The control of host processes which influence cell-cell communication is central to determining the outcome of a virus infection in tissue. Despite this, it remains unclear how cells either in close or distant proximity to an infected cell differentially respond to the primary infection. We established a virus infection microenvironment to resolve molecular features and functional consequences of cell spatial address within this localized niche by proteomic analyses, and identified differential regulations of membrane contact site (MCS) proteins in different cell populations. Here, we conducted targeted mass spectrometry (PRM) analyses for all MCS proteins representing every major organelle-organelle association, and identified elevation of MCS proteins in infected and neighboring cells. To check the contribution of extracellular vesicle (EV) dependent mechanism to the viral proteins detected in the neighboring cells, HCMV protein PRM analyses were conducted with or without EV inhibitors, GW4869 or manumycin A (MA), showing that EVs contribute to the viral protein levels in neighboring population.

Cells were lysed by detergent lysis and proteins were subjected to trypsin digestion using suspension trapping columns (S-Trap, Protifi) for 1 hour. Tryptic peptides unique to MCS proteins were separated by nano-liquid chromatography coupled to tandem mass spectrometry with a Q Exactive HF Hybrid Quadrupole-Orbitrap instrument (Thermo Scientific), using scheduled runs with 5 to 8 minute isolation windows. The MCS PRM panel monitored 207 peptides, the panel was split into 3 separate scheduled PRM methods using the above settings monitoring 69 peptides in each method to ensure as few overlapping transitions as possible. Similarly, The HCMV protein PRM panel was split into two separate methods. Libraries were built with Proteome Discoverer, and PRM data was analyzed in Skyline.

In the MCS PRM experiment, SP-TATκ-mCherry MRC5 human fibroblasts were infected with UL32-GFP HCMV, and co-cultured with WT MRC5s. Infected, neighboring, and distal cell populations were FACS sorted using FACSAria Fusion (BD Biosciences) at 48 hours post infection. In the meantime, mock MRC5s were sorted, serving as negative control samples. Cells were lysed by detergent lysis and proteins were subjected to trypsin digestion. In the experiment of HCMV protein PRM w/wo EV inhibitor treatment, WT MRC5s were treated with DMSO, GW4869, or MA for 6h, infected with HCMV for 1h, and then change the media back to DMSO, GW4869, or MA containing media for 48h. The conditional media were then collected and applied to another set of WT MRC5s for 6h. These cells were then subjected to MS sample prep and HCMV protein PRMs were conducted.