Quantative Proteomics Flashcards
Adenovirus infection Nucleolus proteome
200 proteins removed or targeted to nucleolus
How does Influenza Virus change ADAR1
Shown that sequesters does to nuclelus ADAR1 as changes adenosine to inosine which is guanasine and causes hypermutation in RNA virus
ORF57 of KSHV
Trafficked to nuclelous
SILAC Performed twice for KSHV
Infected cell showed greater than 400 nuclear protein changes
Then did for ORF57 inducible cell line which showed 50-60 nucleolar proteome changes
Showed Cellular proteins for polyadenylation redistributed into the nucleolus during KSHV lytic replication
So viral RNA can be poly A d in protective environment
Analysis of Dengue Virus Type 2
SILAC MOCK V INFECTED
1700 cellular proteins
769 nuclear proteins
3 proteins
p56 ADP-ribosylation factor ARL6PI. EC12
ADPribosylation factor and EC12 in nuclear faction likely due to increased Peri nuclear membrane associated with viral replication complex
Crimean Congo Haemorrhagic Fever
Needs Level 4 containment
So use Hazara virus instead only level 2
CCHV N protein expressed from plasmid and fused to GFP so could be visualised
1st Experiment :
SILAC Then used to identify cell proteins that bind to GFP-N
2nd experiment:
SILAC cell proteins that bind to GFP alone
Then immunoppt using anti-GFP Ab and Mass Spec to show ratio of protein abundance and 1:1 ratio showed not specific to N
Showed N affects HSP70 which was focused on has highest abundance ratio
Also affected p53, cytoskeleton and GAPDH
Showed N sequesters HSP70
Which western blot confirmed
HSP70 is inhibitor
Coronavirus Quant proteomics
Causes GI infection
SILAC
Infected Vero with IBV vs MOCK infected vs Uninfected
Showed to affect NF-kb and AP-1 signalling
And
MAPK AND ERK
IF with luciferase reporter fusion using promoter under control of AP-1 / NF-on
Showed major changes in cell morphology and cytoskeleton proteins
Vine run increased in cytoplasm likely due to role in assembly
Little effect on nucleolus
