging Spike variants and VOC. Accurate monitoring of SARS-CoV-2 antibody responses would be essential for selection of optimal responders and vaccine monitoring and design
Rapid spread of the SARS-CoV-2 JN.1 lineage is associated with increased neutralization evasion
No full textSummary: In July/August 2023, the highly mutated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) BA.2.86 lineage emerged and its descendant JN.1 is on track to become the dominant SARS-CoV-2 lineage globally. Compared to the spike (S) protein of the parental BA.2.86 lineage, the JN.1 S protein contains one mutation, L455S, which may affect receptor binding and antibody evasion. Here, we performed a virological assessment of the JN.1 lineage employing pseudovirus particles bearing diverse SARS-CoV-2 S proteins. Using this strategy, it was found that S protein mutation L455S confers increased neutralization resistance but reduces ACE2 binding capacity and S protein-driven cell entry efficiency. Altogether, these data suggest that the benefit of increased antibody evasion outweighs the reduced ACE2 binding capacity and further enabled the JN.1 lineage to effectively spread in the human population
HIV-1 entry and trans-infection of astrocytes involves CD81 vesicles
No full textAstrocytes are extensively infected with HIV-1 in vivo and play a significant role in the development of HIV-1-associated neurocognitive disorders. Despite their extensive infection, little is known about how astrocytes become infected, since they lack cell surface CD4 expression. In the present study, we investigated the fate of HIV-1 upon infection of astrocytes. Astrocytes were found to bind and harbor virus followed by biphasic decay, with HIV-1 detectable out to 72 hours. HIV-1 was observed to associate with CD81-lined vesicle structures. shRNA silencing of CD81 resulted in less cell-associated virus but no loss of co-localization between HIV-1 and CD81. Astrocytes supported trans-infection of HIV-1 to T-cells without de novo virus production, and the virus-containing compartment required 37°C to form, and was trypsin-resistant. The CD81 compartment observed herein, has been shown in other cell types to be a relatively protective compartment. Within astrocytes, this compartment may be actively involved in virus entry and/or spread. The ability of astrocytes to transfer virus, without de novo viral synthesis suggests they are capable of sequestering and protecting virus and thus, they could potentially facilitate viral dissemination in the CNS.Lachlan R. Gray, Stuart G. Turville, Tina L. HItchen, Wan-Jung Cheng, AnneM. Ellett, Hamid Salimi, Michael J. Roche, Steve L. Wesselingh, Paul R. Gorry, Melissa J. Churchil
HIV-1 infection of human macrophages directly induces viperin which inhibits viral production
No full textMacrophages are key target cells for HIV-1. HIV-1BaL induced a subset of interferon-stimulated genes in monocyte-derived macrophages (MDMs), which differed from that in monocyte-derived dendritic cells and CD4 T cells, without inducing any interferons. Inhibition of type I interferon induction was mediated by HIV-1 inhibition of interferon-regulated factor (IRF3) nuclear translocation. In MDMs, viperin was the most up-regulated interferon-stimulated genes, and it significantly inhibited HIV-1 production. HIV-1 infection disrupted lipid rafts via viperin induction and redistributed viperin to CD81 compartments, the site of HIV-1 egress by budding in MDMs. Exogenous farnesol, which enhances membrane protein prenylation, reversed viperin-mediated inhibition of HIV-1 production. Mutagenesis analysis in transfected cell lines showed that the internal S-adenosyl methionine domains of viperin were essential for its antiviral activity. Thus viperin may contribute to persistent noncytopathic HIV-1 infection of macrophages and possibly to biologic differences with HIV-1–infected T cells.Najla Nasr, Susan Maddocks, Stuart G. Turville, Andrew N. Harman, Natalie Woolger, Karla J.Helbig, John Wilkinson, Chris R. Bye, Thomas K. Wright, Dharshini Rambukwelle, Heather Donaghy, Michael R. Beard and Anthony L. Cunningha
The NRTIs lamivudine, stavudine and zidovudine have reduced HIV-1 inhibitory activity in astrocytes
No full textHIV-1 establishes infection in astrocytes and macroage-lineage cells of the central nervous system (CNS). Certain antiretroviral drugs (ARVs) can penetrate the CNS, and are therefore often used in neurologically active combined antiretroviral therapy (Neuro-cART) regimens, but their relative activity in the different susceptible CNS cell populations is unknown. Here, we determined the HIV-1 inhibitory activity of CNS-penetrating ARVs in astrocytes and macrophage-lineage cells. Primary human fetal astrocytes (PFA) and the SVG human astrocyte cell line were used as in vitro models for astrocyte infection, and monocyte-derived macrophages (MDM) were used as an in vitro model for infection of macrophage-lineage cells. The CNS-penetrating ARVs tested were the nucleoside reverse transcriptase inhibitors (NRTIs) abacavir (ABC), lamivudine (3TC), stavudine (d4T) and zidovudine (ZDV), the non-NRTIs efavirenz (EFV), etravirine (ETR) and nevirapine (NVP), and the integrase inhibitor raltegravir (RAL). Drug inhibition assays were performed using single-round HIV-1 entry assays with luciferase viruses pseudotyped with HIV-1 YU-2 envelope or vesicular stomatitis virus G protein (VSV-G). All the ARVs tested could effectively inhibit HIV-1 infection in macrophages, with EC90s below concentrations known to be achievable in the cerebral spinal fluid (CSF). Most of the ARVs had similar potency in astrocytes, however the NRTIs 3TC, d4T and ZDV had insufficient HIV-1 inhibitory activity in astrocytes, with EC90s 12-, 187- and 110-fold greater than achievable CSF concentrations, respectively. Our data suggest that 3TC, d4T and ZDV may not adequately target astrocyte infection in vivo, which has potential implications for their inclusion in Neuro-cART regimens.Lachlan R. Gray, Gilda Tachedjian, Anne M. Ellett, Michael J. Roche, Wan-Jung Cheng, Gilles J. Guillemin, Bruce J. Brew, Stuart G. Turville, Steve L. Wesselingh, Paul R. Gorry, Melissa J. Churchil
Dynamic imaging of the hepatitis C virus NS5A protein during a productive infection
No full textABSTRACT: Hepatitis C virus (HCV) NS5A is essential for viral genome replication within cytoplasmic replication complexes and virus assembly at the lipid droplet (LD) surface, although its definitive functions are poorly understood. We developed approaches to investigate NS5A dynamics during a productive infection. We report here that NS5A motility and efficient HCV RNA replication require the microtubule network and the cytoplasmic motor dynein and demonstrate that both motile and relatively static NS5A-positive foci are enriched with host factors VAP-A and Rab5A. Pulse-chase imaging revealed that newly synthesized NS5A foci are small and distinct from aged foci, while further studies using a unique dual fluorescently tagged infectious HCV chimera showed a relatively stable association of NS5A foci with core-capped LDs. These results reveal new details about the dynamics and maturation of NS5A and the nature of potential sites of convergence of HCV replication and assembly pathways. IMPORTANCE: Hepatitis C virus (HCV) is a major cause of serious liver disease worldwide. An improved understanding of the HCV replication cycle will enable development of novel and improved antiviral strategies. Here we have developed complementary fluorescent labeling and imaging approaches to investigate the localization, traffic and interactions of the HCV NS5A protein in living, virus-producing cells. These studies reveal new details as to the traffic, composition and biogenesis of NS5A foci and the nature of their association with putative sites of virus assembly.Nicholas S. Eyre, Guillaume N. Fiches, Amanda L. Aloia, Karla J. Helbig, Erin M. McCartney, Christopher S. P. McErlean, Kui Li, Anupriya Aggarwal, Stuart G. Turville, Michael R. Bear
<p>Resolving the Sites of HIV Entry: Dynamin Networks Hold the Key</p>
No full textHIV/AIDS pandemic continues to represent a major health concern worldwide and novel strategies are being devised to develop a cure for HIV infection. Central to this is a deeper understanding of host-virus interactions especially at the point of virus entry. HIV can enter target cells either by direct fusion with the plasma membrane or via endocytosis. Herein, we will review the evidence for and against either pathway especially in the context of quiescent CD4 T cells, arguably the most important cellular latent reservoir for HIV. The use of complementary approaches such as single molecule imaging, molecular techniques and small molecule inhibitors have greatly added to our understanding of HIV fusion sites. Cellular GTPase dynamin has emerged as a key player in the process given its ability to modulate HIV infection during virus entry and at later stages of virus replication cycle. These new insights into HIV fusion process in physiologically relevant target cells may in turn be leveraged to advance not only HIV cure efforts, but also immunotherapy
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