The possibility of HIV-1 eradication has been limited by the existence of latently infected cellular reservoirs. Studies to examine control of HIV latency and potential reactivation have been hindered by the small numbers of latently infected cells found in vivo. Major conceptual leaps have been facilitated by the use of latently infected T cell lines and primary cells. However, notable differences exist among cell model systems. Furthermore, screening efforts in specific cell models have identified drug candidates for “anti-latency” therapy, which often fail to reactivate HIV uniformly across different models. Therefore, the activity of a given drug candidate, demonstrated in a particular cellular model, cannot reliably predict its activity in other cell model systems or in infected patient cells, tested ex vivo. This situation represents a critical knowledge gap that adversely affects our ability to identify promising treatment compounds and hinders the advancement of drug testing into relevant animal models and clinical trials. To begin to understand the biological characteristics that are inherent to each HIV-1 latency model, we compared the response properties of five primary T cell models, four J-Lat cell models and those obtained with a viral outgrowth assay using patient-derived infected cells. A panel of thirteen stimuli that are known to reactivate HIV by defined mechanisms of action was selected and tested in parallel in all models. Our results indicate that no single in vitro cell model alone is able to capture accurately the ex vivo response characteristics of latently infected T cells from patients. Most cell models demonstrated that sensitivity to HIV reactivation was skewed toward or against specific drug classes. Protein kinase C agonists and PHA reactivated latent HIV uniformly across models, although drugs in most other classes did not.

Eradication of HIV-1 with highly active antiretroviral therapy (HAART) is not possible due to the persistence of long-lived, latently infected resting memory CD4 + T cells. We now show that HIV-1 latency can be established in resting CD4+ T cells infected with HIV-1 after exposure to ligands for CCR7 (CCL19), CXCR3 (CXCL9 and CXCL10), and CCR6 (CCL20) but not in unactivated CD4 + T cells. The mechanism did not involve cell activation or significant changes in gene expression, but was associated with rapid dephosphorylation of cofilin and changes in filamentous actin. Incubation with chemokine before infection led to efficient HIV-1 nuclear localization and integration and this was inhibited by the actin stabilizer jasplakinolide. We propose a unique pathway for establishment of latency by direct HIV-1 infection of resting CD4 + T cells during normal chemokine-directed recirculation of CD4 + T cells between blood and tissue.

IntroductionThe major barrier to eradication of HIV-1 is persistent long-lived and latently infected resting CD4 ϩ T cells. 1,2 Preintegration latency refers to unintegrated HIV-1 DNA that is unstable and will either degrade or will integrate into the host cell genome, usually after cell activation. [3][4][5][6] Postintegration latency refers to the presence of integrated HIV-1 DNA in cells that are not actively producing viral particles. One of the paradoxes of postintegration latency is the inefficiency of reverse transcription and integration of HIV-1 into resting CD4 ϩ T cells from the peripheral blood, 7 whereas there is efficient infection of resting CD4 ϩ T cells in lymphoid organ cultures in vitro or in the tissues of HIV-1-infected persons or SIV-infected macaques. [8][9][10][11] The 2 known CCR7 ligands, CCL19 and CCL21, are constitutively expressed in lymphoid organs, particularly by resident stromal cells in the T-zone, and are critical for T-cell and dendritic cell (DC) trafficking within secondary lymphoid organs. [12][13][14] Given that latent HIV-1 infection predominantly occurs in CCR7 expressing resting CD4 ϩ T cells [15][16][17][18] and that infection of resting CD4 ϩ T cells occurs with greater efficiency in lymphoid tissue compared with blood, we hypothesized that CCL19 and CCL21 may be critical factors that condition resting CD4 ϩ T cells to HIV-1 infection, integration and latency. Materials and methods Isolation of CD4 ؉ T cellsPeripheral blood mononuclear cells (PBMCs) were isolated by FicollPaque (GE Healthcare, Chalfont St. Giles, United Kingdom) density centrifugation. Resting CD4 ϩ T cells were obtained by negative selection and magnetic beads ( Figure 1A). HIV-1 infectionPurified resting CD4 ϩ T cells (either total or purified CD45RO Ϫ (naive) and CD45RO ϩ (memory) T cells) were cultured for 3 days in the presence of CCL19 (10-100 nM) or CCL21 (10-100 nM; R&D Systems, Minneapolis, MN), phytohemagglutinin (PHA; 10 g/mL)/interleukin-2 (IL-2) (10 U/mL; Boehringer Mannheim, Mannheim, Germany) or left unactivated. In some experiments, an antibody to CCR7, 3D12 (10 g/mL; BD Biosciences, Franklin Lakes, NJ) or IgG 2a isotype control was added to resting CD4 ϩ T cells 24 hours before addition of CCL19. We then infected the cells with either pNL4.3 (X4 using) or AD8 (R5 using) HIV-1 or HIV-1 containing a deletion in the nef gene (⌬nef) and replaced with enhanced green fluorescent protein (EGFP) [pNL4.3 ⌬nef EGFP and AD8 ⌬nef EGFP] or pNL4.3 with a mutation in integrase D116N 19 ( Figure 1B). All infections were performed at a multiplicity of infection of 1 count per minute reverse transcriptase (RT) per cell. RT concentration was determined as described previously. 20 In some experiments, integrated HIV-1 DNA was quantified using Alu-long terminal repeat (LTR) real-time polymerase chain reaction (PCR) as described previously. 7,21 Flow cytometryCells were washed and stained with anti-CD25-phycoerythrin (PE), anti-CD69-fluorescein isothiocyanate (FITC), human leukocyte antigen (HLA)-DR-PE, an...

Persistence of latent, replication-competent Human Immunodeficiency Virus type 1 (HIV-1) provirus is the main impediment towards a cure for HIV/AIDS (Acquired Immune Deficiency Syndrome). Therefore, different therapeutic strategies to eliminate the viral reservoirs are currently being explored. We here propose a novel strategy to reduce the replicating HIV reservoir during primary HIV infection by means of drug-induced retargeting of HIV integration. A novel class of integration inhibitors, referred to as LEDGINs, inhibit the interaction between HIV integrase and the LEDGF/p75 host cofactor, the main determinant of lentiviral integration site selection. We show for the first time that LEDGF/p75 depletion hampers HIV-1 reactivation in cell culture. Next we demonstrate that LEDGINs relocate and retarget HIV integration resulting in a HIV reservoir that is refractory to reactivation by different latency-reversing agents. Taken together, these results support the potential of integrase inhibitors that modulate integration site targeting to reduce the likeliness of viral rebound.

Synaptogenesis at the neuromuscular junction requires agrin-induced stable localization of acetylcholine receptors (AChRs) at the endplate. The effects of agrin are transduced by the muscle-specific receptor tyrosine kinase (MuSK). This study provides evidence that Src-class protein tyrosine kinases mediate the effects of agrin-activated MuSK to regulate clustering and anchoring of AChRs in skeletal muscle. MuSK was complexed with both Src and Fyn in the C2 mouse muscle cell line. These associations were enhanced by agrin and by increasing protein tyrosine phosphorylation with pervanadate. Coupling between MuSK and the Src-class kinases in vivo appeared to be caused by a phosphotyrosine-SH2 domain interaction because binding of MuSK to the SH2 domains of Fyn and Src in vitro was specific, enhanced by phosphorylation, and dependent on MuSK autophosphorylation. In addition, Src and Fyn phosphorylated MuSK. AChR phosphorylation, stimulated by agrin or pervanadate, was inhibited by blocking Src-class kinases with PP1. Furthermore, agrin-induced clustering and cytoskeletal anchoring of AChRs was dependent on Src-family kinases. These data support the conclusion that Fyn and Src act downstream of MuSK to regulate the stable localization of AChRs at the neuromuscular endplate during agrin-induced synaptogenesis.