HIV signaling through CD4 and CCR5 activates Rho family GTPases that are required for optimal infection of primary CD4+ T cells

Abstract

BackgroundHIV-1 hijacks host cell machinery to ensure successful replication, including cytoskeletal components for intracellular trafficking, nucleoproteins for pre-integration complex import, and the ESCRT pathway for assembly and budding. It is widely appreciated that cellular post-translational modifications (PTMs) regulate protein activity within cells; however, little is known about how PTMs influence HIV replication. Previously, we reported that blocking deacetylation of tubulin using histone deacetylase inhibitors promoted the kinetics and efficiency of early post-entry viral events. To uncover additional PTMs that modulate entry and early post-entry stages in HIV infection, we employed a flow cytometric approach to assess a panel of small molecule inhibitors on viral fusion and LTR promoter-driven gene expression.ResultsWhile viral fusion was not significantly affected, early post-entry viral events were modulated by drugs targeting multiple processes including histone deacetylation, methylation, and bromodomain inhibition. Most notably, we observed that inhibitors of the Rho GTPase family of cytoskeletal regulators—including RhoA, Cdc42, and Rho-associated kinase signaling pathways—significantly reduced viral infection. Using phosphoproteomics and a biochemical GTPase activation assay, we found that virion-induced signaling via CD4 and CCR5 activated Rho family GTPases including Rac1 and Cdc42 and led to widespread modification of GTPase signaling-associated factors.ConclusionsTogether, these data demonstrate that HIV signaling activates members of the Rho GTPase family of cytoskeletal regulators that are required for optimal HIV infection of primary CD4+ T cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-017-0328-7) contains supplementary material, which is available to authorized users.

Global phosphoproteomics of CCR5-tropic HIV-1 signaling reveals reprogramming of cellular protein production pathways and identifies p70-S6K1 and MK2 as HIV-responsive kinases required for optimal infection of CD4+ T cells

“…We analyzed phosphopeptides with large fold-changes at both the 1- and 15-min time points and found nearly all were from proteins with roles in either (1) actin cytoskeletal architecture, endocytosis, and movement of vesicles along actin and tubulin networks, or (2) transcription, mRNA splicing and capping, translation, or acetylation of methionine residues on newly produced proteins. These data support previous studies demonstrating that these pathways are actively modulated by signaling upon HIV binding to the cell surface [ 22 , 26 , 27 , 30 ].…”

“…Rho GTPases regulate a variety of cellular processes including cytoskeletal dynamics, transcription, endosomal trafficking, cell cycle, cell adhesion and cytokinesis. The observation that HIV signaling influences Rho GTPase signaling pathways has been explored by our lab recently, in combination with a small molecule screen that implicated Rho GTPases in regulation of HIV infection [ 30 ]. Briefly, CCR5-tropic signaling was found to dramatically alter the cellular Rho GTPase landscape, regulating not only cdc42 and Rac1 GTPases but also at least 25 guanosine dissociation inhibitors (GDIs), guanosine exchange factors (GEFs) and GTPase activating proteins (GAPs) that influence GTPase signaling.…”

Proteo-Transcriptomic Dynamics of Cellular Response to HIV-1 Infection

“…We observed a pronounced upregulation response at the RNA level, for the most part involving signal transduction pathways, as opposed to the other two omics levels. Our data suggest that HIV-1 influences the signaling mechanisms of infected cells mainly by affecting expression of cell surface receptors, chemokine receptors, G-protein-coupled receptors, or genes involved in signaling of Rho GTPases (Supplementary File S5), recapitulating previous findings 22–25 . At the protein and phosphoprotein levels, the identified differentially expressed genes were mainly enriched in pathways describing immune system, metabolism, gene expression, cell cycle, and HIV-related processes (Fig.…”

All-Round Manipulation of the Actin Cytoskeleton by HIV

“…The authors conclude that the Cdc42-NWASP-Arp2/3 pathway may be equal to or more important than the Rac1 pathway described above [ 137 ]. Recent studies have confirmed this hypothesis, by showing that Env signaling through CD4 and CCR5 leads to activation of both Rac1 and Cdc42 in primary CD4 T-cells ( Figure 2 a) [ 138 ]. Similarly, ingenuity pathway analysis scored Cdc42 and Rac1 pathways within the top-five deregulated cellular networks in T-cells that are exposed to cell-free [ 138 ], or cell-transmitted virus [ 139 ].…”