Coronavirus Molecular Biology and Medicine Fall 2024 Lecture PDF

Coronavirus Molecular Biology and Medicine Fall 2024 Interferons and coronaviruses Ricardo Rajsbaum, PhD [email protected] Department of Medicine Center for Virus-Host-Innate Immunity GSND 5335Q...

Coronavirus Molecular Biology and Medicine Fall 2024 Interferons and coronaviruses Ricardo Rajsbaum, PhD [email protected] Department of Medicine Center for Virus-Host-Innate Immunity GSND 5335Q Lecture 10/01/2024  Virus infection and host defense Type-I IFNs IFN -Recognition of viruses by pattern - Viral evasion of innate immune recognition receptors (PRRs) in cells. response (viral antagonists of IFNs) -Examples: toll like receptors (tlr) -Pattern recognition receptors detect pathogen - Detrimental effects of type-1 associated molecular pattern (PAMPS) interferons (IFNs) -Regulation of antiviral signaling pathways - Effects of type-1 IFNs in cellular - Antiviral effector mechanisms (inducible) immune responses  THE DISCOVERY OF INTERFERON A study collected the supernatant of infected cells Used supernatants to inclubate non- infected cells and found that incubation with supernatants provides cells with protection against infection. There was some sort of “viral interference”. Interferon interferes with viral infection at onset of infection. Isaacs and Lindenmann, 1957. Proc. roy. Soc. B  Pathways of type 1 IFN induction and receptor signaling Lipopolysaccharide (LPS) DNA RNA (Gram-negative bacteria) Viruses Viruses dsRNA ssRNA CpG Localization of different PRRs is critical for proper recognition of pathogen products. Pathways contain signaling molecules that need to localize in particular places in the cell. Depends on how virus enters the cell. Ex. RIG-1 and MDAs located on the mitochondria signal for RNA viruses. Ex. Toll like receptors in endosome gives specific response for viruses that enter the cell via the endosomes. All viruses eventually go through kinases that phosphorylate transcription factors IRF3 and IRF7 that need to translocate to nucleus, required for interferon alpha and interferon beta induction. Interferon signaling activates kinases that phosphorylate different transcription factors. ISGF3 complex is an important complex that contains 3 transcription factors (STAT1, STAT2 and IRF9). Interferon-sensitive response elements (ISREs) are binding sites for the IFN-stimulated gene factor 3 (ISGF3) complex, which is responsible for the induction of IFN-stimulated genes (ISGs).  Interferons and their receptors Each type of interferons use different receptors Type 1 and type 3 interferons use different receptors but will have similar responses because they use the same signaling pathways that forms the ISGF complex. Type 2 Interferons Type 3 Interferon receptors are localized to epithelial. In contrast to the expression of the receptor for IFN-I (IFNAR) in most nucleated cells, the IFN-3 is restricted to tissues of epithelial origin. e.g. gastrointestinal, respiratory and reproductive tracts. IFN𝛌 effects appear less inflammatory - Expression of the receptors in specific cell types - Production of IFNs by specific cell types  IFN receptor binding affinity IFNβ is more potent than IFN⍺ Differences in type 1 IFNs Protein affinity to the receptor can trigger different strengths of the response. IFN beta has high affinity for binding to the type 1 interferon receptor there it can trigger a much stronger response. It has high levels of IFG induction and a much more potent antiviral activity. Situation where interferons are added 24 hours after host as been infected Viral proteins block interferon response. Once virus is already replicating in the cell, interferons have very little antiviral effects. Lack of type-I (or type-II) IFN production or signaling results in inefficient antiviral responses - Examples: many viruses do not replicate in WT (IFN competent mice), but do replicate in IFN Receptor KO - Many human viruses need to be adapted to mice for replication, but replicate well in IFN-deficient mice How would you study whether IFNs are protective against a particular pathogen? - The use of type-I IFN receptor KO cells and/or mice - The use of cells that do not produce active IFN-I (e.g. Vero cells – do not produce but do respond) Effects of type-I IFNs in cellular immune responses  Virus enters the cell, cell recognizes infection and triggers signaling pathways that produce interferons. Type 1 interferons are sensed by neighboring cells, this then produces interferon stimulating genes that can block viral infection. Indirect effects as a result of viral infection- Enhancing immune response via other cells Macrophages and dendritic cells can be activated by interferons. Interferons help B cells with antibody production.  Type-I IFN effects on regulation of other cytokines Interferons can promote induction of other cytokines important in viral infection such as IL-10 IL-10 is a regulatory cytokine. Interferons can induce IL-10 which can result in regulatory functions and antiinflammatory effects. Immune cell types, PRRs and cytokine production What determines the levels of IFN production?  Different types of dendritic cells that contain different expression levels of TLRs pDCs are specialized dendritic cells that can produce high levels of type 1 interferons. quick and high interferon production is possible for 2 reasons. 1. Express high levels of Toll like receptor (tlr) 7 and TLR 9 that can LPS Flagellin IL-1 IL-18 recognize viral RNA. Lipoproteins 2. High constitutive expression of transcription factor IRF7 in Pam3Cys plasmacytoid dendritic cells (pDCs). IL-1R IL-18R TLR1, TLR2 or TLR4 TLR5 TLR6 BM-myeloid DC T cells and macrophages Expression levels of TLRs in dendritic cell subsets Macrophages BM-myeloid DC BM-myeloid DC Splenic DC Splenic DC TLR Human blood Mouse spleen Splenic DC Macrophages Cell CD11c+ pDC CD4+ CD8a+ DN pDC membrane TIR-domain TLR1 +++ ++ ++ ++ ++ ++ MAL TLR2 TRAM ++ +/- ++ ++ ++ ++ MyD88 MyD88 MyD88 MyD88 TLR3 MyD88 +++ - + +++ ++ +/- TLR4 ++ - + + + +/- TRIF TLR5 ++ +/- +++ + ++ +/- CpG dsRNA DNA TLR6 ++ + +++ ++ ++ ++ Endosomes Cytoplasm TLR7 + ++ ++ - + +++ ssRNA TLR8 + - ++ ++ ++ ++ TLR9 pDC (mouse+human) TLR3 BM-myeloid DC (mouse) Splenic DC (mouse) TLR9 - +++ ++ ++ ++ +++ BM-myeloid DC MAC (mouse) Macrophages TLR7,8 (also on B cells-mouse and human) TLR10 ++ +/- N/A N/A N/A N/A Splenic DC Other cells F 8 I TR D8 My pDC (mouse+human) BM-myeloid DC (mouse) 8 MyD8 Splenic DC (mouse) MAC (mouse)  SARS-CoV-2 and the innate immune response - Coronaviridae - Enveloped - Positive-sense single-stranded RNA virus - Virus can trigger cytokine storm (hyper inflammation) which leads to tissue damage. - Interferons are also triggered in COVID-19 viral viral antagonists antagonists ? ? Harrison, Lin, Wang. Trends in Immunology. 2020, Vol. 41, No. 12  Figure 1 SARS-CoV-2 induction of innate immune activation and IFNs Virus more likely to be identified by MDA-5 receptors Various proteins present to be able to inhibit various parts of the pathways. SARS-CoV-2 infects epithelial cells that respond to type 3 interferons and interferon lambda can act as antiviral cytokine to the infection.  Figure 2 IFN actions against SARS-CoV-2 mediated by select ISGs Many ISGs act as antiviral factors attack at the early stages of SARS-CoV-2 viral replication Viruses have evolved to take advantage of antiviral factors such as ISGs to enhance viral replication.  Pathogenic roles of Type-I and type-III IFNs Reports have shown critically ill patients with reduced interferon responses and high levels of inflammatory cytokines. In other cases, high levels of interferons appeared to damage tissue. Some studies have shown that type 3 interferon gamma can promote apoptosis and impaired epithelial proliferation which prevents tissue recovery. Type 1 interferons have their receptors on many cells therefore have more inflammatory effects. - Mechanisms by which IFN-λ responses contribute to pathogenesis in viral pneumonias. - Conversely, sceintists studied peripheral blood responses from a cohort of 50 patients with coronavirus disease 2019 (COVID-19), demonstrating that critically ill patients have reduced IFN responses paired with a proinflammatory response. Are IFNs good or bad for the host?  Protective and Pathogenic Roles of Type I IFNs during Coronavirus Infection The effect of IFN-I in COVID-19 outcome is time dependent. Early IFN-I response can control viral growth yields an antiviral state, so leading to a mild form of disease, while delayed IFN-I expression is associated with rapid SARS-CoV-2 replication, so the patients present more extensive disease and poorer clinical outcomes. Anaeigoudari et al., 2021. Inflammation  Type-I IFNs IFN IFN-I Receptor signaling ISGs Nucleus Antiviral effectors Virus (inducible) Viral evasion or inhibition of the antiviral response How would you study IFN antagonists?  Screening for SARS-CoV-2 IFN antagonists E S N Identifying Interferon antagonists M All viral proteins are cloned in expression vectors. Reporter systems transfects cells that contain an interferon promoter. Reporter system and cell transfection is triggered by over expressing a domain of RIG-I that is active all the time. RIG-I is typically in an inactive form. When RIG-I encounters viral RNA it undergoes a conformational change to constitutively activate the RIG-I domain = over expression = interferons are triggered. RIG-I (2CARD) viral IRF3 antagonists? IFNβ IRF3 Luciferase reporter  Proviral role of caspase-6 (apoptosis) in coronavirus infections SARS can induce via membrane protein & trigger apoptosis pathway that leads to caspase 6 activation. Caspase 6 leads to apoptosis. Caspase 6 can also cleave the nucleocapsid protein of SARS. A cleavage product of nucleocapsid can inhibit interferon 3 response and allows virus to replicate better.