Dynamic Analysis of HIV-Human Protein-Protein Interactions During Infection Jeffrey Johnson, 1 Shannon Eliuk, 2 Amnon Golan, 1 Tasha Johnson, 1 Vlad Zabrouskov, 2 Nevan Krogan 1 1 UCSF, San Francisco, CA; 2 Thermo Fisher Scientific, San Jose, CA
UCSF, San Francisco, CA; Thermo Fisher Abstract The identification of virus-host protein-protein interactions has emerged as a powerful tool to characterize the mechanisms by which viruses evade and usurp host cellular pathways. We previously described a highly successful analysis of HIV-human protein-protein interactions through affinity purification and mass spectrometry (AP-MS) analyses of cell lines expressing affinitytagged HIV proteins. Here, we demonstrate the power of applying deep coverage, quantitative analysis of HIV-human protein-protein interactions over a time course of infection using a novel hybrid instrument, Thermo ScientificTM Orbitrap FusionTM mass spectrometer, based on mass resolving quadrupole, Orbitrap, collision cell, linear ion trap (Q-OT-qIT) architecture. Clustering time-dependent quantitative interaction profiles rapidly separated specific and non-specific interactions and identified novel protein-protein interactions that were not identified in previous studies. Background Strategy HIV VIF Jurkats transfe tagged Infection 2h Time po 8h 16h Reverse transcription (6- Host genome integra Virus rel Jurkat cells stably tra proteins were infecte corresponding HIV pr time points through th affinity purification ma right). This analysis w and VPR. Design of the Orbitra AP-MS samples were 1) a new Orbitrap architecture, and 2) an We previously reported on a comprehensive analysis of HIVhuman protein-protein interactions, derived from affinity purification of individually expressed, tagged HIV proteins from human cell lines and LC-MS/MS analysis of eluates. 2 Dynamic Analysis of HIV-Human Protein-Protein Interactions During Infection Raw data were ana algorithm and the Ma from the two instrume quantification for down
Strategy HIV VIF Infection Jurkats stably transfected with tagged VIF Time points analyzed 2h 8h 16h 24h Reverse transcription (6-12h) Host genome integration (8-2h) Virus release (2-3h) Jurkat cells stably transfected with 2xStrep/3xFLAG-tagged HIV proteins were infected with HIV deletion strains lacking the corresponding HIV protein. Infected cells were harvested at 4 time points through the HIV replication life cycle and subjected to affinity purification mass spectrometry (AP-MS) analysis (above, right). This analysis was performed for 3 HIV proteins: VIF, VPU, and VPR. Design of the Orbitrap Fusion MS AP-MS samples were analyzed on two types of instrumentation: 1) a new Orbitrap Fusion MS system with a Q-OT-qIT architecture, and 2) an Orbitrap Elite with IT-OT architecture. Raw data were analyzed with both the Protein Prospector algorithm and the MaxQuant algorithm in order to compare data from the two instruments and to obtain label-free MS1 precursor quantification for downstream analysis. Thermo Scientifi c Poster Note PN ASMS13_W689_SEliuk_E 7/13S 3
Perform Comparison 25 Unique Protein IDs 14 Unique Peptide IDs 2 12 1 15 8 1 5 Orbi Elite Orbi Fusion 6 4 2 Orbi Elite Orbi Fusion The Orbitrap Fusion MS increased protein and peptide identifications significantly. The increase in identifications was most clearly impacted by a significantly increased scan rate realized by mass selection with the quadrupole and parallelization of mass analysis and injection of the subsequent precursor. Time Point Clustering Keratins / junk VPR and known interactors 45 22 7 Proteins in VPR cluster (blue) Interactions From Jager et al 211 (red) Non-specific interactions Cytoscape network of proteins in the VPR cluster highlighting those identified in Jager et al. Using MS1 precursor quantification and hierarchical clustering, most known VPR interacting proteins fell within a cluster of proteins that were most abundant at 8h. 4 Dynamic Analysis of HIV-Human Protein-Protein Interactions During Infection Whereas the majority of proteins increased over time in the infection (above, right), nearly all known VPR interacting proteins were observed to be most abundant at 8h (above, left).
Identification of a Novel VPR Interacting Protein 8 AP-MS Intensity of CEP78 Global Ub Analysis of HIV 7 6 Intensity 5 4 3 2 1 Infected Neg ctrl Uninfected Time point clustering identified an unknown VPR-interacting protein, CEP78, that was observed to have a time point abundance profile similar to that of knock VPR-interacting proteins. It was also abundantly detected in VPR AP-MS of uninfected cells. Interestingly, CEP78 was also found to be significantly ubiquitinated in a global ubiquitination analysis of HIV-infected Jurkat cells. Summary Analysis of dynamic protein-protein interactions that shape the HIV-human interface have been analyzed using an AP-MS strategy combined with a new mass spectrometry design that significantly increased sensitivity and resulted in more than twofold increases in protein identifications. Clustering by abundance of prey proteins purified in various time points of HIV infection revealed profiles that rapidly separated specific from non-specific interactions. This analysis identified more than 75% of human proteins determined to specifically interact with the HIV protein VPR in a previous systematic analysis of HIV-human interactions. In addition, this analysis identified a novel VPR-interacting protein that was also observed to have a strong ubiquitination response in HIV infection. www.thermoscientific.com 213 Thermo Fisher Scientifi c Inc. All rights reserved. ISO is a trademark of the International Standards Organization. All other trademarks are the property of Thermo Fisher Scientifi c, Inc. and its subsidiaries. Specifi cations, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. Thermo Fisher Scientifi c, San Jose, CA USA is ISO 91:28 Certifi ed. Africa-Other +27 11 57 184 Australia +61 3 9757 43 Austria +43 1 333 5 34 Belgium +32 53 73 42 41 Canada +1 8 53 8447 China +86 1 8419 3588 Denmark +45 7 23 62 6 Europe-Other +43 1 333 5 34 Finland/Norway/Sweden +46 8 556 468 France +33 1 6 92 48 Germany +49 613 48 114 India +91 22 6742 9434 Italy +39 2 95 591 Japan +81 45 453 91 Latin America +1 561 688 87 Middle East +43 1 333 5 34 Netherlands +31 76 579 55 55 New Zealand +64 9 98 67 Russia/CIS +43 1 333 5 34 South Africa +27 11 57 184 Spain +34 914 845 965 Switzerland +41 61 716 77 UK +44 1442 233555 USA +1 8 532 4752 ASMS13_W689_SEliuk_E 7/13S