Proofreading mechanisms of the innate immune receptor RIG-I: distinguishing self and viral RNA.

The RIG-I-like receptors (RLRs), comprising retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2), are pattern recognition receptors belonging to the DExD/H-box RNA helicase family of proteins. RLRs detect viral RNAs in the cytoplasm and respond by initiating a robust antiviral response that up-regulates interferon and cytokine production. RIG-I and MDA5 complement each other by recognizing different RNA features, and LGP2 regulates their activation. RIG-I's multilayered RNA recognition and proofreading mechanisms ensure accurate viral RNA detection while averting harmful responses to host RNAs. RIG-I's C-terminal domain targets 5'-triphosphate double-stranded RNA (dsRNA) blunt ends, while an intrinsic gating mechanism prevents the helicase domains from non-specifically engaging with host RNAs. The ATPase and RNA translocation activity of RIG-I adds another layer of selectivity by minimizing the lifetime of RIG-I on non-specific RNAs, preventing off-target activation. The versatility of RIG-I's ATPase function also amplifies downstream signaling by enhancing the signaling domain (CARDs) exposure on 5'-triphosphate dsRNA and promoting oligomerization. In this review, we offer an in-depth understanding of the mechanisms RIG-I uses to facilitate viral RNA sensing and regulate downstream activation of the immune system.

Cellular Stress Responses against Coronavirus Infection: A Means of the Innate Antiviral Defense.

Cellular stress responses are crucial for maintaining cellular homeostasis. Stress granules (SGs), activated by eIF2α kinases in response to various stimuli, play a pivotal role in dealing with diverse stress conditions. Viral infection, as one kind of cellular stress, triggers specific cellular programs aimed at overcoming virus-induced stresses. Recent studies have revealed that virus-derived stress responses are tightly linked to the host's antiviral innate immunity. Virus infection-induced SGs act as platforms for antiviral sensors, facilitating the initiation of protective antiviral responses called "antiviral stress granules" (avSGs). However, many viruses, including coronaviruses, have evolved strategies to suppress avSG formation, thereby counteracting the host's immune responses. This review discusses the intricate relationship between cellular stress responses and antiviral innate immunity, with a specific focus on coronaviruses. Furthermore, the diverse mechanisms employed by viruses to counteract avSGs are described.

Cellular RNA and DNA sensing pathways are essential for the dose-dependent response of human monocytes to ionizing radiation.

Circulating monocytes are important players of the inflammatory response to ionizing radiation (IR). These IR-resistant immune cells migrate to radiation-damaged tissues and differentiate into macrophages that phagocytize dying cells, but also facilitate inflammation. Besides the effect of damage-associated molecular patterns, released from irradiated tissues, the inflammatory activation of monocytes and macrophages is largely dependent on IR-induced DNA damage and aberrant transcriptional activity, which may facilitate expression of type I interferons (IFN-I) and numerous inflammation-related genes. We analyzed the accumulation of dsRNA, dsDNA fragments, and RNA:DNA hybrids in the context of induction of RNA-triggered MAVS-mediated and DNA-triggered STING-mediated signaling pathways, in primary human monocytes and a monocytic cell line, THP1, in response to various doses of gamma IR. We found that exposure to lower doses (<7.5 Gy) led to the accumulation of dsRNA, along with dsDNA and RNA:DNA hybrids and activated both MAVS and STING pathway-induced gene expression and signaling activity of IFN-I. Higher doses of IR resulted in the reduced dsRNA level, degradation of RNA-sensing mediators involved in MAVS signaling and coincided with an increased accumulation of dsDNA and RNA:DNA hybrids that correlated with elevated STING signaling and NF-κB-dependent gene expression. While both pathways activate IFN-I expression, using MAVS- and STING-knockout THP1 cells, we identified differences in the spectra of interferon-stimulated genes (ISGs) that are associated with each specific signaling pathway and outlined a large group of STING signaling-associated genes. Using the RNAi technique, we found that increasing the dose of IR activates STING signaling through the DNA sensor cGAS, along with suppression of the DDX41 helicase, which is known to reduce the accumulation of RNA:DNA hybrids and thereby limit cGAS/STING signaling activity. Together, these results indicate that depending on the applied dose, IR leads to the activation of either dsRNA-induced MAVS signaling, which predominantly leads to the expression of both pro- and anti-inflammatory markers, or dsDNA-induced STING signaling that contributes to pro-inflammatory activation of the cells. While RNA:DNA hybrids boost both MAVS- and STING-mediated signaling pathways, these structures being accumulated upon high IR doses promote type I interferon expression and appear to be potent enhancers of radiation dose-dependent pro-inflammatory activation of monocytes.

Toll-like receptor 7 and RIG-I-like receptors expression in peripheral blood mononuclear cells of naïve patients with hepatitis C.

BACKGROUND: The proper function of Pattern Recognition Receptors (PRRs) as a part of the host immune system can eliminate numerous pathogens from the body. However, some viruses can manipulate PRRs to escape the innate immune system. As there is controversy in the activation of PRRs in patients infected with HCV, we decided to evaluate the gene expression changes of PRRs in HCV cases compared to the healthy control. METHODS: In this study, the relative expression of Toll-like receptor 7, RIG-I, and MAD-5 in peripheral mononuclear blood cells of twenty HCV patients and twenty healthy controls of the same gender and age were analyzed by quantitative Real-time PCR. RESULTS: Our results showed that the expression of RIG-I and MAD-5 significantly increased in HCV-infected samples compared to the controls (P value:0.01; P value:0.05), while the expression of TLR7 was similar between the case and the control group (P value:0.1). CONCLUSION: It seems in suppressing HCV, RIG-I and MAD-5 receptors are likely to be more activated than TRL7 in HCV patients. The lack of TLR7 gene expression might reflect the defect of the host in the stimulation of the innate immune system through the TLR7 pathway.

Pteropus vampyrus TRIM40 Is an Interferon-Stimulated Gene That Antagonizes RIG-I-like Receptors.

Nipah virus (NiV; genus: Henipavirus; family: Paramyxoviridae) naturally infects Old World fruit bats (family Pteropodidae) without causing overt disease. Conversely, NiV infection in humans and other mammals can be lethal. Comparing bat antiviral responses with those of humans may illuminate the mechanisms that facilitate bats' tolerance. Tripartite motif proteins (TRIMs), a large family of E3-ubiquitin ligases, fine-tune innate antiviral immune responses, and two human TRIMs interact with Henipavirus proteins. We hypothesize that NiV infection induces the expression of an immunosuppressive TRIM in bat, but not human cells, to promote tolerance. Here, we show that TRIM40 is an interferon-stimulated gene (ISG) in pteropodid but not human cells. Knockdown of bat TRIM40 increases gene expression of IFNß, ISGs, and pro-inflammatory cytokines following poly(I:C) transfection. In Pteropus vampyrus, but not human cells, NiV induces TRIM40 expression within 16 h after infection, and knockdown of TRIM40 correlates with reduced NiV titers as compared to control cells. Bats may have evolved to express TRIM40 in response to viral infections to control immunopathogenesis.

Hypoxia-inducible Factor-1α Suppresses the Innate Immune Response in Cultured Human Proximal Tubular Cells.

BACKGROUND/AIM: Retinoic acid-inducible gene (RIG)-I like receptors (RLRs) are expressed on renal proximal tubular epithelial cells (RPTECs) in viral nephropathy, indicating the presence of RLR-mediated innate immune responses in RPTECs. Hypoxia is also known to affect innate immunity. This study investigated the effects of hypoxia, and hypoxia-inducible factor (HIF) on innate immunity in RPTECs. MATERIALS AND METHODS: Primary human RPTECs were cultured under normoxic or hypoxic conditions and treated with a synthetic analog of double-stranded RNA (polyIC). The expression levels of RIG-I and MDA5, as RLRs, and IFNß, IL6, and TNFα, as inflammatory mediators were evaluated using quantitative reverse transcription-polymerase chain reaction, western blotting, and lactate dehydrogenase activity (LDH) assays. To further investigate the role of hypoxia, a small interfering RNA was used to knockdown HIF1α. RESULTS: Under normoxic conditions, polyIC increased RIG-I, MDA5, and IFNß mRNA expression in RPTECs by, 9.4±0.4-, 10.8±0.5-, and 4.0±0.1-fold, respectively, compared to control, and by 5.4±0.1-, 7.4±0.1-, and 2.4±0.3-fold, respectively, under hypoxic conditions, the rate of increase was lower than that under normoxic conditions (p<0.01). Protein expression showed a similar trend. Under hypoxic conditions, polyIC treatment with HIF1α knockdown in RPTECs increased RIG-I, MDA5, and IFNß mRNA expression by 3.1±0.5-, 2.9±0.4-, and 6.1±0.4-fold, respectively, and cytotoxicity, demonstrated by LDH assay, was increased compared to that without knockdown (all p<0.01). CONCLUSION: Hypoxia suppresses polyIC-induced RLRs mediated innate immune responses in RPTECs via HIF1α.

Immune-epithelial cell cross-talk enhances antiviral responsiveness to SARS-CoV-2 in children.

The risk of developing severe COVID-19 rises dramatically with age. Schoolchildren are significantly less likely than older people to die from SARS-CoV-2 infection, but the molecular mechanisms underlying this age-dependence are unknown. In primary infections, innate immunity is critical due to the lack of immune memory. Children, in particular, have a significantly stronger interferon response due to a primed state of their airway epithelium. In single-cell transcriptomes of nasal turbinates, we find increased frequencies of immune cells and stronger cytokine-mediated interactions with epithelial cells, resulting in increased epithelial expression of viral sensors (RIG-I, MDA5) via IRF1. In vitro, adolescent peripheral blood mononuclear cells produce more cytokines, priming A549 cells for stronger interferon responses to SARS-CoV-2. Taken together, our findings suggest that increased numbers of immune cells in the airways of children and enhanced cytokine-based interactions with epithelial cells tune the setpoint of the epithelial antiviral system. Our findings shed light on the molecular basis of children's remarkable resistance to COVID-19 and may suggest a novel concept for immunoprophylactic treatments.

Crosstalk between TLR8 and RIG-I-like receptors enhances antiviral immune responses.

Background: Toll-like receptor (TLR) agonists have been investigated due to their potential dual effects as latency reverting agents and immune modulatory compounds in people living with HIV (PLWH). Here, we investigated whether co-stimulation of TLR7/8 agonists with RIG-I-like receptor (RLR) agonists enhances antiviral immunity. Methods: Peripheral blood mononuclear cells (PBMCs) and monocyte-derived dendritic cells (DCs) were incubated with TLR and RLR-agonists for 24 h and innate and adaptive immune responses were determined (maturation markers, cytokines in supernatant, ISG expression). Results: Both TLR7 and TLR8 agonists induced pro-inflammatory cytokines in DCs as well as PBMCs. TLR8 agonists were more potent in inducing cytokine responses and had a stronger effect on DC-induced immunity. Notably, while all compounds induced IL-12p70, co-stimulation with TLR8 agonists and RLR agonist polyI: C induced significantly higher levels of IL-12p70 in PBMCs. Moreover, crosstalk between TLR8 and RLR agonists induced a strong type I Interferon (IFN) response as different antiviral IFN-stimulated genes were upregulated by the combination compared to the agonists alone. Conclusion: Our data strongly suggest that TLR crosstalk with RLRs leads to strong antiviral immunity as shown by induction of IL-12 and type I IFN responses in contrast to TLRs alone. Thus, co-stimulation of TLRs and RLRs might be a powerful strategy to induce reactivation of latent reservoir as well as antiviral immunity that eliminates the reactivated cells.

Mutations at site 207 of influenza a virus NS1 protein switch its function in regulating RIG-I-like receptors mediated antiviral responses.

RIG-I-like receptors (RLRs), retinoic acid inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5), are pattern recognition receptors through which cells initially sense pathogenic RNA and trigger interferon (IFN) signaling. Herein, we report that interferon induced protein 35 (IFI35) activates the ring finger protein 125 (RNF125)-UbcH5c-dependent degradation of RLRs and represses the recognition by RIG-I and MDA5 of viral RNA to inhibit innate immunity. Furthermore, IFI35 binds selectively to different subtypes of influenza A virus (IAV) nonstructural protein 1 (NS1) with asparagine residue207 (N207). Functionally, the NS1(N207)-IFI35 interaction restores the activity of RLRs, and IAV with NS1(non-N207) showed high pathogenicity in mice. Big data analysis showed that the 21st century pandemic IAV are almost all characterized by NS1 protein with non-N207. Collectively, our data uncovered the mechanism of IFI35 restricting the activation of RLRs and provides a new drug target comprising the NS1 protein of different IAV subtypes.

Exploiting RIG-I-like receptor pathway for cancer immunotherapy.

RIG-I-like receptors (RLRs) are intracellular pattern recognition receptors that detect viral or bacterial infection and induce host innate immune responses. The RLRs family comprises retinoic acid-inducible gene 1 (RIG-I), melanoma differentiation-associated gene 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2) that have distinctive features. These receptors not only recognize RNA intermediates from viruses and bacteria, but also interact with endogenous RNA such as the mislocalized mitochondrial RNA, the aberrantly reactivated repetitive or transposable elements in the human genome. Evasion of RLRs-mediated immune response may lead to sustained infection, defective host immunity and carcinogenesis. Therapeutic targeting RLRs may not only provoke anti-infection effects, but also induce anticancer immunity or sensitize "immune-cold" tumors to immune checkpoint blockade. In this review, we summarize the current knowledge of RLRs signaling and discuss the rationale for therapeutic targeting RLRs in cancer. We describe how RLRs can be activated by synthetic RNA, oncolytic viruses, viral mimicry and radio-chemotherapy, and how the RNA agonists of RLRs can be systemically delivered in vivo. The integration of RLRs agonism with RNA interference or CAR-T cells provides new dimensions that complement cancer immunotherapy. Moreover, we update the progress of recent clinical trials for cancer therapy involving RLRs activation and immune modulation. Further studies of the mechanisms underlying RLRs signaling will shed new light on the development of cancer therapeutics. Manipulation of RLRs signaling represents an opportunity for clinically relevant cancer therapy. Addressing the challenges in this field will help develop future generations of cancer immunotherapy.

Endogenous Retroviruses as Modulators of Innate Immunity.

Endogenous retroviruses (ERVs), or LTR retrotransposons, are a class of transposable elements that are highly represented in mammalian genomes. Human ERVs (HERVs) make up roughly 8.3% of the genome and over the course of evolution, HERV elements underwent positive selection and accrued mutations that rendered them non-infectious; thereby, the genome could co-opt them into constructive roles with important biological functions. In the past two decades, with the help of advances in sequencing technology, ERVs are increasingly considered to be important components of the innate immune response. While typically silenced, expression of HERVs can be induced in response to traumatic, toxic, or infection-related stress, leading to a buildup of viral transcripts and under certain circumstances, proteins, including functionally active reverse transcriptase and viral envelopes. The biological activity of HERVs in the context of the innate immune response can be based on the functional effect of four major viral components: (1) HERV LTRs, (2) HERV-derived RNAs, (3) HERV-derived RNA:DNA duplexes and cDNA, and (4) HERV-derived proteins and ribonucleoprotein complexes. In this review, we will discuss the implications of HERVs in all four contexts in relation to innate immunity and their association with various pathological disease states.

SAMHD1 expression modulates innate immune activation and correlates with ovarian cancer prognosis.

Purpose: SAMHD1 is a deoxynucleotide triphosphate (dNTP) triphosphohydrolase which has been proposed as a putative prognostic factor in haematological cancers and certain solid tumours, although with controversial data. Here, we evaluate SAMHD1 function in ovarian cancer, both in vitro and in ovarian cancer patients. Methods: SAMHD1 expression was downregulated in ovarian cancer cell lines OVCAR3 and SKOV3 by RNA interference. Gene and protein expression changes in immune signalling pathways were assessed. SAMHD1 expression in ovarian cancer patients was evaluated by immunohistochemistry and survival analysis was performed according to SAMHD1 expression. Results: SAMHD1 knockdown induced a significant upregulation of proinflammatory cytokines concomitant to increased expression of the main RNA-sensors, MDA5 and RIG-I, and interferon-stimulated genes, supporting the idea that the absence of SAMHD1 promotes innate immune activation in vitro. To assess the contribution of SAMHD1 in ovarian cancer patients, tumours were stratified in SAMHD1-low and SAMHD1-high expressing tumours, resulting in significantly shorter progression free survival (PFS) and overall survival (OS) in SAMHD1-high expression subgroup (p=0.01 and 0.04, respectively). Conclusions: SAMHD1 depletion correlates with increased innate immune cell signalling in ovarian cancer cells. In clinical samples, SAMHD1-low expressing tumors showed increased progression free survival and overall survival irrespective of BRCA mutation status. These results point towards SAMHD1 modulation as a new therapeutic strategy, able to enhance innate immune activation directly in tumour cells, leading to improved prognosis in ovarian cancer.

N6-methyladenosine modification of viral RNA and its role during the recognition process of RIG-I-like receptors.

N6-methyladenosine (m6A) is the most abundant RNA chemical modification in eukaryotes and is also found in the RNAs of many viruses. In recent years, m6A RNA modification has been reported to have a role not only in the replication of numerous viruses but also in the innate immune escape process. In this review, we describe the viruses that contain m6A in their genomes or messenger RNAs (mRNAs), and summarize the effects of m6A on the replication of different viruses. We also discuss how m6A modification helps viral RNAs escape recognition by exogenous RNA sensors, such as retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), during viral invasion. Overall, the goal of our review is to summarize how m6A regulates viral replication and facilitates innate immune escape. Furthermore, we elaborate on the potential of m6A as a novel antiviral target.

Regulation of Pattern-Recognition Receptor Signaling by HBX During Hepatitis B Virus Infection.

As a small DNA virus, hepatitis B virus (HBV) plays a pivotal role in the development of various liver diseases, including hepatitis, cirrhosis, and liver cancer. Among the molecules encoded by this virus, the HBV X protein (HBX) is a viral transactivator that plays a vital role in HBV replication and virus-associated diseases. Accumulating evidence so far indicates that pattern recognition receptors (PRRs) are at the front-line of the host defense responses to restrict the virus by inducing the expression of interferons and various inflammatory factors. However, depending on HBX, the virus can control PRR signaling by modulating the expression and activity of essential molecules involved in the toll-like receptor (TLR), retinoic acid inducible gene I (RIG-I)-like receptor (RLR), and NOD-like receptor (NLR) signaling pathways, to not only facilitate HBV replication, but also promote the development of viral diseases. In this review, we provide an overview of the mechanisms that are linked to the regulation of PRR signaling mediated by HBX to inhibit innate immunity, regulation of viral propagation, virus-induced inflammation, and hepatocarcinogenesis. Given the importance of PRRs in the control of HBV replication, we propose that a comprehensive understanding of the modulation of cellular factors involved in PRR signaling induced by the viral protein may open new avenues for the treatment of HBV infection.

Cytoplasmic RNA sensors and their interplay with RNA-binding partners in innate antiviral response: theme and variations.

Sensing of pathogen-associated molecular patterns including viral RNA by innate immunity represents the first line of defense against viral infection. In addition to RIG-I-like receptors and NOD-like receptors, several other RNA sensors are known to mediate innate antiviral response in the cytoplasm. Double-stranded RNA-binding protein PACT interacts with prototypic RNA sensor RIG-I to facilitate its recognition of viral RNA and induction of host interferon response, but variations of this theme are seen when the functions of RNA sensors are modulated by other RNA-binding proteins to impinge on antiviral defense, proinflammatory cytokine production and cell death programs. Their discrete and coordinated actions are crucial to protect the host from infection. In this review, we will focus on cytoplasmic RNA sensors with an emphasis on their interplay with RNA-binding partners. Classical sensors such as RIG-I will be briefly reviewed. More attention will be brought to new insights on how RNA-binding partners of RNA sensors modulate innate RNA sensing and how viruses perturb the functions of RNA-binding partners.

Evolution of RNA sensing receptors in birds.

Birds are important hosts for many RNA viruses, including influenza A virus, Newcastle disease virus, West Nile virus and coronaviruses. Innate defense against RNA viruses in birds involves detection of viral RNA by pattern recognition receptors. Several receptors of different classes are involved, such as endosomal toll-like receptors and cytoplasmic retinoic acid-inducible gene I-like receptors, and their downstream adaptor proteins. The function of these receptors and their antagonism by viruses is well established in mammals; however, this has received less attention in birds. These receptors have been characterized in a few bird species, and the completion of avian genomes will permit study of their evolution. For each receptor, functional work has established ligand specificity and activation by viral infection. Engagement of adaptors, regulation by modulators and the supramolecular organization of proteins required for activation are incompletely understood in both mammals and birds. These receptors bind conserved nucleic acid agonists such as single- or double-stranded RNA and generally show purifying selection, particularly the ligand binding regions. However, in birds, these receptors and adaptors differ between species, and between individuals, suggesting that they are under selection for diversification over time. Avian receptors and signalling pathways, like their mammalian counterparts, are targets for antagonism by a variety of viruses, intent on escape from innate immune responses.

Toll-Like Receptors (TLRs), NOD-Like Receptors (NLRs), and RIG-I-Like Receptors (RLRs) in Innate Immunity. TLRs, NLRs, and RLRs Ligands as Immunotherapeutic Agents for Hematopoietic Diseases.

The innate immune system plays a pivotal role in the first line of host defense against infections and is equipped with patterns recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Several classes of PRRS, including Toll-like receptors (TLRs), NOD-like receptors (NLRs), and RIG-I-like receptors (RLRs) recognize distinct microbial components and directly activate immune cells. TLRs are transmembrane receptors, while NLRs and RLRs are intracellular molecules. Exposure of immune cells to the ligands of these receptors activates intracellular signaling cascades that rapidly induce the expression of a variety of overlapping and unique genes involved in the inflammatory and immune responses. The innate immune system also influences pathways involved in cancer immunosurveillance. Natural and synthetic agonists of TLRs, NLRs, or RLRs can trigger cell death in malignant cells, recruit immune cells, such as DCs, CD8+ T cells, and NK cells, into the tumor microenvironment, and are being explored as promising adjuvants in cancer immunotherapies. In this review, we provide a concise overview of TLRs, NLRs, and RLRs: their structure, functions, signaling pathways, and regulation. We also describe various ligands for these receptors and their possible application in treatment of hematopoietic diseases.

Comparative Analysis of Six IRF Family Members in Alveolar Epithelial Cell-Intrinsic Antiviral Responses.

Host cell-intrinsic antiviral responses are largely mediated by pattern-recognition receptor (PRR) signaling and the interferon (IFN) system. The IFN regulatory factor (IRF) family of transcription factors takes up a central role in transcriptional regulation of antiviral innate immunity. IRF3 and IRF7 are known to be key players downstream of PRRs mediating the induction of type I and III IFNs. IFN signaling then requires IRF9 for the expression of the full array of interferon stimulated genes (ISGs) ultimately defining the antiviral state of the cell. Other members of the IRF family clearly play a role in mediating or modulating IFN responses, such as IRF1, IRF2 or IRF5, however their relative contribution to mounting a functional antiviral response is much less understood. In this study, we systematically and comparatively assessed the impact of six members of the IRF family on antiviral signaling in alveolar epithelial cells. We generated functional knockouts of IRF1, -2, -3, -5, -7, and -9 in A549 cells, and measured their impact on the expression of IFNs and further cytokines, ISGs and other IRFs, as well as on viral replication. Our results confirmed the vital importance of IRF3 and IRF9 in establishing an antiviral state, whereas IRF1, 5 and 7 were largely dispensable. The previously described inhibitory activity of IRF2 could not be observed in our experimental system.

Interferon responses to norovirus infections: current and future perspectives.

Human noroviruses (HuNoVs) are increasingly becoming the main cause of transmissible gastroenteritis worldwide, with hundreds of thousands of deaths recorded annually. Yet, decades after their discovery, there is still no effective treatment or vaccine. Efforts aimed at developing vaccines or treatment will benefit from a greater understanding of norovirus-host interactions, including the host response to infection. In this review, we provide a concise overview of the evidence establishing the significance of type I and type III interferon (IFN) responses in the restriction of noroviruses. We also critically examine our current understanding of the molecular mechanisms of IFN induction in norovirus-infected cells, and outline the diverse strategies deployed by noroviruses to supress and/or avoid host IFN responses. It is our hope that this review will facilitate further discussion and increase interest in this area.

Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) variants govern transmissibility, responsiveness to vaccination, and disease severity. In a screen for new models of SARS-CoV-2 infection, we identify human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of angiotensin-converting enzyme 2 (ACE2) expression. Remarkably, H522 infection requires the E484D S variant; viruses expressing wild-type S are not infectious. Anti-S monoclonal antibodies differentially neutralize SARS-CoV-2 E484D S in H522 cells as compared to ACE2-expressing cells. Sera from vaccinated individuals block this alternative entry mechanism, whereas convalescent sera are less effective. Although the H522 receptor remains unknown, depletion of surface heparan sulfates block H522 infection. Temporally resolved transcriptomic and proteomic profiling reveal alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type I interferon signaling. These findings establish an alternative SARS-CoV-2 host cell receptor for the E484D SARS-CoV-2 variant, which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.