Anti-SARS-CoV-2 activity of targeted kinase inhibitors: Repurposing clinically available drugs for COVID-19 therapy

Coronavirus disease 2019 (COVID-19) remains a major public health concern, and vaccine unavailability, hesitancy, or failure underscore the need for discovery of efficacious antiviral drug therapies. Numerous approved drugs target protein kinases associated with viral life cycle and symptoms of infection. Repurposing of kinase inhibitors is appealing as they have been vetted for safety and are more accessible for COVID-19 treatment. However, an understanding of drug mechanism is needed to improve our understanding of the factors involved in pathogenesis. We tested the in vitro activity of three kinase inhibitors against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including inhibitors of AXL kinase, a host cell factor that contributes to successful SARS-CoV-2 infection. Using multiple cell-based assays and approaches, gilteritinib, nintedanib, and imatinib were thoroughly evaluated for activity against SARS-CoV-2 variants. Each drug exhibited antiviral activity, but with stark differences in potency, suggesting differences in host dependency for kinase targets. Importantly, for gilteritinib, the amount of compound needed to achieve 90% infection inhibition, at least in part involving blockade of spike protein-mediated viral entry and at concentrations not inducing phospholipidosis (PLD), approached a clinically achievable concentration. Knockout of AXL, a target of gilteritinib and nintedanib, impaired SARS-CoV-2 variant infectivity, supporting a role for AXL in SARS-CoV-2 infection and supporting further investigation of drug-mediated AXL inhibition as a COVID-19 treatment. This study supports further evaluation of AXL-targeting kinase inhibitors as potential antiviral agents and treatments for COVID-19. Additional mechanistic studies are needed to determine underlying differences in virus response.     

HIV-1 incorporation of host-cell-derived glycosphingolipid GM3 allows for capture by mature dendritic cells

The interaction between HIV and dendritic cells (DCs) is an important early event in HIV-1 pathogenesis that leads to efficient viral dissemination. Here we demonstrate a HIV gp120-independent DC capture mechanism that uses virion-incorporated host-derived gangliosides with terminal α2-3-linked sialic acid linkages. Using exogenously enriched virus and artificial liposome particles, we demonstrate that both α2-3 gangliosides GM1 and GM3 are capable of mediating this interaction when present in the particle at high levels. In the absence of overexpression, GM3 is the primary ligand responsible for this capture mechanism, because siRNA depletion of GM3 but not GM1 from the producer cell and hence virions, resulted in a dramatic decrease in DC capture. Furthermore, HIV-1 capture by DCs was competitively inhibited by targeting virion-associated GM3, but was unchanged by targeting GM1. Finally, virions were derived from monocytoid THP-1 cells that constitutively display low levels of GM1 and GM3, or from THP-1 cells induced to express high surface levels of GM1 and GM3 upon stimulation with the TLR2/1 ligand Pam3CSK4. Compared with untreated THP-1 cells, virus produced from Pam3CSK4-stimulated THP-1 cells incorporated higher levels of GM3, but not GM1, and showed enhanced DC capture and trans-infection. Our results identify a unique HIV-1 DC attachment mechanism that is dependent on a host-cell-derived ligand, GM3, and is a unique example of pathogen mimicry of host-cell recognition pathways that drive virus capture and dissemination in vivo.     

Immature dendritic cell-derived exosomes can mediate HIV-1 trans infection

Immature dendritic cells (DCs) capture HIV type 1 (HIV-1) and can transmit captured virus particles to T cells. In this report, we show that HIV-1 particles captured by DCs can be transmitted to T cells by exocytosis without de novo infection. Captured HIV-1 particles were rapidly endocytosed to tetraspan protein (CD9, CD63)-positive endocytic compartments that were reminiscent of multivesicular endosomal bodies. Furthermore, some of the endocytosed virus particles were constitutively released into the extracellular milieu in association with HLA-DR1(+), CD1b(+), CD9(+), and CD63(+) vesicles (exosomes) and could initiate productive infections of CD4(+) target cells. Surprisingly, the exocytosed vesicle-associated HIV-1 particles from DCs were 10-fold more infectious on a perparticle basis than cell-free virus particles. These studies describe a previously undescribed mechanism of DC-mediated HIV-1 transmission and suggest that virus particle trafficking to multivesicular endosomal bodies and subsequent exocytosis can provide HIV-1 particles captured by DCs an avenue for immune escape.     

Capture and transfer of HIV-1 particles by mature dendritic cells converges with the exosome-dissemination pathway

Exosomes are secreted cellular vesicles that can be internalized by dendritic cells (DCs), contributing to antigen-specific naive CD4(+) T-cell activation. Here, we demonstrate that human immunodeficiency virus type 1 (HIV-1) can exploit this exosome antigen-dissemination pathway intrinsic to mature DCs (mDCs) for mediating trans-infection of T lymphocytes. Capture of HIV-1, HIV-1 Gag-enhanced green fluorescent protein (eGFP) viral-like particles (VLPs), and exosomes by DCs was up-regulated upon maturation, resulting in localization within a CD81(+) compartment. Uptake of VLPs or exosomes could be inhibited by a challenge with either particle, suggesting that the expression of common determinant(s) on VLP or exosome surface is necessary for internalization by mDCs. Capture by mDCs was insensitive to proteolysis but blocked when virus, VLPs, or exosomes were produced from cells treated with sphingolipid biosynthesis inhibitors that modulate the lipid composition of the budding particles. Finally, VLPs and exosomes captured by mDCs were transmitted to T lymphocytes in an envelope glycoprotein-independent manner, underscoring a new potential viral dissemination pathway.     

Expression of pro‐ and anti‐apoptosis gene products in brains from paediatric patients with HIV‐1 encephalitis

We have previously demonstrated the presence of DNA fragmentation in neurons, macrophages and microglia consistent with apoptosis, but not in reactive astrocytes in brain tissue from paediatric patients with HIV‐1 encephalitis (HIVE). To further understand the underlying mechanism(s) for these findings as they relate to gene‐directed neural cell death, we studied the in‐situ expression of the Bcl‐2 family of proteins, including the pro‐apoptosis gene product Bax, the anti‐apoptosis gene product Bcl‐2, and Bcl‐x. We demonstrate significantly elevated numbers of Bax‐positive microglia and macrophages immunoreactive in basal ganglia and cerebral cortex of children who had HIVE, in comparison to HIV‐1 infected children without encephalitis or children who were seronegative for HIV‐1. In contrast, patients with HIVE, but not HIV‐1 without encephalitis, or seronegative controls, had increased expression of Bcl‐2 and Bcl‐x in reactive astrocytes in cortex and basal ganglia. In vitro studies using Western blot analysis demonstrated an up‐regulation in the levels of Bax, and phosphorylated (i.e. inactive) Bcl‐2 in HIV‐1 infected macrophages, and in LPS‐activated macrophages, relative to levels in virus‐negative unstimulated macrophages. These results suggest that productive HIV‐1 infection, or cellular activation, renders macrophages more vulnerable to apoptosis. Taken together, these findings suggest that brain‐resident macrophages and microglia in patients with HIV‐1 encephalitis are more prone to undergo apoptosis and that astrocytes in contrast may be resistant to apoptosis. This may represent a mechanism to limit microglial activation and the spread of productive HIV‐1 infection in the CNS of children with HIV‐1 encephalitis.