Methods for characterizing protein acetylation during viral infection

Abstract

Lysine acetylation is a prevalent posttranslational modification that acts as a regulator of protein function, subcellular localization, and interactions. A growing body of work has highlighted the importance of temporal alterations in protein acetylation during infection with a range of human viruses. It has become clear that both cellular and viral proteins are decorated by lysine acetylations, and that these modifications contribute to core host defense and virus replication processes. Further defining the extent and dynamics of protein acetylation events during the progression of an infection can provide an important new perspective on the intricate mechanisms underlying the biology and pathogenesis of virus infections. Here, we provide protocols for identifying, quantifying, and probing the regulation of lysine acetylations during viral infection. We describe the use of acetyl-lysine immunoaffinity purification and quantitative mass spectrometry for assessing the cellular acetylome at different stages of an infection. As an alternative to traditional antibody-mediated western blotting, we discuss the benefits of targeted mass spectrometry approaches for detecting and quantifying site-specific acetylations on proteins of interest. Specifically, we provide a protocol using parallel reaction monitoring (PRM). We further discuss experimental considerations that are specific to studying viral infections. Finally, we provide a brief overview of the types of assays that can be employed to characterize the function of an acetylation event in the context of infection. As a method to interrogate the regulation of acetylation, we describe the Fluor de Lys assay for monitoring the enzymatic activities of deacetylases.

Effect of statins on SARS-CoV-2 infection

“…Interestingly, gene ontology annotation suggests that 80% of proteins downregulated exclusively in fluvastatin treated SARS-CoV-2 infected cells are post-translationally acetylated. Protein acetylation regulates both cellular and viral protein function, subcellular localization, and interaction 50 . Whether or not protein acetylation plays a role during SARS-CoV-2 infection requires experimental clarification.…”

Lamin B1 acetylation slows the G1 to S cell cycle transition through inhibition of DNA repair

“…To quantify the abundance of the K134 endogenous acetylation in cells enriched in the G2 phase of the cell cycle, six 70% confluent plates of MRC5 cells were treated with 10 μM CDK1 inhibitor RO-3306 (Sigma Aldrich) for 48 h. Samples were then collected, and acetylated peptides were enriched and prepared for mass spectrometry analysis as previously described ( 26 , 36 ). Briefly, cell pellets were lysed, proteins were reduced and alkylated, proteins were precipitated by methanol/chloroform extraction, and proteins were digested with trypsin overnight.…”

The Pleiotropic Function of Human Sirtuins as Modulators of Metabolic Pathways and Viral Infections

“…SIRT1 inhibitors affect the replication and growth of many viruses including Mers-CoV, HIV, Epathitis B, Vesicular Stomatitis Virus, flu strains, adenovirus and others [ 126 , 135 , 136 , 137 , 138 , 139 ]. Notably, SIRT1 affects angiotensin-converting enzyme 2 receptor (ACE2R) expression [ 140 , 141 , 142 ].…”