1. Host Response to HIV-1 Infection: Quantitative Proteomics & Allied Approaches Eric Chan

2. Overview of Projects I. HIV-1 infection of primary CD4 cells Quantitative proteomic analysis of HIV-1 LAI infection of CD4 cells Quantitative lipidomic analysis of HIV-1 LAI infection of CD4 cells II. Influenza A infection of human cells 1.Quantitative proteomic analysis of primary human macrophages ± infection 2.Quantitative proteomic analysis of A549 cells ± infection III. Miscellaneous 1.Macaque tissue sample collection 2.Human-viral protein interactions 3.Effects of opioids on sAIDS progression (P20/P01) Dr. Yu Li Univ. HK

3. Overview of Projects I. HIV-1 infection of primary CD4 cells Quantitative proteomic analysis of HIV-1 LAI infection of CD4 cells Quantitative lipidomic analysis of HIV-1 LAI infection of CD4 cells II. Influenza A infection of human cells 1.Quantitative proteomic analysis of primary human macrophages ± infection 2.Quantitative proteomic analysis of A549 cells ± infection III. Miscellaneous 1.Macaque tissue sample collection 2.Human-viral protein interactions 3.Effects of opioids on sAIDS progression Dr. Yu Li Univ. HK

4. Design: Total protein quantification from HIV-1-infected primary CD4 cells 5x CD4 cells HIV-1 LAI 2 TCID50 /cell Primary CD4 cell isolation HIV- vs. Mock-infection (Time-course) Mock Protein extraction Peptide digestion Liquid chromatography- Mass spectrometry (13  g per analysis) Trypsin digestion Global quantification of >74,000 peptides 24h p.i. (peak gag p24) 8h p.i.

5. Quantitative proteomic analysis of HIV-1 LAI 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

6. 2,618 peptides (3.5% of total quantified) changed in abundance following HIV-1-infection ANOVA p*<0.05 (FDR-adjusted); invariant among mock-infections 8h HIV 24h HIV 24h Mock 8h Mock Replicate Experiments (Z score-transformed abundance) Low High 2,618 peptides (3.5% of 74,314 peptides quantified)

7. Three patterns of expression 8h HIV 24h HIV (Z score-transformed abundance) Low High Low/Unchanged 24h Mock 8h Mock Progressive Decrease Kinetic increase 8h High Unchanged Kinetic increase 24h Low/Unchanged HighVery Low

8. 2,613 peptides >> 750 “biomarker” proteins Pattern# ProteinsViral life cycle 1. Kinetic ↑, 8h97Integration 2. Kinetic ↑, 24h390Full replication 3. Progressive ↓263Full replication

9. Protein-interaction network (Ingenuity PA) 8h p.i. Up No change Down

10. 24h p.i. Up No change Down Protein-interaction network (Ingenuity PA)

11. Functional interpretation: HIV integration coincided with the promotion of Iκβ-degradation and alterations in nuclear envelope structures 8h p.i. Up No change Down Iκβ degradation Nuclear pore complex

12. Protein expression pattern resembled 8h p.i. in the presence of RT inhibitor (Efavirenz) 8h HIV 24h HIV 24h Mock 8h Mock (Z score-transformed abundance) Low High 8h HIV 24h HIV

13. Down-regulation of select karyopherins (importins and exportins) also evident at 24h p.i. (but not at 8h p.i.) 24h p.i. Up No change Down Karyopherins

14. Prediction: Decreased importin α4 abundance would reduce nuclear localization of its cargoes CASP2 (Cargo #1) SGK1 (Cargo #2) Oct-1 (Nuclear Marker; Loading Control) Mock HIV Western blots: 10 5 primary CD4 cells per lane, nuclear fraction KPNA4 (Carrier)

15. Nuclear abundance of KPNA4 cargoes reduced at 24h p.i. in the presence of stimulus CASP2 (Cargo #1) SGK1 (Cargo #2) Oct-1 (Nuclear Marker; Loading Control) Mock HIV Western blots: 10 5 primary CD4 cells per lane, nuclear fraction KPNA4 (Carrier)

16. Peptide concentrations as proxy for protein abundance measurements Intensity (relative to most abundant peptide) Mass Peaks = peptides

17. Intensity >> peptide abundance ??? >> peptide identity Intensity (relative to most abundant peptide) Mass Peaks = peptides

18. 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

19. 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

21. Step 1: Label-free LC-MS data

22. Step 2: Convert to VIPER-readable format

23. Step 3: Import VIPER outputs back into Elucidator

26. Overview of Projects I. HIV-1 infection of primary CD4 cells Quantitative proteomic analysis of HIV-1 LAI infection of CD4 cells Quantitative lipidomic analysis of HIV-1 LAI infection of CD4 cells II. Influenza A infection of human cells 1.Quantitative proteomic analysis of primary human macrophages ± infection 2.Quantitative proteomic analysis of A549 cells ± infection III. Miscellaneous 1.Macaque tissue sample collection 2.Human-viral protein interactions 3.Effects of opioids on sAIDS progression (P20/P01) Dr. Yu Li Univ. HK

27. Label-free quantification of lipid abundance: HIV vs. Mock ABI Q-STAR

28. Overview of Projects I. HIV-1 infection of primary CD4 cells Quantitative proteomic analysis of HIV-1 LAI infection of CD4 cells Quantitative lipidomic analysis of HIV-1 LAI infection of CD4 cells II. Influenza A infection of human cells 1.Quantitative proteomic analysis of primary human macrophages ± infection 2.Quantitative proteomic analysis of A549 cells ± infection III. Miscellaneous 1.Macaque tissue sample collection 2.Human-viral protein interactions 3.Effects of opioids on sAIDS progression (P20/P01) Dr. Yu Li Univ. HK

29. Samples Donors: #1 and #2 Fractions: Cytoplasmic and Nuclear Protocol: i) Peiris - Original ii) Katze – Modified

30. Peptide abundance from mock-infected primary human lung macrophages Blue: Below average Magenta: Above average

31. 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

32. Overview of Projects I. HIV-1 infection of primary CD4 cells Quantitative proteomic analysis of HIV-1 LAI infection of CD4 cells Quantitative lipidomic analysis of HIV-1 LAI infection of CD4 cells II. Influenza A infection of human cells 1.Quantitative proteomic analysis of primary human macrophages ± infection 2.Quantitative proteomic analysis of A549 cells ± infection III. Miscellaneous 1.Macaque tissue sample collection 2.Human-viral protein interactions 3.Effects of opioids on sAIDS progression (P20/P01) Dr. Yu Li Univ. HK

33. Label-free LC-MS peptide abundance