citations

                    Global post-translational modification profiling of HIV-1-infected cells reveals mechanisms of host cellular pathway remodeling
                

Authors:

Jeffrey R. Johnson, David C. Crosby, Judd F. Hultquist, Donna Li, John Marlett, Justine Swann, Ruth Hüttenhain, Erik Verschueren, Tasha L. Johnson, Billy W. Newton, Michael Shales, View ORCID ProfilePedro Beltrao, Alan D. Frankel, Alexander Marson, Oliver I. Fregoso, John A. T. Young, Nevan J. Krogan

In:

Source: BioResearch Open Access
Publication Date: (2020)
Issue: 1: 1

Research Area:

Immunotherapy / Hematology

Cells used in publication:

CD4+, human: Species: human; Tissue Origin: blood

Platform:

4D-Nucleofector® 96-well Systems

4D-Nucleofector® X-Unit

Experiment

Cas9 RNPs were prepared fresh for each experiment. crRNA and tracrRNA were first mixed 1:1 and incubated for 30 minutes at 37°C to generate 80 µM crRNA:tracrRNA duplexes. An equal volume of 40 µM S. pyogenes Cas9-NLS was slowly added to the crRNA:tracrRNA and incubated for 15 minutes at 37°C to generate 20 µM Cas9 RNPs. For each reaction, approximately 105 T cells were pelleted and resuspended in 20 µL P3 buffer. 4 µL of 20 µM Cas9 RNP mix
was added directly to these cells and the entire volume was transferred to a 96-well
reaction cuvette. Cells were electroporated using program EH-115 on the Amaxa 4D Nucleofector (Lonza). 80 µL pre-warmed complete RPMI-1640 was added to each well and the cells were allowed to recover for 30 minutes at 37°C. Cells were then restimulated using CD2/CD3/CD28 flow cytometry-compatible stimulation beads (Miltenyi Biotec) in complete RPMI-1640 supplemented with 80 U/ml IL-2-IS (Miltenyi Biotec) and cultured in 96-well V-bottom plates.

Abstract

Viruses must effectively remodel host cellular pathways to replicate and evade immune defenses, and they must do so with limited genomic coding capacity. Targeting post-translational modification (PTM) pathways provides a mechanism by which viruses can broadly and rapidly transform a hostile host environment into a hospitable one. We used quantitative proteomics to measure changes in two PTM types – phosphorylation and ubiquitination – in response to HIV-1 infection with viruses harboring targeted deletions of a subset of HIV-1 genes. PTM analysis revealed a requirement for Aurora kinase A activity in HIV-1 infection and furthermore revealed that AMP-activated kinase activity is modulated during infection via HIV-1 Vif-mediated degradation of B56-containing protein phosphatase 2A (PP2A). Finally, we demonstrated that the Cullin4A-DDB1-DCAF1 E3 ubiquitin ligase ubiquitinates histone H1 somatic isoforms and that HIV-1 Vpr inhibits this process, leading to defects in DNA repair. Thus, global PTM profiling of infected cells serves as an effective tool for uncovering specific mechanisms of host pathway modulation.