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Respiratory infections cause significant morbidity and mortality, yet it is unclear why some individuals succumb to severe disease. In patients hospitalized with avian A(H7N9) influenza, we investigated early drivers underpinning fatal disease. Transcriptomics strongly linked oleoyl-acyl-carrier-protein (ACP) hydrolase (OLAH), an enzyme mediating fatty acid production, with fatal A(H7N9) early after hospital admission, persisting until death. Recovered patients had low OLAH expression throughout hospitalization. High OLAH levels were also detected in patients hospitalized with life-threatening seasonal influenza, COVID-19, respiratory syncytial virus (RSV), and multisystem inflammatory syndrome in children (MIS-C) but not during mild disease. In olah-/- mice, lethal influenza infection led to survival and mild disease as well as reduced lung viral loads, tissue damage, infection-driven pulmonary cell infiltration, and inflammation. This was underpinned by differential lipid droplet dynamics as well as reduced viral replication and virus-induced inflammation in macrophages. Supplementation of oleic acid, the main product of OLAH, increased influenza replication in macrophages and their inflammatory potential. Our findings define how the expression of OLAH drives life-threatening viral disease.
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Myxovirus resistance (Mx) proteins are products of interferon stimulated genes (ISGs) and Mx proteins of different species have been reported to mediate antiviral activity against a number of viruses, including influenza A viruses (IAV). Ferrets are widely considered to represent the 'gold standard' small animal model for studying pathogenesis and immunity to human IAV infections, however little is known regarding the antiviral activity of ferret Mx proteins. Herein, we report induction of ferret (f)Mx1/2 in a ferret lung cell line and in airway tissues from IAV-infected ferrets, noting that fMx1 was induced to higher levels that fMx2 both in vitro and in vivo. Overexpression confirmed cytoplasmic expression of fMx1 as well as its ability to inhibit infection and replication of IAV, noting that this antiviral effect of fMx1was modest when compared to cells overexpressing either human MxA or mouse Mx1. Together, these studies provide the first insights regarding the role of fMx1 in cell innate antiviral immunity to influenza viruses. Understanding similarities and differences in the antiviral activities of human and ferret ISGs provides critical context for evaluating results when studying human IAV infections in the ferret model.
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Hurones , Virus de la Influenza A , Proteínas de Resistencia a Mixovirus , Infecciones por Orthomyxoviridae , Animales , Proteínas de Resistencia a Mixovirus/genética , Proteínas de Resistencia a Mixovirus/metabolismo , Virus de la Influenza A/inmunología , Humanos , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/virología , Replicación Viral/efectos de los fármacos , Antivirales/farmacología , Línea Celular , Ratones , Inmunidad Innata , Pulmón/virología , Pulmón/inmunologíaRESUMEN
Influenza A virus (IAV) is one of the major global public health concerns but the emerging resistance of IAV to currently available antivirals requires the need to identify potential alternatives. Polyphenol rich sugarcane extract (PRSE) is an extract prepared from the sugarcane plant Saccharum Officinarum. Herein we aimed to determine if PRSE had antiviral activity against IAV. We showed that treatment of IAV-infected cells with PRSE results in a dose-dependent inhibition of virus infection at concentrations that were non-cytotoxic. PRSE treatment limited the early stages of infection, reducing viral genome replication, mRNA transcription and viral protein expression. PRSE did not affect the ability of IAV to bind sialic acid or change the morphology of viral particles. Additionally, PRSE treatment attenuated the replication of multiple IAV strains of the H3N2 and H1N1 subtype. In conclusion, we show that PRSE displays antiviral activity against a broad range of IAV strains, in vitro.
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Subtipo H1N1 del Virus de la Influenza A , Virus de la Influenza A , Gripe Humana , Saccharum , Humanos , Polifenoles/farmacología , Subtipo H1N1 del Virus de la Influenza A/fisiología , Subtipo H3N2 del Virus de la Influenza A , Replicación Viral , Extractos Vegetales/farmacología , Antivirales/farmacologíaRESUMEN
Host cell restriction factors are intracellular proteins that can inhibit virus replication. Characterisation of novel host cell restriction factors can provide potential targets for host-directed therapies. In this study, we aimed to assess a member of the Tripartite-motif family protein (TRIM) family, TRIM16, as a putative host cell restriction factor. To this end, we utilized constitutive or doxycycline-inducible systems to overexpress TRIM16 in HEK293T epithelial cells and then tested for its ability to inhibit growth by a range of RNA and DNA viruses. In HEK293T cells, overexpression of TRIM16 resulted in potent inhibition of multiple viruses, however, when TRIM16 was overexpressed in other epithelial cell lines (A549, Hela, or Hep2), virus inhibition was not observed. When investigating the antiviral activity of endogenous TRIM16, we report that siRNA-mediated knockdown of TRIM16 in A549 cells also modulated the mRNA expression of other TRIM proteins, complicating the interpretation of results using this method. Therefore, we used CRISPR/Cas9 editing to knockout TRIM16 in A549 cells and demonstrate that endogenous TRIM16 did not mediate antiviral activity against the viruses tested. Thus, while initial overexpression in HEK293T cells suggested that TRIM16 was a host cell restriction factor, alternative approaches did not validate these findings. These studies highlight the importance of multiple complementary experimental approaches, including overexpression analysis in multiple cell lines and investigation of the endogenous protein, when defining host cell restriction factors with novel antiviral activity.
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Antiviral CD8+ T cell immunity depends on the integration of various contextual cues, but how antigen-presenting cells (APCs) consolidate these signals for decoding by T cells remains unclear. Here, we describe gradual interferon-α/interferon-ß (IFNα/ß)-induced transcriptional adaptations that endow APCs with the capacity to rapidly activate the transcriptional regulators p65, IRF1 and FOS after CD4+ T cell-mediated CD40 stimulation. While these responses operate through broadly used signaling components, they induce a unique set of co-stimulatory molecules and soluble mediators that cannot be elicited by IFNα/ß or CD40 alone. These responses are critical for the acquisition of antiviral CD8+ T cell effector function, and their activity in APCs from individuals infected with severe acute respiratory syndrome coronavirus 2 correlates with milder disease. These observations uncover a sequential integration process whereby APCs rely on CD4+ T cells to select the innate circuits that guide antiviral CD8+ T cell responses.
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Antivirales , COVID-19 , Humanos , Calibración , Células Presentadoras de Antígenos , Linfocitos T CD8-positivos , Antígenos CD40 , Interferón-alfa , Linfocitos T CD4-PositivosRESUMEN
Ectopic protein overexpression in immortalised cell lines is a commonly used method to screen host factors for their antiviral activity against different viruses. However, the question remains as to what extent such artificial protein overexpression recapitulates endogenous protein function. Previously, we used a doxycycline-inducible overexpression system, in conjunction with approaches to modulate the expression of endogenous protein, to demonstrate the antiviral activity of IFITM1, IFITM2, and IFITM3 against influenza A virus (IAV) but not parainfluenza virus-3 (PIV-3) in A549 cells. We now show that constitutive overexpression of the same IFITM constructs in A549 cells led to a significant restriction of PIV-3 infection by all three IFITM proteins. Variable IFITM mRNA and protein expression levels were detected in A549 cells with constitutive versus inducible overexpression of each IFITM. Our findings show that overexpression approaches can lead to levels of IFITM1, IFITM2, and IFITM3 that significantly exceed those achieved through interferon stimulation of endogenous protein. We propose that exceedingly high levels of overexpressed IFITMs may not accurately reflect the true function of endogenous protein, thus contributing to discrepancies when attributing the antiviral activity of individual IFITM proteins against different viruses. Our findings clearly highlight the caveats associated with overexpression approaches used to screen cellular host proteins for antiviral activity.
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Macrophages are key cellular contributors to the pathogenesis of COVID-19, the disease caused by the virus SARS-CoV-2. The SARS-CoV-2 entry receptor ACE2 is present only on a subset of macrophages at sites of SARS-CoV-2 infection in humans. Here, we investigated whether SARS-CoV-2 can enter macrophages, replicate, and release new viral progeny; whether macrophages need to sense a replicating virus to drive cytokine release; and, if so, whether ACE2 is involved in these mechanisms. We found that SARS-CoV-2 could enter, but did not replicate within, ACE2-deficient human primary macrophages and did not induce proinflammatory cytokine expression. By contrast, ACE2 overexpression in human THP-1-derived macrophages permitted SARS-CoV-2 entry, processing and replication, and virion release. ACE2-overexpressing THP-1 macrophages sensed active viral replication and triggered proinflammatory, antiviral programs mediated by the kinase TBK-1 that limited prolonged viral replication and release. These findings help elucidate the role of ACE2 and its absence in macrophage responses to SARS-CoV-2 infection.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/fisiología , Enzima Convertidora de Angiotensina 2/genética , Citocinas , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Macrófagos/metabolismo , Virión/metabolismoRESUMEN
Many interferon (IFN)-stimulated genes are upregulated within host cells following infection with influenza and other viruses. While the antiviral activity of some IFN-stimulated genes, such as the IFN-inducible GTPase myxoma resistance (Mx)1 protein 1, has been well defined, less is known regarding the antiviral activities of related IFN-inducible GTPases of the guanylate-binding protein (GBP) family, particularly mouse GBPs, where mouse models can be used to assess their antiviral properties in vivo. Herein, we demonstrate that mouse GBP1 (mGBP1) was upregulated in a mouse airway epithelial cell line (LA-4 cells) following pretreatment with mouse IFNα or infection by influenza A virus (IAV). Whereas doxycycline-inducible expression of mouse Mx1 (mMx1) in LA-4 cells resulted in reduced susceptibility to IAV infection and reduced viral growth, inducible mGBP1 did not. Moreover, primary cells isolated from mGBP1-deficient mice (mGBP1-/- ) showed no difference in susceptibility to IAV and mGBP1-/- macrophages showed no defect in IAV-induced NLRP3 (NLR family pyrin domain containing 3) inflammasome activation. After intranasal IAV infection, mGBP1-/- mice also showed no differences in virus replication or induction of inflammatory responses in the airways during infection. Thus, using complementary approaches such as mGBP1 overexpression, cells from mGBP1-/- mice and intranasal infection of mGBP1-/- we demonstrate that mGBP1 does not play a major role in modulating IAV infection in vitro or in vivo.
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Proteínas de Unión al GTP , Gripe Humana , Animales , Humanos , Ratones , Antivirales/metabolismo , Virus de la Influenza A , Gripe Humana/genética , Interferones/metabolismo , Macrófagos/metabolismo , Proteínas de Unión al GTP/metabolismoRESUMEN
MARCH1 and MARCH8 are closely related E3 ubiquitin ligases that ubiquitinate an overlapping spectrum of host proteins and restrict replication of certain viruses. While the antiviral activity of MARCH8 has been intensively studied, less is known regarding virus inhibition by MARCH1. Isoforms 1 and 2 of MARCH1 are very similar in overall structure but show major differences in their N-terminal cytoplasmic domain (N-CT). Herein, we used a doxycycline-inducible overexpression system to demonstrate that MARCH1.1 reduces titres of influenza A virus (IAV) released from infected cells whereas MARCH1.2 does not. The deletion of the entire N-CT of MARCH1.2 restored its ability to restrict IAV infectivity and sequential deletions mapped the restoration of IAV inhibition to delete the 16 N-terminal residues within the N-CT of MARCH1.2. While only MARCH1.1 mediated anti-IAV activity, qPCR demonstrated the preferential expression of MARCH1.2 over MARCH1.1 mRNA in unstimulated human peripheral blood mononuclear cells and also in monocyte-derived macrophages. Together, these studies describe the differential ability of MARCH1 isoforms to restrict IAV infectivity for the first time. Moreover, as published immunological, virological and biochemical studies examining the ability of MARCH1 to target particular ligands generally use only one of the two isoforms, these findings have broader implications for our understanding of how MARCH1 isoforms might differ in their ability to modulate particular host and/or viral proteins.
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Virus de la Influenza A , Gripe Humana , Humanos , Virus de la Influenza A/genética , Leucocitos Mononucleares , Isoformas de Proteínas/genética , AntiviralesRESUMEN
Intracellular RIG-I receptors represent key innate sensors of RNA virus infection, and RIG-I activation results in the induction of hundreds of host effector genes, including interferon-stimulated genes (ISGs). Synthetic RNA agonists targeting RIG-I have shown promise as antivirals against a broad spectrum of viruses, including influenza A virus (IAV), in both in vitro and mouse models of infection. Herein, we demonstrate that treatment of a ferret airway epithelial (FRL) cell line with a RIG-I agonist rapidly and potently induced expression of a broad range of ISGs and resulted in potent inhibition of growth of different IAV strains. In ferrets, a single intravenous injection of RIG-I agonist was associated with upregulated ISG expression in peripheral blood mononuclear cells and lung tissue, but not in nasal tissues. In a ferret model of viral contact transmission, a single treatment of recipient animals 24 h prior to cohousing with IAV-infected donors did not reduce virus transmission and shedding but did result in reduced lung virus titers 6 days after treatment. A single treatment of the IAV-infected donor animals also resulted in reduced virus titers in the lungs 2 days later. Thus, a single intravenous treatment with RIG-I agonist prior to infection or to ferrets with an established IAV infection can reduce virus growth in the lungs. These findings support further development of RIG-I agonists as effective antiviral treatments to limit the impact of IAV infections, particularly in reducing virus replication in the lower airways. IMPORTANCE RIG-I agonists have shown potential as broad-spectrum antivirals in vitro and in mouse models of infection. However, their antiviral potential has not been reported in outbred animals such as ferrets, which are widely regarded as the gold standard small animal model for human IAV infections. Herein, we demonstrate that RIG-I agonist treatment of a ferret airway cell line resulted in ISG induction and inhibition of a broad range of human influenza viruses. A single intravenous treatment of ferrets also resulted in systemic induction of ISGs, including in lung tissue, and when delivered to animals prior to IAV exposure or to animals with established IAV infection treatment resulted in reduced virus replication in the lungs. These data demonstrate the effectiveness of single RIG-I treatment against IAV in the ferret model and highlight the importance of future studies to optimize treatment regimens and delivery routes to maximize their ability to ameliorate IAV infections.
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Virus de la Influenza A , Gripe Humana , Animales , Antivirales/farmacología , Hurones/metabolismo , Humanos , Inmunidad Innata , Virus de la Influenza A/genética , Interferones/metabolismo , Leucocitos Mononucleares/metabolismo , Pulmón , Ratones , Replicación Viral/genéticaRESUMEN
RIG-I is an innate sensor of RNA virus infection and its activation induces interferon-stimulated genes (ISGs). In vitro studies using human cells have demonstrated the ability of synthetic RIG-I agonists (3pRNA) to inhibit IAV replication. However, in mouse models of IAV the effectiveness of 3pRNA reported to date differs markedly between studies. Myxoma resistance (Mx)1 is an ISG protein which mediates potent anti-IAV activity, however most inbred mouse strains do not express a functional Mx1. Herein, we utilised C57BL/6 mice that do (B6.A2G-Mx1) and do not (B6-WT) express functional Mx1 to assess the ability of prophylactic 3pRNA treatment to induce ISGs and to protect against subsequent IAV infection. In vitro, 3pRNA treatment of primary lung cells from B6-WT and B6.A2G-Mx1 mice resulted in ISG induction however inhibition of IAV infection was more potent in cells from B6.A2G-Mx1 mice. In vivo, a single intravenous injection of 3pRNA resulted in ISG induction in lungs of both B6-WT and B6.A2G-Mx1 mice, however potent and long-lasting protection against subsequent IAV challenge was only observed in B6.A2G-Mx1 mice. Thus, despite broad ISG induction, expression of a functional Mx1 is critical for potent and long-lasting RIG-I agonist-mediated protection in the mouse model of IAV infection.
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Proteína 58 DEAD Box , Proteínas de Resistencia a Mixovirus , Infecciones por Orthomyxoviridae , Animales , Antivirales , Virus de la Influenza A , Interferones , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Proteínas de Resistencia a Mixovirus/genética , ProteínasRESUMEN
Infections caused by human respiratory syncytial virus (RSV) are associated with substantial rates of morbidity and mortality. Treatment options are limited, and there is urgent need for the development of efficient antivirals. Pattern recognition receptors such as the cytoplasmic helicase retinoic acid-inducible gene (RIG) I can be activated by viral nucleic acids, leading to activation of interferon-stimulated genes and generation of an "antiviral state." In the current study, we activated RIG-I with synthetic RNA agonists (3pRNA) to induce resistance to RSV infection in vitro and in vivo. In vitro, pretreatment of human, mouse, and ferret airway cell lines with RIG-I agonist before RSV exposure inhibited virus infection and replication. Moreover, a single intravenous injection of 3pRNA 1 day before RSV infection resulted in potent inhibition of virus replication in the lungs of mice and ferrets, but not in nasal tissues. These studies provide evidence that RIG-I agonists represent a promising antiviral drug for RSV prophylaxis.
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Infecciones por Virus Sincitial Respiratorio , Virus Sincitial Respiratorio Humano , Animales , Humanos , Virus Sincitial Respiratorio Humano/fisiología , Hurones , Pulmón , Replicación Viral , Antivirales/farmacología , TretinoinaRESUMEN
Respiratory tract infection with SARS-CoV-2 results in varying immunopathology underlying COVID-19. We examine cellular, humoral and cytokine responses covering 382 immune components in longitudinal blood and respiratory samples from hospitalized COVID-19 patients. SARS-CoV-2-specific IgM, IgG, IgA are detected in respiratory tract and blood, however, receptor-binding domain (RBD)-specific IgM and IgG seroconversion is enhanced in respiratory specimens. SARS-CoV-2 neutralization activity in respiratory samples correlates with RBD-specific IgM and IgG levels. Cytokines/chemokines vary between respiratory samples and plasma, indicating that inflammation should be assessed in respiratory specimens to understand immunopathology. IFN-α2 and IL-12p70 in endotracheal aspirate and neutralization in sputum negatively correlate with duration of hospital stay. Diverse immune subsets are detected in respiratory samples, dominated by neutrophils. Importantly, dexamethasone treatment does not affect humoral responses in blood of COVID-19 patients. Our study unveils differential immune responses between respiratory samples and blood, and shows how drug therapy affects immune responses during COVID-19.
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COVID-19 , Anticuerpos Antivirales , Humanos , Inmunidad , Inmunoglobulina G , Inmunoglobulina M , Sistema Respiratorio , SARS-CoV-2 , Índice de Severidad de la Enfermedad , Glicoproteína de la Espiga del CoronavirusRESUMEN
Myxovirus resistance (Mx) proteins are dynamin-like GTPases that are inducible by interferons (IFNs) following virus infections. Most studies investigating Mx proteins have focused on their activity against influenza A viruses (IAV), although emerging evidence suggests that some Mx proteins may exhibit broader antiviral activity. Herein, we demonstrate that in addition to IAV, overexpression of mouse Mx1 (mMx1), but not mMx2, resulted in potent inhibition of growth of the human alphaherpesviruses herpes simplex virus 1 (HSV-1) and HSV-2, whereas neither inhibited the mouse betaherpesvirus murine cytomegalovirus (MCMV) in vitro. IFN induction of a functional endogenous mMx1 in primary mouse fibroblasts ex vivo was also associated with inhibition of HSV-1 growth. Using an in vitro overexpression approach, we demonstrate that mutations that result in redistribution of mMx1 from the nucleus to the cytoplasm or in loss of its combined GTP binding and GTPase activity also abrogated its ability to inhibit HSV-1 growth. Overexpressed mMx1 did not inhibit early HSV-1 gene expression but was shown to inhibit both replication of the HSV-1 genome as well as subsequent late gene expression. In a mouse model of cutaneous HSV-1 infection, mice expressing a functional endogenous mMx1 showed significant reductions in the severity of skin lesions as well as reduced HSV-1 titers in both the skin and dorsal root ganglia (DRG). Together, these data demonstrate that mMx1 mediates potent antiviral activity against human alphaherpesviruses by blocking replication of the viral genome and subsequent steps in virus replication. Moreover, endogenous mMx1 potently inhibited pathogenesis in the zosteriform mouse model of HSV-1 infection. IMPORTANCE While a number of studies have demonstrated that human Mx proteins can inhibit particular herpesviruses in vitro, we are the first to report the antiviral activity of mouse Mx1 (mMx1) against alphaherpesviruses both in vitro and in vivo. We demonstrate that both overexpressed mMx1 and endogenous mMx1 potently restrict HSV-1 growth in vitro. mMx1-mediated inhibition of HSV-1 was not associated with inhibition of virus entry and/or import of the viral genome into the nucleus, but rather with inhibition of HSV-1 genomic replication as well as subsequent late gene expression. Therefore, inhibition of human alphaherpesviruses by mMx1 occurs by a mechanism that is distinct from that reported for human Mx proteins against herpesviruses. Importantly, we also provide evidence that expression of a functional endogenous mMx1 can limit HSV-1 pathogenesis in a mouse model of infection.
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Herpes Simple , Herpesvirus Humano 1 , Proteínas de Resistencia a Mixovirus , Replicación Viral , Animales , Modelos Animales de Enfermedad , Regulación Viral de la Expresión Génica , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiología , Interferones/metabolismo , Ratones , Muromegalovirus , Proteínas de Resistencia a Mixovirus/metabolismoRESUMEN
Better methods to interrogate host-pathogen interactions during Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections are imperative to help understand and prevent this disease. Here we implemented RNA-sequencing (RNA-seq) using Oxford Nanopore Technologies (ONT) long-reads to measure differential host gene expression, transcript polyadenylation and isoform usage within various epithelial cell lines permissive and non-permissive for SARS-CoV-2 infection. SARS-CoV-2-infected and mock-infected Vero (African green monkey kidney epithelial cells), Calu-3 (human lung adenocarcinoma epithelial cells), Caco-2 (human colorectal adenocarcinoma epithelial cells) and A549 (human lung carcinoma epithelial cells) were analyzed over time (0, 2, 24, 48 hours). Differential polyadenylation was found to occur in both infected Calu-3 and Vero cells during a late time point (48 hpi), with Gene Ontology (GO) terms such as viral transcription and translation shown to be significantly enriched in Calu-3 data. Poly(A) tails showed increased lengths in the majority of the differentially polyadenylated transcripts in Calu-3 and Vero cell lines (up to ~101 nt in mean poly(A) length, padj = 0.029). Of these genes, ribosomal protein genes such as RPS4X and RPS6 also showed downregulation in expression levels, suggesting the importance of ribosomal protein genes during infection. Furthermore, differential transcript usage was identified in Caco-2, Calu-3 and Vero cells, including transcripts of genes such as GSDMB and KPNA2, which have previously been implicated in SARS-CoV-2 infections. Overall, these results highlight the potential role of differential polyadenylation and transcript usage in host immune response or viral manipulation of host mechanisms during infection, and therefore, showcase the value of long-read sequencing in identifying less-explored host responses to disease.
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COVID-19 , Animales , COVID-19/genética , Células CACO-2 , Chlorocebus aethiops , Humanos , Poliadenilación , ARN Mensajero/metabolismo , Proteínas Ribosómicas/metabolismo , SARS-CoV-2 , Análisis de Secuencia de ARN , Células VeroRESUMEN
Membrane-associated RING-CH8 (MARCH8) impairs the cell surface expression of envelope glycoproteins from different viruses, reducing their incorporation into virions. Using stable cell lines with inducible MARCH8 expression, we show that MARCH8 did not alter susceptibility to influenza A virus (IAV) infection, but virions released from infected cells were markedly less infectious. Knockdown of endogenous MARCH8 confirmed its effect on IAV infectivity. The expression of MARCH8 impaired the infectivity of both H3N2 and H1N1 strains and was dependent on its E3 ligase activity. Although virions released in the presence of MARCH8 expressed smaller amounts of viral hemagglutinin (HA) and neuraminidase (NA) proteins, there was no impact on levels of the viral HA, NA, or matrix 2 (M2) proteins detected on the surface of infected cells. Moreover, mutation of lysine residues in the cytoplasmic tails of HA, NA, and/or M2, or in the viral M1 protein, did not abrogate MARCH8-mediated restriction. While MARCH1 and -8 target similar immunological ligands and both restrict HIV-1, only MARCH8 inhibited IAV infectivity. Deletion of the N-terminal cytoplasmic (N-CT) domain of MARCH8 confirmed it to be a critical determinant of IAV inhibition. Of interest, deletion of the MARCH1 N-CT or its replacement with the MARCH8 N-CT resulted in acquisition of IAV restriction. Together, these data demonstrate that MARCH8 restricts a late stage in IAV replication by a mechanism distinct to its reported activity against other viruses. Moreover, we show that the N-CT of MARCH8 is essential for anti-IAV activity, whereas the MARCH1 N-CT inhibits its ability to restrict IAV. IMPORTANCE The antiviral activity of MARCH8 has been associated with the downregulation of envelope glycoproteins from a range of different viruses, resulting in reduced incorporation into nascent virions. Here, we show that MARCH8 restricts IAV at a late stage in virus replication, but this was not associated with reduced expression of IAV envelope glycoproteins on the surface of infected cells, pointing to a distinct mechanism of antiviral activity. Our studies also demonstrate the differential ability of MARCH1 and -8 to restrict IAV infectivity, highlighting the critical role of the N-CT domain of each protein in modulating IAV restriction. Overall, these studies provide novel insights regarding the mechanisms by which MARCH proteins contribute to cell-intrinsic immunity against IAV.
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Expresión Génica , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/genética , Ubiquitina-Proteína Ligasas/genética , Replicación Viral/genética , Animales , Perros , Regulación hacia Abajo , Células HEK293 , Humanos , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Subtipo H3N2 del Virus de la Influenza A/patogenicidad , Células de Riñón Canino Madin DarbyRESUMEN
Interferon-induced transmembrane (IFITM) proteins inhibit a broad range of enveloped viruses by blocking entry into host cells. We used an inducible overexpression system to investigate if IFITM1, IFITM2, and IFITM3 could modulate early and/or late stages of influenza A virus (IAV) or parainfluenza virus 3 (PIV-3) infection in human A549 airway epithelial cells. IAV and PIV-3 represent respiratory viruses which utilize distinct cellular entry pathways. We verify entry by endocytosis for IAV, whereas PIV-3 infection was consistent with fusion at the plasma membrane. Following induction prior to infection, all three IFITM proteins restricted the percentage of IAV-infected cells at 8 hours postinfection. In contrast, prior induction of IFITM1 and IFITM2 did not inhibit PIV-3 infection, although a modest reduction was observed with IFITM3. Small interfering RNA (siRNA)-mediated knockdown of endogenous IFITM1, IFITM2, and IFITM3 expression, in the presence or absence of pretreatment with type I interferon, resulted in increased IAV, but not PIV-3, infection. This finding suggests that while all three IFITMs display antiviral activity against IAV, they do not restrict the early stages of PIV-3 infection. IAV and PIV-3 infection culminates in viral egress through budding at the plasma membrane. Inducible expression of IFITM1, IFITM2, or IFITM3 immediately after infection did not impact titers of infectious virus released from IAV- or PIV-3-infected cells. Our findings show that IFITM proteins differentially restrict the early stages of infection of two respiratory viruses with distinct cellular entry pathways but do not influence the late stages of replication for either virus. IMPORTANCE Interferon-induced transmembrane (IFITM) proteins restrict the initial stages of infection for several respiratory viruses; however, their potential to modulate the later stages of virus replication has not been explored. In this study, we highlight the utility of an inducible overexpression system to assess the impact of IFITM proteins on either early- or late-stage replication of two respiratory viruses. We demonstrate antiviral activity by IFITM1, IFITM2, and IFITM3 against influenza A virus (IAV) but not parainfluenza virus 3 (PIV-3) during the early stages of cellular infection. Furthermore, IFITM induction following IAV or PIV-3 infection does not restrict the late stages of replication of either virus. Our findings show that IFITM proteins can differentially restrict the early stages of infection of two viruses with distinct cellular entry pathways and yet do not influence the late stages of replication for either virus.
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Virosis/metabolismo , Replicación Viral/fisiología , Células A549 , Antígenos de Diferenciación/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Endocitosis/fisiología , Interacciones Huésped-Patógeno/fisiología , Humanos , Virus de la Influenza A/metabolismo , Virus de la Influenza A/patogenicidad , Interferones/metabolismo , Proteínas de la Membrana/metabolismo , Virus de la Parainfluenza 3 Humana/metabolismo , Virus de la Parainfluenza 3 Humana/patogenicidad , Proteínas de Unión al ARN/metabolismo , Internalización del VirusRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses subgenomic RNA (sgRNA) to produce viral proteins for replication and immune evasion. We apply long-read RNA and cDNA sequencing to in vitro human and primate infection models to study transcriptional dynamics. Transcription-regulating sequence (TRS)-dependent sgRNA upregulates earlier in infection than TRS-independent sgRNA. An abundant class of TRS-independent sgRNA consisting of a portion of open reading frame 1ab (ORF1ab) containing nsp1 joins to ORF10, and the 3' untranslated region (UTR) upregulates at 48 h post-infection in human cell lines. We identify double-junction sgRNA containing both TRS-dependent and -independent junctions. We find multiple sites at which the SARS-CoV-2 genome is consistently more modified than sgRNA and that sgRNA modifications are stable across transcript clusters, host cells, and time since infection. Our work highlights the dynamic nature of the SARS-CoV-2 transcriptome during its replication cycle.
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COVID-19/genética , SARS-CoV-2/genética , Transcripción Genética/genética , Animales , Células CACO-2 , Línea Celular , Chlorocebus aethiops , Epigénesis Genética , Genoma Viral/genética , Humanos , Evasión Inmune , Sistemas de Lectura Abierta , ARN Viral/genética , Transcriptoma , Células Vero , Proteínas Virales/genéticaRESUMEN
The impact of respiratory virus infections on global health is felt not just during a pandemic, but endemic seasonal infections pose an equal and ongoing risk of severe disease. Moreover, vaccines and antiviral drugs are not always effective or available for many respiratory viruses. We investigated how induction of effective and appropriate antigen-independent innate immunity in the upper airways can prevent the spread of respiratory virus infection to the vulnerable lower airways. Activation of TLR2, when restricted to the nasal turbinates, resulted in prompt induction of innate immune-driven antiviral responses through action of cytokines, chemokines, and cellular activity in the upper but not the lower airways. We have defined how nasal epithelial cells and recruitment of macrophages work in concert and play pivotal roles to limit progression of influenza virus to the lungs and sustain protection for up to 7 days. These results reveal underlying mechanisms of how control of viral infection in the upper airways can occur and support the implementation of strategies that can activate TLR2 in nasal passages to provide rapid protection, especially for at-risk populations, against severe respiratory infection when vaccines and antiviral drugs are not always effective or available.