Virus+Host cell

Question 1

cell intrinsic antiviral pathways

Answer 1

not multicellular immune responses

-CRISPR
-RNAi
-Type I IFN

Question 2

Type I IFN response basics

Answer 2

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)

Question 3

viral sensing

Answer 3

PRRs in cells
recognise PAMPs

how innate response detects pathogens

Question 4

viral PAMPs

Answer 4

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)

Question 5

cytoplasmic dsRNA sensors

Answer 5

MDA5 receptor
RIG-I receptor

signalg transduced by MAVS

activate TFs that drive expression of IRF3/7, NFKB (type I IFNs - pro-inflammatory cytokines)

Question 6

cytoplasmic dsDNA sensor

Answer 6

cGAS receptor

produces cGAMP metabolite signal
signal transduced by STING

activates same TFs as dsRNA receptors (ones that drive IRF3/7 and NFKB)

Question 7

MDA5 dsRNA sensing

Answer 7

recognises >1kb stretches of dsRNA

Question 8

RIG-I dsRNA sensing

Answer 8

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

Question 9

endosomal viral nucleic acid sensing

Answer 9

Toll like receptors
TLRs

membrane bound
in endosomes

typically more expressed in immune cells but also present in all cell types

Question 10

properties of common lab cell lines

Answer 10

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

Question 11

antiviral state

Answer 11

activation triggered by IFNs
secreted IFNs activate signalling cascade in infected cell and cells surrounding it

Question 12

antiviral state signalling pathway

Answer 12

IFNs bind an IFN TM receptor

activates JAK/STAT signalling
activates transcription of ~500 antiviral genes (ISGs)
creates non-permissive state for viral replication

Question 13

ISG properties

Answer 13

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

Question 14

different responses to viral nucleic acids

Answer 14

IFN and ISG production

mRNA translational shutdown
RNA degradation
(these two are part of ISG pathways, BUT are also activated directly by dsRNA)

Question 15

mRNA translational shutdown in direct response to dsRNA

Answer 15

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

Question 16

PKR translational shutdown in response to dsRNA

Answer 16

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

Question 17

widespread RNA degradation in presence of dsRNA

Answer 17

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

Question 18

IFN repsonse downside

Answer 18

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

Question 19

mis-recognition of TEs as foreign

Answer 19

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

Question 20

Viral evasion of IFN response basic

Answer 20

all componenrs of dsRNA sensinf pathway can be inhibited by viral proteins

Question 21

influenza IFN response evasion

Answer 21

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

Question 22

mumps IFN response evasion

Answer 22

mumps V protein blocks MDA5 from signalling

Question 23

ebola evasion of IFN response

Answer 23

Ebola VP35 blocks the IRF3/7 and NFkB TFs

Question 24

Zika evasion of IFN response

Answer 24

inhibits thew JAK kinases in the JAK/STAT signal transduction from TM IFN receptors

Question 25

herpesvirus evasion of IFN response

Answer 25

creates pseudosubstrate for PKR

PKR phosphorylates that instead of elF2-alpha

elF2-alpha can remain unphosphorylated/uninhibited and keep recruiting ribosomes to mRNA

Question 26

SARS-CoV-2 IFN evasion

Answer 26

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

Question 27

impaired IFN response and SARS-CoV-2 pathology

Answer 27

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

Question 28

IFN discovery

Answer 28

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

Question 29

viral-host evolution

Answer 29

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