PNNL

Abstract

We report on a proteomic analysis of ex vivo HIV-1 infection in human primary CD4 cells by shotgun LC-MS/MS analysis, revealing two distinct proteomic profiles at two phases of virus replication. Relative to mock-infected cells, 168 signature proteins exhibited abundance changes at the first sign of gag p24 production (8h post-infection, p.i.) or the peak of virus replication (24h p.i.); interestingly, most of the changes were exclusive to only one phase of virus replication. Based on characterization by functional ontology and known human-HIV protein interactions, we observed the enrichment for protein abundance increases pertaining to protein synthesis and nucleasomal reorganization amidst an otherwise placid cellular proteome at the first sign of HIV replication. In contrast, we observed indications of decreased protein turnover, concomitant with heightened DNA repair activities and preludes to apoptosis, in the presence of robust virus replication. We also observed hints of disruptions in protein and small molecule trafficking. Our label-free proteomics strategy allowed us to perform multiplexed comparisons—we buttressed our detection specificity with the use of a reverse-transcriptase inhibitor as a counter-screen, enabling for highlighting cellular protein abundance changes unique to robust virus replication as opposed to viral entry. In conjunction with complementary high-throughput screens for cellular partners of HIV, we put forth a model pinpointing specific rerouting of cellular biosynthetic, energetic and trafficking pathways as HIV replication accelerates in human primary CD4 cells.