Everything you wanted to know about viral immunology' No Holds Barred

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Everything you wanted to know about viral
immunology.No Holds Barred
Or..
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EB meets CB
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Up to date chapter on viral immunology

• Yewdell, Pierson, Bennink
• email me jyewdell _at_nih.gov

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Must read

• Evolution and Emergence
• Principles of Viral Pathogenesis

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What is a virus?

• My definition obligate intracellular parasite
  that uses host ribosomes to produce all of its
  proteins

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Leading Virus Associated Diseases  
1 Disability adjusted life years - disability
years lost in 1998 due to the disease 2 Includes
non-viral infectious disease mortality 3 Chronic
Hepatitis B contributes to these deaths from
liver cirrhosis and cancer
 
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Types of Viruses

• Can group by nucleic acid
• Or by structure
• This makes more sense for immunologists

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Enveloped vs Non-enveloped

• Envelope is derived from cellular membranes (most
  often the plasma membrane, but also ER and GC,
  and complicated combinations)
• Both have highly repetitive structures on their
  surfaces
• Susceptible to low affinity (high avidity)
  interactions

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Enveloped vs Non-enveloped

• Envelope is derived from cellular membranes (most
  often the plasma membrane, but also ER and GC,
  and complicated combinations)
• Both have highly repetitive structures on their
  surfaces
• Susceptible to low affinity (high avidity)
  interactions

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Common strategies of replication

• Must be transferred from host to host
• Must bind to cells and penetrate
• Cell surface
• Endosomal
• Enveloped viruses fuse
• Non-enveloped viruses poke holes

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Mutability and Plasticity

• Have phenomenally high mutation rates
• No matter how simple the virus, each virion in
  population is unique
• Poses a major challenge for the immune system
• Large DNA viruses have a palate for hijacking
  host genes and modifying their function

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Tropism

• Tropism
• Level of organism
• Many viruses only infect humans (HBV)
• Some infect almost anything (rabies)
• Level of organ
• Often governed by the receptor specificity

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Pathogenicity

• Pathogenicity is usually unrelated (or inversely
  related) to viral transmission
• Selection pressure is against pathogenicity
• Ebola virus
• The most pathogenic viruses are introduced from
  other species
• Ebola
• Herpes B
• Hanta
• Exception smallpox

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Can Directly Disable Immune Cells

• EBV
• HIV
• adenovirus
• HHV6
• HHV7
• HTLV

• B-cells (C3d receptor)
• T-cells (CD4)
• tonsils adenoids
• T-cells
• T-cells
• T-cells

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Viruses are dangerous and ubiquitous foes
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Viruses offer myriad opportunities for
computational biologists
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e.g.

• Infection outcome host polymorphism
• Sequence diversity
• Evolution in individuals and species
• New ORFs
• Other information
• Codon usage
• Host
• Organ

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Viruses are dangerous and ubiquitous foes
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100 MYR Is A Long Time
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Levels of Defense

• Innate versus adaptive
• Humoral versus cellular
• These arent absolute distinctions
• There are two types of scientists those who
  divide everything into two, and those who dont

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Innate Immunity Physical Barriers

• First and most important components of the immune
  system are the skin and mucosa and its secretions

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Innate Immunity Humoral

• Defensins
• Complement
• natural antibodies
• Anti- terminal 1-3a-Gal

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Innate Immunity Cellular

• Cells innately sense viral infections
• DS RNA triggers nucleases esp. RNAase
• TLRs recognize viral nucleic acids
• a- and b-IFNprobably the most important element
  in anti-viral immunity

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IMPORTANT message from your sponsor

• Every cell is part of the immune system!!!

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Innate Immunity Professional Cells

• NK
• Cytokines esp. interferons
• Can be specific for viruses (sort of)
• May demonstrate memory
• DC/Monocytes, granulocytes, mast cells
• NO2 other ROS
• GKW

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This is your skin.

Neutrophils and macrophages Autofluorescence
(hair)
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This is your skin on Vac.
Neutrophils and macrophages VV and
autofluorescence (hair)
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Adaptive Immunity

• Antibodies
• Absolutely key for protective immunity
• Likely mechanism for most effective vaccines
• Mechanisms
• Neutralize infectivityprevent penetration
• Target virions or virus infected cells for
  cellular annihilation
• Lyse membrane virions with complement

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Epitope

• Physical area on antigen that interacts with
  antibody
• Immunogenicity of antibody epitopes is a topic of
  near total ignorance
• Opportunity for CB
• Other CB opportunities
• Complexity of Ab responses
• Specificity of Ab responses
• Genetic difference in Ab responses

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Adaptive Immunity T cells
TCD4
TCD8

• CD4 positive
• Enhance antibody responses and inflammation.
• Antigen recognition associated with class II MHC
  molecules.
• Induced by noninfectious and infectious virus
• Peptides recognized are primarily produced in
  endosomes

• CD8 positive
• Destroy infected cells or impair pathogen
  replication.
• Antigen recognition associated with class I MHC
  molecules.
• Induced by infectious virus.
• Peptides recognized are primarily 8-10 residues
  produced in the cytosol.

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CTL in flagrante delicto
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Fundamental feature of T cell recognition is
MHC-restricted recognition of oligopeptidesthis
greatly simplifies defining epitopes
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Also recall

• Humans have hundreds of alleles at each class I
  and class II locus
• As a species, we can bind a very wide array of
  peptides for T cell recognition
• This may have evolved as a consequence of viral
  mutability and introduction

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Now a commercial break
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TCR
How Are MHC Class I Peptide Ligands Generated
From Biosynthesized Proteins?
8-10 residue peptide
MHC
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Party Line 1986-1996
Peptides derive from the standard degradation of
total pool of biosynthesized proteins
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But..

• Viral proteins are generally very stable
• And in 1992 we reported that VSV makes enough
  nucleocapsid protein to enable T cell recognition
  within 45 min of adding virus to cells

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So.

• How can this small amount of VSV nucleocapsid
  compete with the enormous amount of old
  cellular proteins (billions of copies) that are
  degraded?

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Defective Ribosomal Productsa.k.a.
DRiPs
1
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Simple Idea Protean sin thesis Kant bee prefect
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Murphys Law as Applied to Protein Biogenesis

• Errors in transcription, splicing
• Errors in translation
• Inappropriate initiation
• Premature termination, read-thru
• Alternate reading frames
• Misincorporation of amino acids
• Errors in post-translational modifications,
  protein assembly or folding
• Imbalances in subunits
• Significant fraction of protein folding is
  chaperone dependent

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DRiPs Appear to be the Rule Rather than the
Exception

• One example

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Complex generation from highly stable viral
protein reaches Vmax rapidly following vaccinia
virus infection and ceases within 60 min of
adding proteasome or protein synthesis inhibitors
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CB opportunityModeling biological processes
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Immunoribosome Hypothesis
Peptide-Generating Surveillance Pathway
DRiP
20S
Immunoribosome
TAP-ER
Class I
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Searching for DRiPs
strikes again
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Ideas are good, even bad ones, as long as they
lead to novel approaches

• Could tRNA misacylation be a source of DRiPs?

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It is well established1 that aminoacylation by
aminoacyl synthetases is super accurate one
error per 10,000-40,000 couplings
1Or is it?
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tRNA misacylation

• Of 451 tRNA genes in humans (270 unique species),
  151 are located in the HLA!!!

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HypothesisHLA-encoded tRNAs are dedicated to
making DRiPs by deliberately delivering the
wrong amino acids to immunoribosomes
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The First tRNA Microarray
Tao tRNA Pan University of Chicago
Each array contains18 replicates of 136 tRNA
probes
Dittmar et al. EMBO Reports 6, 151-157 (2005).
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Direct Misacylation Assay

• HeLa cells infected with Flu (4 hours) or
  Adenovirus (14 hours)
• Cells pulse-labeled with 35S-met.
• Charged tRNAs extracted
• Hybridize tRNAs directly on microarrays
• Measure radioactivity at non-cognate tRNA spots

Nir Netzer
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Surprise 111 (not .01) of Met is on the
wrong tRNA
tRNAMet included
tRNAMet eliminated
1I did warn you
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Surprise 2 following virus infection up to 13
of 35S-Met is on non-cognate tRNA!
Flu
Untreated
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Misacylation is highly specific I.
Red 0.4-1 Orange 0.1-0.4Blue lt0.1

• Mitochondrial tRNA are not misacylated
• Met misacylation is specific for subset of
  nuclear encoded tRNA

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Dont see with Cys
Misacylation is highly specific II
Untreated
LPS
poly-IC
Or I, F, V, Y
Val
Tyr
Phe
Ile
Met
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Misacylation occurs in liver too!

• Inject 35S-Met directly into portal vein of live
  mouse
• Harvest liver 60 seconds later isolate tRNA

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CB opportunity

• Differences between in vivo and in vitro biology

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Evidence for 35S-Met mis-incorporation into
proteins
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Who is winning? Cell vs. Virus

• Virus is inducing misacylation?
• Or a cellular response to viral infection?

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Cell is winning

• Misacylation is triggered within an hour of flu
  infection also by UV inactivated virus
• Misacylation is triggered by other TLR ligands
• Poly IC
• LPS (!)

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What does it all mean?
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Getting by with a little help from your friends
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Met Provides Protection from Inflammatory
Oxidative Damage
Akiko Iwasaki
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Which led us to friend 2
Rod Levine
Replace Met with NorLeu in e. Coli broth Cells
are more sensitive to oxidative damage!
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Remarkably, Met-Misacylation is Induced by
Oxidative Stress
Untreated
H2O2
arsenite
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DPI inhibits cellular generation by NADPH oxidase
and other ROS generators
Sebastian Amigorena
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DPI blocks TLR- oxidative stress-induced
misacylation
Untreated
H2O2
arsenite
arsenite DPI
Poly-IC DPI
Poly-IC
AP, Met-oligos
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SerenDRiPity

• Search for DRiPs led to discovery of oxidative
  stress induced Met misacylation
• Links innate immune recognition to ROS generation
  in non-professional APCs
• Most important conclusion
• genetic code is conditional
• Met is encoded by certain codons under stress
  conditions

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Working hypothesis Met replacement facilitates
handling ROS by generating novel translation
products protected from ROS damage or adept at
minimizing its effects
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SerenDRiPity

• But this is, of course, only a hypothesis
• Uncertainty lies at the heart of good science.

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CB lesson

• Dont believe everything you read
• Amended.Dont believe anything your read

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CB Opportunities

• Genetic code
• Genetic code
• Genetic code