HIV-1 induces cytoskeletal alterations and Rac1 activation during monocyte-blood–brain barrier interactions: modulatory role of CCR5

Abstract

BackgroundMost HIV strains that enter the brain are macrophage-tropic and use the CCR5 receptor to bind and infect target cells. Because the cytoskeleton is a network of protein filaments involved in cellular movement and migration, we investigated whether CCR5 and the cytoskeleton are involved in endothelial-mononuclear phagocytes interactions, adhesion, and HIV-1 infection.ResultsUsing a cytoskeleton phospho-antibody microarray, we showed that after co-culture with human brain microvascular endothelial cells (HBMEC), HIV-1 infected monocytes increased expression and activation of cytoskeleton-associated proteins, including Rac1/cdc42 and cortactin, compared to non-infected monocytes co-cultured with HBMEC. Analysis of brain tissues from HIV-1-infected patients validated these findings, and showed transcriptional upregulation of Rac1 and cortactin, as well as increased activation of Rac1 in brain tissues of HIV-1-infected humans, compared to seronegative individuals and subjects with HIV-1-encephalitis. Confocal imaging showed that brain cells expressing phosphorylated Rac1 were mostly macrophages and blood vessels. CCR5 antagonists TAK-799 and maraviroc prevented HIV-induced upregulation and phosphorylation of cytoskeleton-associated proteins, prevented HIV-1 infection of macrophages, and diminished viral-induced adhesion of monocytes to HBMEC. Ingenuity pathway analysis suggests that during monocyte-endothelial interactions, HIV-1 alters protein expression and phosphorylation associated with integrin signaling, cellular morphology and cell movement, cellular assembly and organization, and post-translational modifications in monocytes. CCR5 antagonists prevented these HIV-1-induced alterations.ConclusionsHIV-1 activates cytoskeletal proteins during monocyte-endothelial interactions and increase transcription and activation of Rac1 in brain tissues. In addition to preventing macrophage infection, CCR5 antagonists could diminish viral-induced alteration and phosphorylation of cytoskeletal proteins, monocyte adhesion to the brain endothelium and viral entry into the central nervous system.

Toll-like receptor-3 mediates HIV-1 transactivation via NFκB and JNK pathways and histone acetylation, but prolonged activation suppresses Tat and HIV-1 replication

“…), was used in a RT assay to quantify RT enzyme levels. The RT assay method has been described in detail in previous publications [31, 33]. …”

All-Round Manipulation of the Actin Cytoskeleton by HIV

“…In contrast, the role of ERMs for outbound virus is less well understood. HIV infection has been shown to increase expression of moesin or ezrin in various cell types, including CEM, HeLa and monocytes [ 106 , 107 , 108 ]. However, silencing of either protein increased HIV replication in infected Jurkat cells [ 107 , 109 ], suggesting a rather restrictive role of ERMs in outbound HIV spread.…”

“…Both Rac1 and Abl activate WAVE2 to prompt actin polymerization by the Arp2/3 complex (see Section 5.1.3 ). Since disruption of any of these proteins resulted in decreased HIV entry and arrested fusion at the hemi-fusion stage, the authors conclude that this signaling pathway ultimately leads to actin remodeling changes that facilitate HIV fusion-pore formation and enlargement ( Figure 2 b) [ 7 , 108 , 119 ]. In agreement, studies by Pontow et al confirmed that interfering with Rac1 function reduces viral fusion, leading to decreased infection rates [ 120 , 121 ].…”

“…In agreement, studies by Pontow et al confirmed that interfering with Rac1 function reduces viral fusion, leading to decreased infection rates [ 120 , 121 ]. While these mechanisms were elucidated mainly in adherent cell lines, other studies support Env-dependent activation of Rac1 in primary T-cells and monocytes ex vivo, as well as brain macrophages in vivo [ 108 ]. Of note, soluble Tat is also able to activate Rac1 in uninfected endothelial and HeLa cells, although the corresponding mechanisms are less well understood [ 122 , 123 , 124 ].…”

“…On the other hand, HIV-infected cells also display dramatic Rac1 activation, and this is mainly mediated by Nef. Nef expression increased Rac1 activity in a wide range of cell types, including podocytes [ 125 , 126 ], T-cells [ 127 ], monocytes [ 108 , 128 ] and DCs [ 129 ]. The mechanism of Nef-mediated Rac1 activation is currently controversial but most likely involves direct binding of Nef to the leukocyte-enriched GPD-exchange factor Vav1 (see Section 5.3.1 ) [ 129 , 130 ].…”