Innate Host Responses to Viral Infection 1. MICR3002. Dr Nigel McMillan Centre for Immunology and Cancer Research. Complete lecture with notes and summary lecture podcast* (with slides) available at www.cicr.uq.edu.au/students * Plays in iTunes or Quicktime (free from apple.com) Peloponnesian War and Thucydides Quic kTi me™ a nd a TIFF (Un com pres sed ) dec omp ress or are nee ded to see this pic ture. QuickTime™ and a TI FF (Uncompressed) decompressor are needed to see thi s pi ct ure. Q u i k Ti m e ™ a n d a TI F (U n c o m p re s s e d ) d e c o m p re s s o r a re n e e d e d to s e e th i s p i c tu re . c F Q u i k Ti m e ™ a n d a TI F (U n c o m p re s s e d ) d e c o m p re s s o r a re n e e d e d to s e e th i s p i c tu re . c F What happens when a virus infects a host? Innate Crosstalk Immediate Adaptive Delayed (1-2 weeks) The innate immune responses are •present from birth •non-specific •do not become more efficient over time (but there is evolutionary selection) Outcome of Infection • Infection wins • Infection loses death live (innate plus adaptive) Studied intensely Best outcome is • Infections killed quickly (innate only) Not studied How is it that most people are not perpetually sick? (Innate immunity squelching most infections) Process of infection Initial 0-4 hrs Innate 4-96 hrs Cytokines x Clonal expansion T-cells B-cells Adaptive >96 hrs x NK Cell Memory Do we need an innate response? • Yes - major role is limiting initial infection to a manageable level • Adaptive not quick enough to protect – Young unimmunized children (no memory) – Viruses that replicate quickly -Ebola • Becoming clear innate response required for proper development of adaptive response Mice with No Innate response to Virus KO Heterozygote Normal Transgenic mice missing the gene required for innate response infected with SFV What is Innate Immunity? • Initial recognition of infection (Toll-like receptors, dsRNA-based defenses • Response - Cytokine-mediated responses (Interferon, TNF, IL-1, etc) • cell-mediated (NK cells) • Mechanical - mucus etc Why bother studying this? Viral drugs - how many? Could be the best way to deal with infection New viruses Recognition of viral infection How does a cell know its infected and what can it do about it? How are pathogens recognized? i.e. what turns on innate responses? Toll-like receptors (TLRs) • pattern recognition receptors • Can identify a foreign invader (virus, bacterial, etc) via a conserved microbial product and initiate the innate response • 9 identified so far Toll-like receptor pairs Cell surfaces a. b. c. d. TLR-1/TLR-2 -bacterial lipopeptides and GPI-anchored proteins in parasites; TLR-2/TL6 - lipoteichoic acid from grampositive cell walls and zymosan from fungi; TLR-4/TLR-4 -LPS from gram-negative cell walls TLR-5* - bacterial flagellin Endosomes a. TLR-3* - dsRNA b. TLR-7* -uracil-rich single-stranded viral RNA (HIV) c. TLR-8* - single-stranded viral RNA d. TLR-9* - unmethylated CpG DNA found in bacterial and viral genomes. *other member of pair is unknown Key: Different combinations of TLRs appear in different cell types and seem to appear as TLR pairs. dsRNA TLR3 IRF7 Interferon Regulatory Factor 3 IFNb Antiviral Response dsRNA appears to do something else RNA-interference • • • • RNA-based gene regulation and defense mechanism used for control of our own genes (miRNA) recognition of viruses (dsRNA) Controls gene expression by – Degrading mRNA complimentary to dsRNA – Inhibit mRNA translation into protein – Turn off gene by heterochromatin remodeling • Many plant viruses encode suppressors of RNAi • Important in antiviral defense? QuickTime™ and a H.263 decompressor are needed to see this picture. http://www.nature.com/focus/rnai/animations/ RNA Interference C 1nM 5nM 10nM 20nM 40nM Lung Metastasis Con E7 Tubulin HPV16E6 Seq. 10 Cancer Cancer + con siRNA Cancer + siRNA How do we recognize dsRNA as viral rather than endogenous? The 3’, 2bp overhang of siRNA/miRNAs are “self” signals April 30, 2006 Advanced online publication Response: Cytokines • Small soluble proteins made by one cell that can effect the behavior of that or other cells • interact with specific receptors and signal transduction pathways to elicit major changes in cell behavior INTERFERON, TNF, IL-1 Important cytokines IFN-a/b TNF-a Infection IL-1 IL-12 Fever, Direct antiviral effects Activates NK cells Fever, shock Fever, IL-6 production Activates NK cells, signals adaptive immune response Interferon “Interferons are protein components of animal cells which are synthesized and excreted under a variety of stimuli and make other cells of the same species incapable of replicating virus”. DeSomer and Cocito 1968 Issac and Linderman’s Discovery (1957) Cells plus heat-inactivated Influenza virus Discard cells and transfer supernatant onto new cells Incubate Overnight Incubate Overnight then add live virus NO INFECTION IFN Protect cells against viral infection IFN-deficient patient. Before IFN therapy What induces IFN? • Something in viruses – Influenza virus - heat and UV treated – DNA viruses inactivated normal DNA viruses need to replicate: RNA viruses do not dsRNA is best activator of IFN genes. IFN is induced by many other substances • viruses DNA (active) and RNA (active and inactive) • rickettsia • bacteria (esp. gram-negative) • live/killed mycoplasma • protozoa • nucleic acids esp. dsRNA Biological Activities of IFN Signal ISG IFN-induced protein Inhibition of viral replication Inhibition of Cell Growth Regulation of Cell differentiation Activation of Immune System What is the basis of species specificity? Add Human IFa Bind? Activity? Mouse Cell +Hu IFNa/bR + Hu IFNgR -IFNa/b share receptor, IFNg has its own, & receptor is species barrier dsRNA TLR3 IRF7 Interferon Regulatory Factor 3 IFNb JAK/STAT Mx Antiviral proteins Virus Death PKR 2-5A Virus sensitivity to IFNs Small RNA viruses - picornaviruses Large RNA viruses - Flu, rotovirus Small DNA viruses - papillomavirus Large DNA viruses - Herpes, poxvirus Clinical Use of IFN • Viral Infections – Hepatitis B and C – HPV warts – RSV • Cancer Other conditions – Hairy cell Leukemia (90% effective) – Follicular lymphoma – cervical (HPV) – basal cell cancer (8090%) – Kaposi’s sarcoma (HHV type 8) - chronic granulomatous disease (IFN-g) - multiple sclerosis Clinical use cont. • A powerful drug – acts like a blunderbuss – Not a magic bullet – Overused - oral verses systemic • us$4 billion in 1999 • Confounded by non-responders – 1/3 respond – 1/3 respond and relapse – 1/3 non-response. Learning Objectives • Understand the parts of the innate immune system against viruses • Mechanism of RNA interference • Specificity of IFN • Viral sensitivity to the IFN systems Next time The viruses fight back!