The Autophagy Cargo Receptor SQSTM1 Inhibits Infectious Bursal Disease Virus Infection through Selective Autophagic Degradation of Double-Stranded Viral RNA

Selective autophagy mediates the degradation of cytoplasmic cargos, such as damaged organelles, invading pathogens, and protein aggregates. However, whether it targets double-stranded RNA (dsRNA) of intracellular pathogens is still largely unknown. Here, we show that selective autophagy regulates the degradation of the infectious bursal disease virus (IBDV) dsRNA genome. The amount of dsRNA decreased greatly in cells that overexpressed the autophagy-required protein VPS34 or autophagy cargo receptor SQSTM1, while it increased significantly in SQSTM1 or VPS34 knockout cells or by treating wild-type cells with the autophagy inhibitor chloroquine or wortmannin. Confocal microscopy and structured illumination microscopy showed SQSTM1 colocalized with dsRNA during IBDV infection. A pull-down assay further confirmed the direct binding of SQSTM1 to dsRNA through amino acid sites R139 and K141. Overexpression of SQSTM1 inhibited the replication of IBDV, while knockout of SQSTM1 promoted IBDV replication. Therefore, our findings reveal the role of SQSTM1 in clearing viral dsRNA through selective autophagy, highlighting the antiviral role of autophagy in the removal of the viral genome.

Untangling the roles of RNA helicases in antiviral innate immunity

One of the first layers of protection that metazoans put in place to defend themselves against viruses rely on the use of proteins containing DExD/H-box helicase domains. These members of the duplex RNA–activated ATPase (DRA) family act as sensors of double-stranded RNA (dsRNA) molecules, a universal marker of viral infections. DRAs can be classified into 2 subgroups based on their mode of action: They can either act directly on the dsRNA, or they can trigger a signaling cascade. In the first group, the type III ribonuclease Dicer plays a key role to activate the antiviral RNA interference (RNAi) pathway by cleaving the viral dsRNA into small interfering RNAs (siRNAs). This represents the main innate antiviral immune mechanism in arthropods and nematodes. Even though Dicer is present and functional in mammals, the second group of DRAs, containing the RIG-I-like RNA helicases, appears to have functionally replaced RNAi and activate type I interferon (IFN) response upon dsRNA sensing. However, recent findings tend to blur the frontier between these 2 mechanisms, thereby highlighting the crucial and diverse roles played by RNA helicases in antiviral innate immunity. Here, we will review our current knowledge of the importance of these key proteins in viral infection, with a special focus on the interplay between the 2 main types of response that are activated by dsRNA.

Infection of Brain Pericytes Underlying Neuropathology of COVID-19 Patients

A wide range of neurological manifestations have been associated with the development of COVID-19 following SARS-CoV-2 infection. However, the etiology of the neurological symptomatology is still largely unexplored. Here, we used state-of-the-art multiplexed immunostaining of human brains (n = 6 COVID-19, median age = 69.5 years; n = 7 control, median age = 68 years) and demonstrated that expression of the SARS-CoV-2 receptor ACE2 is restricted to a subset of neurovascular pericytes. Strikingly, neurological symptoms were exclusive to, and ubiquitous in, patients that exhibited moderate to high ACE2 expression in perivascular cells. Viral dsRNA was identified in the vascular wall and paralleled by perivascular inflammation, as signified by T cell and macrophage infiltration. Furthermore, fibrinogen leakage indicated compromised integrity of the blood–brain barrier. Notably, cerebrospinal fluid from additional 16 individuals (n = 8 COVID-19, median age = 67 years; n = 8 control, median age = 69.5 years) exhibited significantly lower levels of the pericyte marker PDGFRβ in SARS-CoV-2-infected cases, indicative of disrupted pericyte homeostasis. We conclude that pericyte infection by SARS-CoV-2 underlies virus entry into the privileged central nervous system space, as well as neurological symptomatology due to perivascular inflammation and a locally compromised blood–brain barrier.

Structure of the poxvirus decapping enzyme D9 reveals its mechanism of cap recognition and catalysis

SUMMARYPoxviruses encode decapping enzymes that remove the protective 5’ cap from both host and viral mRNAs to commit transcripts for decay by the cellular exonuclease Xrn1. Decapping by these enzymes is critical for poxvirus pathogenicity by means of simultaneously suppressing host protein synthesis and limiting the accumulation of viral dsRNA, a trigger for antiviral responses. Here we present the first high resolution structural view of the vaccinia virus decapping enzyme D9. This Nudix enzyme contains a novel domain organization in which a three-helix bundle is inserted into the catalytic Nudix domain. The 5’ mRNA cap is positioned in a bipartite active site at the interface of the two domains. Specificity for the methylated guanosine cap is achieved by stacking between conserved aromatic residues in a manner similar to that observed in canonical cap binding proteins VP39, eIF4E, and CBP20 and distinct from eukaryotic decapping enzyme Dcp2.

On-chip Paper Electrophoresis for Ultrafast Screening of Infectious Diseases

The outbreak of new viral strains promotes advances in universal diagnostic techniques for detecting infectious diseases with unknown viral sequence. Long double-stranded RNA (dsRNA), a hallmark of infections, serves as a virus marker for prompt detection of viruses with unknown genomes. Here, we report on-chip paper electrophoresis for ultrafast screening of infectious diseases. Negatively charged RNAs pass through the micro and nanoscale pores of cellulose in order of size under an external electric field applied to the paper microfluidic channel. Quantitative separation of long dsRNA mimicking poly I:C was analyzed from 1.67 to 33 ng·μL−1, which is close to the viral dsRNA concentration in infected cells. This paper-based capillary electrophoresis chip (paper CE chip) can provide a new diagnostic platform for ultrafast viral disease detection at the point-of-care (POC) level.

ADAR mediated A-to-I RNA editing affects SARS-CoV-2 characteristics and fuels its evolution

Upon SARS-CoV-2 infection, viral intermediates activate the Type I interferon (IFN) response through MDA5-mediated sensing and accordingly induce ADAR1 p150 expression, which might lead to A-to-I RNA editing of SARS-CoV-2. Here, we developed an RNA virus-specific editing identification pipeline, surveyed 7622 RNA-seq data from diverse types of samples infected with SARS-CoV-2, and constructed an atlas of A-to-I RNA editing sites in SARS-CoV-2. We found that A-to-I editing was dynamically regulated, and on average, approximately 91 editing events were deposited at viral dsRNA intermediates per sample. Moreover, editing hotspots were observed, including recoding sites in the spike gene that affect viral infectivity and antigenicity. Finally, we provided evidence that RNA editing accelerated SARS-CoV-2 evolution in humans. Collectively, our data suggest that SARS-CoV-2 hijacks components of the host antiviral machinery to edit its genome and fuel its evolution.

Innate Immune Responses to Wildtype and Attenuated Sheeppox Virus Mediated Through RIG-1 Sensing in PBMC In-Vitro

Sheeppox (SPP) is a highly contagious disease of small ruminants caused by sheeppox virus (SPPV) and predominantly occurs in Asia and Africa with significant economic losses. SPPV is genetically and immunologically closely related to goatpox virus (GTPV) and lumpy skin disease virus (LSDV), which infect goats and cattle respectively. SPPV live attenuated vaccines (LAVs) are used for vaccination against SPP and goatpox (GTP). Mechanisms related to innate immunity elicited by SPPV are unknown. Although adaptive immunity is responsible for long-term immunity, it is the innate responses that prevent viral invasion and replication before LAVs generate specific long-term protection. We analyzed the relative expression of thirteen selected genes that included pattern recognition receptors (PRRs), Nuclear factor-κβ p65 (NF-κβ), and cytokines to understand better the interaction between SPPV and its host. The transcripts of targeted genes in sheep PBMC incubated with either wild type (WT) or LAV SPPV were analyzed using quantitative PCR. Among PRRs, we observed a significantly higher expression of RIG-1 in PBMC incubated with both WT and LAV, with the former producing the highest expression level. However, there was high inter-individual variability in cytokine transcripts levels among different donors, with the expression of TNFα, IL-15, and IL-10 all significantly higher in both PBMC infected with either WT or LAV compared to control PBMC. Correlation studies revealed a strong significant correlation between RIG-1 and IL-10, between TLR4, TNFα, and NF-κβ, between IL-18 and IL-15, and between NF-κβ and IL-10. There was also a significant negative correlation between RIG-1 and IFNγ, between TLR3 and IL-1 β, and between TLR4 and IL-15 (P< 0.05). This study identified RIG-1 as an important PRR in the signaling pathway of innate immune activation during SPPV infection, possibly through intermediate viral dsRNA. The role of immunomodulatory molecules produced by SPPV capable of inhibiting downstream signaling activation following RIG-1 upregulation is discussed. These findings advance our knowledge of the induction of immune responses by SPPV and will help develop safer and more potent vaccines against SPP and GTP.

Mouse primary microglia respond differently to LPS and poly(I:C) in vitro

Microglia, CNS resident innate immune cells, respond strongly to activation of TLR3 and TLR4, which recognize viral dsRNA poly(I:C) and bacterial endotoxin LPS, respectively. However, few studies have thoroughly and parallelly compared functional phenotypes and downstream mechanisms between LPS- and poly(I:C)-exposed primary microglia. Here, we investigated the responses of mouse primary microglia upon LPS and poly(I:C) stimulation by detecting various phenotypes ranging from morphology, proliferation, secretion, chemotaxis, to phagocytosis. Furthermore, we explored their sequential gene expression and the downstream signal cascades. Interestingly, we found that the microglial activation pattern induced by LPS was distinguished from that induced by poly(I:C). Regarding microglial morphology, LPS caused an ameboid-like shape while poly(I:C) induced a bushy shape. Microglial proliferation was also facilitated by LPS but not by poly(I:C). In addition, LPS and poly(I:C) modulated microglial chemotaxis and phagocytosis differently. Furthermore, genome-wide analysis provided gene-level support to these functional differences, which may be associated with NF-κb and type I interferon pathways. Last, LPS- and poly(I:C)-activated microglia mediated neurotoxicity in a co-culture system. This study extends our understanding of TLR roles in microglia and provides insights into selecting proper inflammatory microglial models, which may facilitate identification of new targets for therapeutic application.

The innate immune kinase TBK1 directly increases mTORC2 activity and downstream signaling to Akt

TBK1 (TANK-binding kinase 1) responds to microbial pathogens to initiate cellular responses critical for host innate immune defense. We found previously that TBK1 phosphorylates mTOR (mechanistic target of rapamycin) (on S2159) to increase mTOR complex 1 (mTORC1) activity and signaling in response to the growth factor EGF and the viral dsRNA mimetic poly(I:C). mTORC1 and the less well studied mTORC2 respond to diverse cues to control cellular metabolism, proliferation, and survival. Here we demonstrate that TBK1 activates mTOR complex 2 (mTORC2) directly to increase Akt phosphorylation at physiological levels of protein expression. We find that TBK1 phosphorylates mTOR S2159 within mTORC2 in vitro, phosphorylates mTOR S2159 in cells, and interacts with mTORC2 in cells. By studying MEFs lacking TBK1, as well as MEFs, macrophages, and mice bearing an Mtor S2159A knock-in allele (MtorA/A), we show that TBK1 and mTOR S2159 phosphorylation increase mTORC2 catalytic activity and promote mTOR-dependent downstream signaling to Akt in response to several growth factors and poly(I:C). While microbial-derived stimuli activate TBK1, we find that growth factors fail to activate TBK1 or increase mTOR S2159 phosphorylation in MEFs. Thus, we propose that basal TBK1 activity cooperates with growth factors in parallel to increase mTORC2 (and mTORC1) signaling. Collectively, these results reveal crosstalk between TBK1 and mTOR complexes (mTORCs), key nodes within two major signaling systems. As TBK1 and mTORCs have each been linked to tumorigenesis and metabolic disorders, these kinases may work together in a direct manner in a variety of physiological and pathological settings.