Abstract
The mechanisms by which human immunodeficiency virus (HIV) circumvents and coopts cellular machinery to replicate and persist in cells are not fully understood. HIV accessory proteins play key roles in the HIV life cycle by altering host pathways that are often dependent on post-translational modifications (PTMs). Thus, the identification of HIV accessory protein host targets and their PTM status is critical to fully understand how HIV invades, avoids detection and replicates to spread infection. To date, a comprehensive characterization of HIV accessory protein host targets and modulation of their PTM status does not exist. The significant gap in knowledge regarding the identity and PTMs of HIV host targets is due, in part, to technological limitations. Here, we applied current mass spectrometry techniques to define mechanisms of viral protein action by identifying host proteins whose abundance is affected by the accessory protein Vpr and the corresponding modulation of down-stream signaling pathways, specifically those regulated by phosphorylation. By utilizing a novel, inducible HIV-1 CD4+ T-cell model system expressing either the wild type or a vpr-negative viral genome, we overcame challenges associated with synchronization and infection-levels present in other models. We report identification and abundance dynamics of over 7000 proteins and 28,000 phospho-peptides. Consistent with Vpr's ability to impair cell-cycle progression, we observed Vpr-mediated modulation of spindle and centromere proteins, as well as Aurora kinase A and cyclin-dependent kinase 4 (CDK4). Unexpectedly, we observed evidence of Vpr-mediated modulation of the activity of serine/arginine-rich protein-specific kinases (SRPKs), suggesting a possible role for Vpr in the regulation of RNA splicing. This study presents a new experimental system and provides a data-resource that lays the foundation for validating host proteins and phosphorylation-pathways affected by HIV-1 and its accessory protein Vpr.
Highlights
From the ‡Department of Pharmacology, §Skaggs School of Pharmacy and Pharmaceutical Sciences, ¶San Diego Veterans Affairs Healthcare System, San Diego, California 92161, and the ʈDepartment of Medicine, University of California San Diego, La Jolla, California 92093
The known cellular targets of Vpr have not defined a clear role for this gene product in viral replication or pathogenesis, but its ability to induce the degradation of the cellular proteins uracil DNA glycosylase (UNG), the endonuclease MUS81, and the DNA helicase HLTF suggest that modulation of DNA-repair is somehow important to human immunodeficiency virus (HIV) replication (9 –12)
Inducible HIV-1 CD4ϩ T-Cell Model Characterization—A well-known phenotype associated with HIV Vpr is delay of the cell cycle in G2/M phase [15]
Summary
From the ‡Department of Pharmacology, §Skaggs School of Pharmacy and Pharmaceutical Sciences, ¶San Diego Veterans Affairs Healthcare System, San Diego, California 92161, and the ʈDepartment of Medicine, University of California San Diego, La Jolla, California 92093. Quantitative Temporal Viromics of Inducible HIV licate and persist are not fully known; elucidating these mechanisms could lead to the identification of new drug-targets. HIV is a retrovirus containing nine protein-encoding genes [5] These gene products include structural proteins, essential regulatory proteins, and so-called accessory proteins [6]. The accessory proteins Vif, Vpr and Vpu direct host proteins that interfere with viral replication to a degradation pathway by hijacking the host ubiquitin-proteasome system [6]. The known cellular targets of Vpr have not defined a clear role for this gene product in viral replication or pathogenesis, but its ability to induce the degradation of the cellular proteins uracil DNA glycosylase (UNG), the endonuclease MUS81, and the DNA helicase HLTF suggest that modulation of DNA-repair is somehow important to HIV replication (9 –12)
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