Antiviral Responses

• • RIG1/MDA5
  • RLRs (RigI-like receptors)Alternative group of intracellular sensors (receptors) for viruses
  • RIG-I, MDA5 & LGP2=RLRs (common protein domains DEAD box helicase and CARD domain-no CARD in LGP2)
  • RIG-I responsive to dsRNA <300bp
  • MDA5 responsive to dsRNA >300bp
  • RIG-I responsive to 5’ phosphate (uncapped RNA)
  • Activation of the RIG-I and MDA5 sensors leads to activation of NFkB and type 1 IFN signalling.
  • LGP2- may act to amplify RIG-I/MDA5 signal.
• • 2’,5’-oligoadenylate synthase
  • 2’,5’-OAS is activated by dsRNA and synthesizes 2-5A (mainly 5’pppAAA)
  • 2-5A activates RNase L which then cleaves after UU and UA nucleotides in ssRNA
  • RNase L is expressed across most (all) tissues but is not activated without the action of 2’5’-OAS
• • cGAS-STING
  • cGAS= cyclic GMP-AMP synthase (an enzyme).
  • Activated by binding to dsDNA (in the cytoplasm)
  • Catalyses the formation of 2’-3’ Cyclic Guanosine monophosphate-Adenosine monophosphate (cGMP-AMP) from GMP and AMP.
  • STING= protein resident in the endoplasmic reticulum membrane.
  • Stimulator of type I interferon genes.
  • Activated by cGMP-AMP
  • Active STING translocates to the golgi and activates TBK1 (which in turn) phosphorylates the transcription factor IRF3
  • IRF3 translocates to the nucleus (stimulates expression of type I interferon genes).
• • RNAi
  • Sequence specific RNA surveillance mechanism triggered by dsRNA
  • RNase III (Dicer)
  • RNA induced silencing complex (RISC includes AGO2)
  • mRNA
• • Protein Kinase R
  • Ubiquitously expressed protein kinase
  • PKR expression is upregulated by type I interferons
  • PKR is activated by dsRNA and phosphroylates eIF2a which shuts down host cell translation.
• • NFkappaB
  • Transcription factor, ubiquitously expressed.
  • Heterodimer p50/p65 although homodimers also exist.
  • Before activation NFkB is in association with an inhibitor (IkB).
  • >150 different extracellular stimuli can lead to the phosphorylation of IkB (by
  • IKK), causing it do dissociate for NFkB, which then translocates to the nucleus.
  • NFkB transcription factor induces genes involved in cellular stress responses, including apoptosis, although depending on the system NFkB can either be protective or necessary with respect to apoptosis.
• • Type 1 IFN
  • Type I interferons are cytokines involved in antiviral responses to virus infections
  • Several subtypes, of which IFNa and IFNb have been the most extensively studied
  • Signal via the IFNAR triggers activation of a signalling pathway that results in expression of a whole range of interferon induced genes (>300)
• IFN-1

  • Many of the genes activated by type I IFN increase sensitivity of antiviral sensors, a minority are involved directly in inducing an antiviral state.
  • Types of genes activated by IFN include sensors (RLRs, 2’5’-OAS, PKR) signalling pathway modifiers (ISG15), or intracellular vesicle budding (Mx) .
• • Interferon inducible transmembrane proteins 
  • Induced by type I interferons in vertebrates
  • IFITM study re-initiated with screens for genes affecting replication of influenza A.
  • There is some evidence that IFITM3 alleles may be linked to disease severity for influenza A in humans.
  • Effect seems to be very broad IFITM proteins seem to affect entry for a whole range of different RNA viruses (not all).
  • Mechanism of action not completely clear but may be linked to changes in the fluidity of endosomal membranes linked to changes in cholesterol content.
• • Double stranded RNA and uncapped RNA are major triggers of antiviral responses in eukaryotic cells (not limited to cells in the immune system).
  • Normal eukaryotic gene expression 
  • Key features 
  • DNA in Nucleus
  • Transcribed by RNA polymerase II to make pre-mRNA
  • RNA processed in nucleus (removal of introns, addition of methylated cap and polyA tail)
  • mRNA exported to the cytoplasm
  • Translation
• • Dengue replication cycle Baltimore group 4
  • No Nuclear phase to infection cycle
  • No opportunity to interact with cellular mRNA capping machinery
  • Antiviral responses summary 
  • All nucleated cells have a series of pathways that recognise and control viruses
  • Since viruses hijack the host cell translation machinery viral proteins are indistinguishable from host proteins
  • Key triggers for innate antiviral response pathways are the presence of dsRNA, uncapped RNA and DNA in the cytoplasm.
• • Mechanisms that different viruses have to solve the capping problem, 
  • Cap snatching 
  • Virus encodes a nuclease that cleaves downstream of the cap in the cellular messenger RNA, the 5’ cap is then used to prime the synthesis of viral mRNA.
  • This mechanism can occur in the nucleus (influenza A, Baltimore V), or in the cytoplasm (bunyaviruses, baltimore V)
• Solving capping problem

  • VpG
  •  The 5’ end of the messenger sense RNA of some positive sense RNA viruses (Baltimore group IV, e.g. polio, caliciviruses) is covalently attached to a viral protein.
  • These viruses often have RNA secondary structures that allow the ribosome to bypass the cap for the translation of the viral RNA.
• Solving capping problem

  • Encode a capping enzyme 
  •  Some viruses that replicate in the cytoplasm encode an enzyme (or enzymes) that cap their RNA (e.g. Bluetongue virus, Baltimore III; Dengue virus, Baltimore IV; Vaccinia virus, Baltimore I)
• capping problem solutions

  • Use the cellular transcription machinery
  • Some viruses that have DNA genomes or which have a DNA phase to their replication cycle express their genes using cellular RNA polymerase II (transcripts are produced and processed as if they were made from a cellular gene).
  • Examples most baltimore class I (e.g. Herpesvirus), Class II (e.g. circoviruses, parvoviruses), Class VI (retroviruses e.g. HIV) and class VII (pararetroviruses, e.g. hepatitis B virus)
• • Capping problem solutions
  • Stabilise the RNA by binding cellular microRNA 
  • Hepatitis C virus (Baltimore class IV) is restricted to growth in cells expressing liver specific microRNA miR-122. Current evidence is that the binding of the miRNA to the viral genome prevents degradation of the RNA and enhances translation of the virus polyprotein.
  • HCV lacks a cap on it’s genomic RNA so one hypothesis is that recruitment of the miRISC complex to the 5’ end of the RNA prevents recognition of the RNA by cellular nucleases.
• • Double stranded RNA and uncapped RNA are major triggers of antiviral responses in eukaryotic cells (not limited to cells in the immune system).
• • Mechanism that viruses use to counteract DsRNA based cellular surveillance systems 
  • Sequester dsRNA- virus replicates within membrane bound vesicles called ‘spherules’ inside the cell (effectively shielding virus dsRNA replication intermediates from the cellular surveillance machinery)
  • Example- Coronaviruses (most positive sense RNA viruses?)
  • Or virus produces a dsRNA binding protein that shields virus dsRNA from the cellular surveillance machinery.
  • Examples- filovirus VP35 or 1A protein from Drosophila C virus
• counteracting DsRNA surveillance

  •  Limit the length of detectable dsRNA
  • Example- most Baltimore group V viruses (negative sense ssRNA)
  • Interfere with effector proteins.
  • Polio (Baltimore IV) produces an RNA that binds to and inhibits RNaseL
• counteract surveillance

  • Interfere with ‘pinch points’ cellular signalling pathways.
  • Example- Influenza NS1
  • MDA5- Polio infections triggers degradation of MDA5 by cellular proteases
  • IRF3- Ebola VP35 prevents phosphorylation of IRF-3 by interacting with upstream kinases IKK-e and TBK-1
  • STAT1- Dengue and west Nile NS4B prevent phosphorylation of STAT1, and Ebola VP24 prevents nuclear import of phosphorylated STAT1 (nuclear STAT1 is a transcription factor that activates expression of type I interferons)
• • DNA viruses 
  • Some evidence for dsRNA from bidirectional overlapping transcription
• • DNA sensors seem to be more cell type specific an often target DNA in the ‘wrong’ place 
  • TLR9- (mainly B cells and dendritic cells) detects DNA in endosomes ( evidence it detect cytoplasmic DNA)- Signals through NFkB and results in type I interferon expression
• DNA sensors

  • cGAS- detects DNA in cytoplasm makes cyclic-di-GMP and cyclic-di-AMP that activates STING which is localised to the outer membrane of mitochondria and activates IFN1 signalling (interferon production).
  • IFI16 – has a NLS but can be detected in the nucleus and the cytoplasm in some cell types. Recruits STING following DNA binding to activate type I interferon production. Not entirely clear how IFI16 in the nucleus distinguishes between viral and cellular DNA
• • DNA viruses also encode proteins that interfere with key cellular pathways that detect viruses 
  • Karposi’s sarcoma associated herpesvirus (Baltimore 1) encodes a transcription factor that can dimerise with IRF7 preventing activation of genes downstream of IRF7
  • KHSV also encodes a protein (K13) that activates NFkappaB (promoting cell survivial)
  • Vaccinia virus ( Baltimore I) produces multiple proteins that interfere with IRF3 based signalling due to detection of cytoplasmic dsRNA and DNA.
• DNA virus proteins that interfere with key cell pathways

  • HSV ( Baltimore I) produces multiple proteins that interfere with cytoplasmic DNA sensors, ie. ICP0 promotes degradation of IFI16 and VP22 prevents activation of cGAS by cytoplasmic dsDNA.
• • Summary 
  • Antiviral immunity includes intracellular responses in every cell (first line of defence?)
  • Knock-out of cellular antiviral detection mechanisms leads to increased virus replication.
  • Viruses encode proteins and RNAs that disrupt cellular antiviral surveillance and defences.