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Jameson Moore
Jameson Moore

Lewin's CELLS Download \/\/TOP\\\\

Lewin's Cells 3rd Edition by George Plopper & David sharp is a great book for study of Cell Biology available in (eBook) PDF download. This 3rd edition, expanded and updated from the second, is geared for advanced undergraduate and graduate students taking a first course in cell biology. A key objective in developing this Lewin's Cell book was to present mechanisms and the concepts underlying cell structure and function, gleaned from decades of research, in a format that provides the information necessary with students for cell biology solid foundation, without overwhelming them with too much detail. The major goal of the team of 29 expert authors and lead editors has been to incorporate the current research in the field, thoroughly provide and cover each topic ample cellular processes illustrations at the molecular level but without being unwieldy.

Lewin's CELLS Download

Eradication of HIV cannot be achieved with combination antiretroviral therapy (cART) because of the persistence of long-lived latently infected resting memory CD4+ T cells. We previously reported that HIV latency could be established in resting CD4+ T cells in the presence of the chemokine CCL19. To define how CCL19 facilitated the establishment of latent HIV infection, the role of chemokine receptor signalling was explored.

HIV integration in CCL19-treated resting CD4+ T cells depends on NF-κB signalling and increases the stability of HIV integrase, which allow subsequent integration and establishment of latency. These findings have implications for strategies needed to prevent the establishment, and potentially reverse, latent infection.

The major barrier to HIV cure in patients receiving combination antiretroviral therapy (cART) is the persistence of long-lived, latently infected, resting memory CD4+ T cells [1]. Understanding how HIV latency is established and maintained is critical to the development of novel approaches to eradicate HIV. Direct infection of resting CD4+ T cells in vitro is inefficient [2, 3]. In contrast, HIV integration and latency is established in resting CD4+ T cells in vivo [4], in tonsil explants [5], or following co-culture with endothelial cells [6] or dendritic cells [7], or following culture with chemokines that bind to the chemokine receptors CCR7, CXCR3 and CCR6 expressed on resting CD4+ T cells [8, 9].

The chemokine CCR7 is expressed on naïve and central memory CD4+ T cells and culturing with the CCR7-ligands CCL19 or CCL21 facilitates entry and integration of HIV into these resting CD4+ T cells with minimal virus production. In this model, resting CD4+ T cells 5 day post infection represent a stable latently infected population that respond to latency-reversing agents (LRA) in a similar pattern to CD4+ T cells from HIV-infected individuals on cART [10]. We therefore believe this is an ideal model to study the early events required for establishing latency.

Binding of chemokines to their specific Gi-coupled receptors leads to activation of the RhoA/GTPase, phosphatidylinositol 3-kinase (PI3K) and phospholipase C (PLC) pathways [11]. Binding of HIV glycoprotein (gp)120 to the HIV co-receptor CXCR4 can also activate the RhoA pathway in resting CD4+ T cells, activating cofilin leading to depolymerization of the cortical actin cytoskeleton, thus facilitating nuclear entry [3, 12]. We demonstrated similar cellular changes with the exogenous chemokine CCL19 [9]. However, in contrast to HIV infection of resting CD4+ T cells, where there is minimal integration of HIV [2, 3, 9], in chemokine treated cells there was efficient nuclear localization and HIV integration [8, 9].

Activation of PI3K leads to changes in nuclear factor kappa B (NF-κB), the Mitogen-Activated Protein kinases (MAPK), extra-cellular signal-regulated kinases 1 and 2 (ERK1/2) [13], p38, c-Jun N-terminal kinase (JNK) [14] and serine/threonine protein kinase (Akt) phosphorylation (Additional file 1: Figure S1). Although NF-κB is a potent activator of HIV transcription, previous studies have demonstrated that latent or productive infection may depend on the relative amount of active NF-κB. In T cell lines, high levels of NF-κB enhance HIV transcription, but low levels of NF-κB are important for HIV integration [15]. Furthermore, generating latent infection by direct infection of Jurkat T cells requires low levels of NF-κB [16]. However, the role of NF-κB in latency in primary resting CD4+ T cells remains unknown.

Other downstream signalling products of chemokine receptor signalling may also impact HIV integration and latent infection of resting CD4+ T cells. Activating protein-1 transcription factors (AP-1) [17] may play a role in establishing and maintaining HIV latency [18]. The Ras/Raf/MEK pathway has previously been associated with the nuclear import of the HIV reverse transcriptase complex (RTC) [19] and thus may enhance HIV nuclear entry required for infection of resting CD4+ T cells. In the present study, we asked whether the PI3K signalling pathway, induced by ligation of CCL19 to CCR7, played a key role in the establishment of latent infection in CCL19-treated resting CD4+ T cells. We demonstrated that activation of the NF-κB pathway is critical for efficient integration of HIV in CCL19-treated resting CD4+ T cells and that the sites of HIV integration depended on the activation state of the cell at the time of infection.

Activation of CCR7 has been shown to induce multiple modules of signalling leading to cell survival, chemotaxis, endocytosis and others (see review in [11, 20]). To determine which pathway of CCL19-mediated signalling was important for establishment of latency, pharmacological agents that inhibit various signalling molecules were used (Additional file 1: Figure S1). Inhibition of CCL19-induced phosphorylation of Akt, NF-κB, ERK and p38 were observed using specific inhibitors to PI3K (LY294002 and Wortmannin), NF-κB (SC-514) (Fig. 1a, c-I, c-II); MEK1/ERK1/2 (PD980509), p38 (SB203580) (Fig. 1b, c-III, c-IV); and a broad-spectrum inhibitor of Akt and NF-κB (Bay11-7082) (Fig. 1a, c-II). Phosphorylation of JNK was not consistently observed in response to CCL19. Since phosphorylation of JNK is required for cell migration [21], the lack of pJNK in CCL19-treated cells is likely due to timing of detection, as CD4+ T cell migration is normally measured at least 3 h after the addition of CCL19 [22]. However, the effect of JNK (SP600125) and AP-1 (SR11302) inhibitors on CCL19-treated cells were indirectly observed (Fig. 1c). SP600125 markedly inhibited phosphorylation of Akt, but increased p38 phosphorylation, while SR11302 inhibited the phosphorylation of both ERK and p38.

We used inhibitors of the PI3K pathway, to determine if activation of this pathway was critical for HIV integration in CCL19-treated resting CD4+ T cells (Fig. 2). In HIV-infected CCL19-treated resting CD4+ T cells, treatment of cells prior to infection with the inhibitors of PI3K, LY294002 and Wortmannin, significantly reduced HIV integration as measured by Alu-LTR PCR, and reduced the levels of 2-LTR circles back to the level of unactivated cells (Fig. 2b, c).

Treatment with PI3K and Ras/Raf/MEK inhibitors, eliminates HIV integration in CCL19-treated resting CD4+ T cells. a Resting CD4+ T-cells were pre-incubated with inhibitors of specific signalling pathways for 1 h before addition of CCL19, PHA-IL2 or DMSO and then infected with HIV NL4-3 for 2 h and cultured with media containing IL2 (1 U/mL) for up to 4 days following infection. HIV integration was measured by qPCR for Alu-LTR (b, d) and nuclear entry was measured by qPCR for 2-LTR circles (c, e). Experiments were also performed in the presence of inhibitors to PI3K (LY294002 and Wortmannin; b, c). d, e Further experiments were conducted in the presence of inhibitors of p38 (SB203580), ERK1/2 (PD980509), JNK (SP600125), AP-1 (SR11302), or NF-κB (SC-514) activation. Each column represents the mean copy number and the symbols represent individual donors. The detection limit for the Alu-LTR was 300 copies/106 cells and is shown as a dashed line. *p

To define the specific pathways downstream of the chemokine receptor important for HIV integration, we next tested inhibitors of p38, MEK1/ERK1/2, JNK, AP-1 and NF-κB, prior to infection of CCL19-treated resting CD4+ T cells. HIV integration was reduced to undetectable levels in the presence of inhibitors to MEK1/ERK1/2, JNK, AP-1 and NF-κB (Fig. 2d). In comparison, the p38 inhibitor SB203580 had a mild inhibitory (but not significant) effect on HIV integration (Fig. 2d), despite being able to fully inhibit p38 phosphorylation (Fig. 1). These inhibitors also reduced the level of 2-LTR circles back to unactivated cells level, although the reduction did not reach statistical significance (Fig. 2e). The effect of these inhibitors on HIV integration and 2-LTR circles were not likely due to the cytotoxicity of these drugs (Additional file 3: Figure S3). We found that the broad-spectrum inhibitor of Akt and NF-κB, Bay11-7082, was highly toxic to resting CD4+ T cells and was removed from further analysis.

Since JNK activation phosphorylates and stabilizes HIV integrase via an interaction with peptidyl prolyl-isomerase (Pin1) in activated CD4+ T cells [23], we used a co-immunoprecipitation (IP) assay to analyse the formation of Pin1/integrase-containing complexes in resting CCL19-treated CD4+ T cells. Like PHA-IL2 activation, CCL19 induced formation of Pin1/integrase complexes and this was impaired in the presence of the JNK inhibitor SP600125 (Fig. 3a). We also used siRNA knockdown of Pin1 to determine whether Pin1 was required for integration and establishment of latency in CCL19-treated resting CD4+ T cells. Expression of Pin1 in CD4+ T cells was similar after stimulation with PHA-IL2 or CCL19, but was greatly reduced in siRNA-transfected cells compared to the scrambled control (Fig. 3b, upper panel). Following HIV infection of these cells, we found a statistically significant reduction in both 2-LTR circles and integrated HIV DNA (Fig. 3b), consistent with a previous report [23]. These data suggest that Pin1 may affect pre-integration events in resting CD4+ T cells as well as PHA-IL2 activated cells. These experiments confirmed that interactions of JNK, integrase and Pin1 are likely similar following infection of resting and activated CD4+ T cells and are critical for HIV infection.


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