Host Response to HIV-1 Infection:Host Response to HIV-1 Infection:Quantitative ProteomicsQuantitative Proteomics
& Allied Approaches& Allied Approaches
Eric Chan
Overview of Projects
I. HIV-1 infection of primary CD4 cells• Quantitative proteomic analysis of
HIV-1LAI infection of CD4 cells• Quantitative lipidomic analysis of
HIV-1LAI infection of CD4 cellsII. Influenza A infection of human cells1. Quantitative proteomic analysis of primary human
macrophages ± infection2. Quantitative proteomic analysis of A549 cells ±
infectionIII. Miscellaneous1. Macaque tissue sample collection2. Human-viral protein interactions3. Effects of opioids on sAIDS progression (P20/P01)
Dr. Yu Li
Univ. HK
Overview of Projects
I. HIV-1 infection of primary CD4 cells• Quantitative proteomic analysis of
HIV-1LAI infection of CD4 cells• Quantitative lipidomic analysis of
HIV-1LAI infection of CD4 cellsII. Influenza A infection of human cells1. Quantitative proteomic analysis of primary human
macrophages ± infection2. Quantitative proteomic analysis of A549 cells ±
infectionIII. Miscellaneous1. Macaque tissue sample collection2. Human-viral protein interactions3. Effects of opioids on sAIDS progression
Dr. Yu Li
Univ. HK
Design: Total protein quantification fromDesign: Total protein quantification fromHIV-1-infected primary CD4 cellsHIV-1-infected primary CD4 cells
5xCD4 cells
HIV-1LAI
2 TCID50/cell
Primary CD4 cell isolation
HIV- vs. Mock-infection(Time-course)
Mock
Protein extraction
Peptide digestion
Liquid chromatography-Mass spectrometry(13g per analysis)
Trypsin digestion
Global quantificationof >74,000 peptides
24h p.i.(peak gag p24)
8h p.i.
Quantitative proteomic analysis ofHIV-1LAI infection of CD4 cells
Goals
• Identify significant (p<0.05) abundance changes of individual proteins bracketing the period of robust HIV-1 replication
• Make inferences about cellular functions impacted by protein abundance changes
• Demonstrate functional consequence of abundance changes on HIV-1 replication
2,618 peptides (3.5% of total quantified) changed 2,618 peptides (3.5% of total quantified) changed in abundance following HIV-1-infectionin abundance following HIV-1-infection
ANOVA p*<0.05 (FDR-adjusted);invariant among mock-infections
8h
HIV
24h
HIV
24h
Moc
k8h
Moc
k
Re
plic
ate
Exp
erim
en
ts
(Z score-transformed abundance) Low High
2,618 peptides (3.5% of 74,314 peptides quantified)
Three patterns of expressionThree patterns of expression8h
H
IV24
h H
IV
(Z score-transformed abundance) Low High
Low/Unchanged
24h
Moc
k8h
Moc
kProgressiveDecrease
Kinetic increase8h
High
Unchanged
Kinetic increase24h
Low/Unchanged
HighVery Low
2,613 peptides >>750 “biomarker” proteins
Pattern # Proteins Viral life cycle
1. Kinetic ↑, 8h 97 Integration
2. Kinetic ↑, 24h 390 Full replication
3. Progressive ↓ 263 Full replication
Protein expression pattern resembled 8h p.i. in Protein expression pattern resembled 8h p.i. in the presence of RT inhibitor (Efavirenz)the presence of RT inhibitor (Efavirenz)
8h
HIV
24h
HIV
24h
Moc
k8h
Moc
k
(Z score-transformed abundance) Low High
8h
HIV
24h
H
IV
Down-regulation of select Down-regulation of select karyopherinskaryopherins((importinsimportins and exportins) also evident at 24h p.i. (but and exportins) also evident at 24h p.i. (but
not at 8h p.i.)not at 8h p.i.)
24h p.i.
UpNo change
DownKaryopherins
Prediction: Prediction: DecreasedDecreased importin importin αα44 abundance abundance would would reducereduce nuclear localization of its cargoes nuclear localization of its cargoes
CASP2(Cargo #1)
SGK1(Cargo #2)
Oct-1(Nuclear Marker;Loading Control)
Mock HIV
Western blots:105 primary CD4 cells per lane, nuclear fraction
KPNA4(Carrier)
Nuclear abundance of KPNA4 cargoes Nuclear abundance of KPNA4 cargoes reducedreduced at 24h p.i.at 24h p.i. in the presence of stimulus in the presence of stimulus
CASP2(Cargo #1)
SGK1(Cargo #2)
Oct-1(Nuclear Marker;Loading Control)
Mock HIV
Western blots:105 primary CD4 cells per lane, nuclear fraction
KPNA4(Carrier)
Peptide concentrations as proxy for protein abundance measurements
Inte
nsity
(re
lativ
e to
mos
t ab
unda
nt p
eptid
e)
Mass
Peaks = peptides
Intensity >> peptide abundance??? >> peptide identity
Inte
nsity
(re
lativ
e to
mos
t ab
unda
nt p
eptid
e)
Mass
Peaks = peptides
Identification of peptides(& proteins)
• Online LC-MS peptide identification– Data-dependent LC-MS/MS
• Offline/de-coupled LC-MS peptide identification– AMT tag approach
• VIPER Elucidator
– Targeted LC-MS/MS• Target “biomarker” peptide peaks for LC-MS/MS
analysis
Identification of peptides(& proteins)
• Online LC-MS peptide identification– Data-dependent LC-MS/MS
• Offline/de-coupled LC-MS peptide identification– AMT tag approach
• VIPER Elucidator
– Targeted LC-MS/MS• Target “biomarker” peptide peaks for LC-MS/MS
analysis
Step 1: Label-free LC-MS data
Step 2: Convert to VIPER-readable format
Step 3: Import VIPER outputs back into Elucidator
Overview of Projects
I. HIV-1 infection of primary CD4 cells• Quantitative proteomic analysis of
HIV-1LAI infection of CD4 cells• Quantitative lipidomic analysis of
HIV-1LAI infection of CD4 cellsII. Influenza A infection of human cells1. Quantitative proteomic analysis of primary human
macrophages ± infection2. Quantitative proteomic analysis of A549 cells ±
infectionIII. Miscellaneous1. Macaque tissue sample collection2. Human-viral protein interactions3. Effects of opioids on sAIDS progression (P20/P01)
Dr. Yu Li
Univ. HK
Label-free quantification of lipid abundance: HIV vs. Mock
ABI Q-STAR
Overview of Projects
I. HIV-1 infection of primary CD4 cells• Quantitative proteomic analysis of
HIV-1LAI infection of CD4 cells• Quantitative lipidomic analysis of
HIV-1LAI infection of CD4 cellsII. Influenza A infection of human cells1. Quantitative proteomic analysis of primary human
macrophages ± infection2. Quantitative proteomic analysis of A549 cells ±
infectionIII. Miscellaneous1. Macaque tissue sample collection2. Human-viral protein interactions3. Effects of opioids on sAIDS progression (P20/P01)
Dr. Yu Li
Univ. HK
Samples
• Donors:#1 and #2
• Fractions:Cytoplasmic and Nuclear
• Protocol:i) Peiris - Originalii) Katze – Modified
Peptide abundance from mock-infected primary human lung macrophages
Blue:
Below average
Magenta:
Above average
Next steps:
• $$$
• Use Peiris fractionation protocol
• Waiting for USDA permit
• Infections– Mock– H1N1 (low-path)– H5N1 (high-path)
• Compare with accompanying (Affymetrix) mRNA profiling expression analysis
Overview of Projects
I. HIV-1 infection of primary CD4 cells• Quantitative proteomic analysis of
HIV-1LAI infection of CD4 cells• Quantitative lipidomic analysis of
HIV-1LAI infection of CD4 cellsII. Influenza A infection of human cells1. Quantitative proteomic analysis of primary human
macrophages ± infection2. Quantitative proteomic analysis of A549 cells ±
infectionIII. Miscellaneous1. Macaque tissue sample collection2. Human-viral protein interactions3. Effects of opioids on sAIDS progression (P20/P01)
Dr. Yu Li
Univ. HK
Label-free LC-MS peptide abundance
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