Virus+Host cell Flashcards
cell intrinsic antiviral pathways
not multicellular immune responses
-CRISPR
-RNAi
-Type I IFN
Type I IFN response basics
Cytokines produced by any cell type infected by virus
starts orchestrating immune response
first layer of defence secreted by infected cell
part of innate response
mobilises adaptive immune cells/Ab production for longer immunity
IFNs
can be used as treatment for viral infections (eg Hep B/C)
though many 2’ effects (fever, muscle aches, chills, fatigue)
viral sensing
PRRs in cells
recognise PAMPs
how innate response detects pathogens
viral PAMPs
their nucleic acids
mammalian DNA only in nucleus and Mt
DNA in cytoplasm = assumed to be presence of pathogen
RNA more complicated
mammalian mRNA in cytoplasm
so cell detects different RNA structures not normally present
eg dsRNA (viruses need to make this to copy genome)
cytoplasmic dsRNA sensors
MDA5 receptor
RIG-I receptor
signalg transduced by MAVS
activate TFs that drive expression of IRF3/7, NFKB (type I IFNs - pro-inflammatory cytokines)
cytoplasmic dsDNA sensor
cGAS receptor
produces cGAMP metabolite signal
signal transduced by STING
activates same TFs as dsRNA receptors (ones that drive IRF3/7 and NFKB)
MDA5 dsRNA sensing
recognises >1kb stretches of dsRNA
RIG-I dsRNA sensing
recognises much shorter fragments
that also lack 5’ m7 guanosine cap typical of mammalian mRNA
viruses replicating in cytoplasm cannot access the nuclear 5’ cap machinery
exploited by RIG-I
endosomal viral nucleic acid sensing
Toll like receptors
TLRs
membrane bound
in endosomes
typically more expressed in immune cells but also present in all cell types
properties of common lab cell lines
many cell lines are tumour derived
so most have lost ability to produce interferons
this helps in lab context when adding nucleic acids to them (eg for transformations)
eg HeLa cells have dsRNA sensing and can make IFNs when challenged
but not for cytoplasmic DNA
HEK-293T cells cannot sense either
need to consider this when working with cells
IFN response can affect results
antiviral state
activation triggered by IFNs
secreted IFNs activate signalling cascade in infected cell and cells surrounding it
antiviral state signalling pathway
IFNs bind an IFN TM receptor
activates JAK/STAT signalling
activates transcription of ~500 antiviral genes (ISGs)
creates non-permissive state for viral replication
ISG properties
either directly or indirectly interfere w viral replication:
direct:
>RNA degradation and mRNA translation shutdown - prevents production of new viral genomes/proteins
>inhibition of viral particle formation
indirect:
via amplifying the antiviral response:
>increase PRR expression-more of them to sense viruses
>more signalling molecules
>more IFN production - positive feedback as this will lead to more PRRs
>also triggers adaptive immunity
different responses to viral nucleic acids
IFN and ISG production
mRNA translational shutdown
RNA degradation
(these two are part of ISG pathways, BUT are also activated directly by dsRNA)
mRNA translational shutdown in direct response to dsRNA
intiation factors bind 5’ cap of mRNA to recruit ribosome loading to mRNA
40s then 60s subunits of ribosome load onto mRNA
get polysomes
this is interrupted in presence of dsRNA
prevents translation of all mRNA
done by PKR
PKR translational shutdown in response to dsRNA
PKR binds dsRNA
autophosphorylates
then phosphorylates elF2-alpha
this prevents loading of ribosomes onto mRNA
can see this when:
looking at sucrose density gradients of cells’ cytoplasm (cytosplasmic nucleic acids marks ribosomes [made of rRNA-95% of RNA in cell])
when plot presence of RNA in diff fractions
see massive decrease of RNA in denser fractions in presence of dsRNA => less polysomes (more ribosomes on one mRNA=denser)
instead see accumulation of single subunits of ribosomes
widespread RNA degradation in presence of dsRNA
OAS binds dsTNA
this causes it to produce 2’-5’ oligo-adenylates
these bind RNaseL
which now degrades all RNA in cell - viral and host
cen see with northern blot from cells
in presence of dsRNA
see rRNA gets degraded
IFN repsonse downside
too much IFNs = not good
activation of IFN response in absence of infection
=>autoimmune disease (systemic lupus, Type I interferonopathies)
from mis-recognition of endogenous nucleic acids by sensing machinery
mis-recognition of TEs as foreign
make up almost 1/2 genome
DNA transposons small proportion of this
mostly due to RNA intermediate retrotransposons
each cell has millions of copies of TEs
can be incorporated in sense and antisense directions
a sense and antisense RNA intermediate can bind each other in cytoplasm
or mRNA intermediate to its cDNA made from itself
this forms dsRNA that can be aberrantly recognised by viral dsRNA sensors
Viral evasion of IFN response basic
all componenrs of dsRNA sensinf pathway can be inhibited by viral proteins
influenza IFN response evasion
influenza MS1 protein coats the RNA making it unrecognisable (to MDA5/RIG-I)
dampens ISG activation
if remove this protein from influenza
can get full ISG activation in cells
mumps IFN response evasion
mumps V protein blocks MDA5 from signalling
ebola evasion of IFN response
Ebola VP35 blocks the IRF3/7 and NFkB TFs
Zika evasion of IFN response
inhibits thew JAK kinases in the JAK/STAT signal transduction from TM IFN receptors
herpesvirus evasion of IFN response
creates pseudosubstrate for PKR
PKR phosphorylates that instead of elF2-alpha
elF2-alpha can remain unphosphorylated/uninhibited and keep recruiting ribosomes to mRNA
SARS-CoV-2 IFN evasion
can manipulate many steps
good at dampening IFN response
stimulate infected cell to IFN stimulated non-infected cell
compare which ISGs are active
non-infected - all ISGs active
SARS-CoV-2 infected - much lower
impaired IFN response and SARS-CoV-2 pathology
individuals that can raise a good IFN response in early infection clear it quicker
if IFN response is delayed until later
end up w more inflammatory long lasting response
could use IFNs as possible treatment
BUT would need to give them to patients BEFORE they were acc infected
many individuals that did badly w SARS-CoV-2 had a lot of auto-Ab to IFNs
dampening their response
IFN discovery
chicken cells
infect w influenza
centrifuge to sediment cells
take supernatant
add to media of uninfected cells
these uninfected cells were now protected against infection
viral-host evolution
hosts have proteins/sensors that target viral traits
causes pressure for evolution of viral proteins to evade them
so pressure on antiviral proteins causing them to mutate and evolve at higher rate than housekeeping genes
