Roles of mammalian sterile 20‐like kinase 2‐dependent phosphorylations of Mps one binder 1B in the activation of nuclear Dbf2‐related kinases

Mammalian nuclear Dbf2‐related (NDR) kinases (LATS1, LATS2, NDR1 and NDR2) play a role in cell proliferation, apoptosis and morphological changes. Mammalian sterile 20‐like (MST) kinases and Mps one binder (MOB) proteins are important in the activation of NDR kinases. MOB1 is phosphorylated by MST1 and MST2 and this phosphorylation enhances the ability of MOB1 to activate NDR kinases. The phosphorylated MOB1 can be more effective as a scaffold protein to facilitate the MST‐dependent phosphorylation of NDR kinases and/or as a direct activator of NDR kinases. We previously reported that Thr74 of MOB1B is phosphorylated by MST2. Thr12 and Thr35 have also been identified as phosphorylation sites. In this study, we quantified the phosphorylation of Thr74 using the phosphorylated Thr74‐specific antibody. Thr74 is indeed phosphorylated by MST2, but the efficiency is low, suggesting that MOB1B can activate NDR kinases without the phosphorylation of Thr74. We also showed that the phosphorylated MOB1B activates NDR1 T444D and LATS2 T1041D, in which threonine residues phosphorylated by MST kinases are replaced with phosphorylation‐mimicking aspartic acid, more efficiently than the unphosphorylated MOB1B does. This finding supports that the phosphorylation of MOB1B enhances its ability as a direct activator of NDR kinases.     

Phosphorylation-dependent regulation of the F-BAR protein Hof1 during cytokinesis

Spatial and timely coordination of cytokinesis is crucial for the maintenance of organelle inheritance and genome integrity. The mitotic exit network (MEN) pathway controls both the timely initiation of mitotic exit and cytokinesis in budding yeast. Here we identified the conserved F-BAR protein Hof1 as a substrate of the MEN kinase complex Dbf2-Mob1 during cytokinesis. We show that polo-like kinase Cdc5 first phosphorylates Hof1 to allow subsequent phosphorylation by Dbf2-Mob1. This releases Hof1 from the septin ring and facilitates Hof1 binding to the medial actomyosin ring (AMR), where Hof1 promotes AMR contraction and membrane ingression. Domain structure analysis established that the central, unstructured, region of Hof1, named the ring localization sequence (RLS), is sufficient to mediate Hof1's binding to the medial ring in a cell cycle-dependent manner. Genetic and functional data support a model in which Dbf2-Mob1 regulates Hof1 by inducing domain rearrangements, leading to the exposure of the Hof1 RLS domain during telophase.     

Inhibition of HIV-1 replication by caffeine and caffeine-related methylxanthines

Human immunodeficiency virus type I (HIV-1) DNA integration is an essential step of viral replication. We have suggested recently that this stage of HIV-1 life-cycle triggers a cellular DNA damage response and requires cellular DNA repair proteins for its completion. These include DNA-PK (DNA-dependent protein kinase), ATR (ataxia telangiectasia and Rad3-related), and, at least in some circumstances, ATM (ataxia telangiectasia mutated). Host cell proteins may constitute an attractive target for anti-HIV-1 therapeutics, since development of drug resistance against compounds targeting these cellular cofactor proteins is unlikely. In this study, we show that an inhibitor of ATR and ATM kinases, caffeine, can suppress replication of infectious HIV-1 strains, and provide evidence that caffeine exerts its inhibitory effect at the integration step of the HIV-1 life-cycle. We also demonstrate that caffeine-related methylxanthines including the clinically used compound, theophylline, act at the same step of the HIV-1 life-cycle as caffeine and efficiently inhibit HIV-1 replication in primary human cells. These data reveal the feasibility of therapeutic approaches targeting host cell proteins and further support the hypothesis that ATR and ATM proteins are involved in retroviral DNA integration.     

ATR–Chk1–APC/CCdh1-dependent stabilization of Cdc7–ASK (Dbf4) kinase is required for DNA lesion bypass under replication stress

Cdc7 kinase regulates DNA replication. However, its role in DNA repair and recombination is poorly understood. Here we describe a pathway that stabilizes the human Cdc7–ASK (activator of S-phase kinase; also called Dbf4), its regulation, and its function in cellular responses to compromised DNA replication. Stalled DNA replication evoked stabilization of the Cdc7–ASK (Dbf4) complex in a manner dependent on ATR–Chk1-mediated checkpoint signaling and its interplay with the anaphase-promoting complex/cyclosome^Cdh1 (APC/C^Cdh1) ubiquitin ligase. Mechanistically, Chk1 kinase inactivates APC/C^Cdh1 through degradation of Cdh1 upon replication block, thereby stabilizing APC/C^Cdh1 substrates, including Cdc7–ASK (Dbf4). Furthermore, motif C of ASK (Dbf4) interacts with the N-terminal region of RAD18 ubiquitin ligase, and this interaction is required for chromatin binding of RAD18. Impaired interaction of ASK (Dbf4) with RAD18 disables foci formation by RAD18 and hinders chromatin loading of translesion DNA polymerase η. These findings define a novel mechanism that orchestrates replication checkpoint signaling and ubiquitin–proteasome machinery with the DNA damage bypass pathway to guard against replication collapse under conditions of replication stress.     

Rif1 controls DNA replication by directing Protein Phosphatase 1 to reverse Cdc7-mediated phosphorylation of the MCM complex

Initiation of eukaryotic DNA replication requires phosphorylation of the MCM complex by Dbf4-dependent kinase (DDK), composed of Cdc7 kinase and its activator, Dbf4. We report here that budding yeast Rif1 (Rap1-interacting factor 1) controls DNA replication genome-wide and describe how Rif1 opposes DDK function by directing Protein Phosphatase 1 (PP1)-mediated dephosphorylation of the MCM complex. Deleting RIF1 partially compensates for the limited DDK activity in a cdc7-1 mutant strain by allowing increased, premature phosphorylation of Mcm4. PP1 interaction motifs within the Rif1 N-terminal domain are critical for its repressive effect on replication. We confirm that Rif1 interacts with PP1 and that PP1 prevents premature Mcm4 phosphorylation. Remarkably, our results suggest that replication repression by Rif1 is itself also DDK-regulated through phosphorylation near the PP1-interacting motifs. Based on our findings, we propose that Rif1 is a novel PP1 substrate targeting subunit that counteracts DDK-mediated phosphorylation during replication. Fission yeast and mammalian Rif1 proteins have also been implicated in regulating DNA replication. Since PP1 interaction sites are evolutionarily conserved within the Rif1 sequence, it is likely that replication control by Rif1 through PP1 is a conserved mechanism.     

HLA-DR, ICAM-1, CD40, CD40L, and CD86 are incorporated to a similar degree into clinical human immunodeficiency virus type 1 variants expanded in natural reservoirs such as peripheral blood mononuclear cells and human lymphoid tissue cultured ex vivo

To provide additional information on the acquisition of host cell membrane proteins by human immunodeficiency virus type 1 (HIV-1) produced by natural cellular reservoirs, two different field isolates were used to infect ex vivo expanded peripheral blood mononuclear cells (PBMCs) and human lymphoid tissue histocultures. The insertion of host-derived HLA-DR, intercellular adhesion molecule-1 (ICAM-1), CD40, CD40L, and CD86 within HIV-1 particles was evaluated by using specific antibodies linked to a solid matrix to capture ultrafiltrated viral progeny. Overall, our data indicate that neither the HIV-1 co-receptor usage (i.e., T-tropic or macrophage-tropic) nor the cellular source of HIV-1 has an impact on the incorporation process but it was found to be under the influence of the donor source. Given that most viral replication is thought to occur in lymphoid tissues and previous works have shown that HIV-1 life cycle is affected by several virus-anchored host proteins, our results suggest that this phenomenon is likely to contribute to the pathogenesis of this retroviral infection.     

Abrogation of Vif function by peptide derived from the N-terminal region of the human immunodeficiency virus type 1 (HIV-1) protease

The human immunodeficiency virus type 1 (HIV-1) auxiliary gene vif is essential for virus propagation in peripheral blood lymphocytes, macrophages, and in some T-cell lines. Previously, it was demonstrated that Vif inhibits the autoprocessing of truncated HIV-1 Gag-Pol polyproteins expressed in bacterial cells, and that purified recombinant Vif and Vif-derived peptides inhibit and bind HIV-1 protease (PR). Here we show that Vif interacts with the N-terminal region of HIV-1 PR, and demonstrate that peptide derived from the N-terminal region of PR abrogates Vif function in non-permissive cells. Specifically, we show that (i) Vif protein binds HIV-1 PR, but not covalently linked tethered PR-PR; (ii) the four amino acids residing at the N terminus of HIV-1 PR are essential for Vif/PR interaction; (iii) synthetic peptide derived from the N terminus of HIV-1 PR inhibits Vif/PR binding; and (iv) this peptide inhibits the propagation of HIV-1 in restrictive cells. Based on these data, we suggest that Vif interacts with the dimerization sites of the viral protease, and that peptide residing at the N terminus of PR abrogates Vif function(s).     

NHERF1 regulates gp120-induced internalization and signaling by CCR5, and HIV-1 production

The scaffolding protein Na(+) /H(+) exchanger regulator factor 1 (NHERF1) plays an important role in the trafficking of G protein-coupled receptors. We previously demonstrated that NHERF1 is involved in chemokine receptor CCR5 homodimer internalization and signal transduction. Given the importance of CCR5 internalization during HIV-1 infection, we evaluated NHERF1's contribution in HIV-1 infection. We challenged human osteosarcoma cells coexpressing CD4 and CCR5 cells expressing either NHERF1 fragment domains or WT NHERF1 with an HIV-1 strain to examine the effects of NHERF1 on HIV-1 entry and replication. WT NHERF1 potentiates HIV-1 envelope gp120-induced CCR5 internalization, and promotes the replication of HIV-1. In order to better understand how NHERF1 affects signal transduction, different domains of NHERF1 were overexpressed in cells to analyze their effect on the different signaling pathways. Here, we show that NHERF1 can associate with CCR5, and promote activation of the gp120-induced MAPK/ERK, focal adhesion kinase and RhoA (Ras homolog gene family member A) signaling pathways. NHERF1 overexpression also increases HIV-1 host cell migration triggered by gp120 via focal adhesion kinase (FAK) signaling. Finally, NHERF1 enhanced actin filament rearrangement in host cells, an important step in post-entry HIV-1 replication events. While postsynaptic density 95/disk-large/zonula occludens 2 (PDZ2) appears to be the major contributor in those events, other domains also participate in the regulation of gp120-induced signaling pathways. Altogether, our results suggest a very important role of the scaffold NHERF1 in the regulation of HIV-1 entry and replication.     

Galactosyl ceramide expressed on dendritic cells can mediate HIV-1 transfer from monocyte derived dendritic cells to autologous T cells

Mucosa, comprising epithelial and dendritic cells, are the major sites for Human Immunodeficiency Virus type 1 (HIV-1) transmission. There, DCs can capture incoming HIV-1 and in turn transfer virus to CD4(+) T lymphocytes in a two-phase process, thereby initiating HIV-1 dissemination. We show that the glycosphingolipid Galactosyl Ceramide (GalCer), acting as mucosal epithelial receptor for HIV-1, was expressed by human monocyte derived immature DCs (iDCs), human primary DCs isolated from blood and mucosal tissue and in situ on mucosal tissue and acts as HIV-1-gp41 receptor. Blocking both GalCer and CD4 with specific mAbs results in a >95% transfer inhibition of HIV-1 from human monocyte-derived iDCs to autologous resting T cells. GalCer interaction with HIV-1 controls the early infection-independent phase of HIV-1 transfer to T cells. Thus, GalCer appears as an initial receptor for HIV-1, common to both mucosal epithelial cells and iDCs.     

Genetic interactions between CDC7 and CDC28: growth inhibition of cdc28-1N by Cdc7 point mutants

Background: Cdc7 kinase of Saccharomyces cerevisiae , a nuclear phosphoprotein, regulates initiation of chromosomal DNA replication. Overexpression of kinase-negative Cdc7 point mutants (T281E, D182N and D163N) arrests the cell cycle of the wild-type Saccharomyces cerevisiae cells at the G1/S boundary. This is caused by titration of a regulatory protein, Dbf4, from the wild-type Cdc7, which leads to inactivation of its kinase activity.
Results: We report here that kinase-negative Cdc7 mutants, when overexpressed in cdc28-1N (ts) at a permissive temperature, not only inhibit DNA replication by inactivating the wild-type Cdc7 but may also disturb coordination between DNA replication and cell division. Suppression of growth inhibition under this condition requires co-expression of both Dbf4 and Cdc28, whereas Dbf4 alone can counteract the growth inhibition in the wild-type cells. In cdc28-1N (ts), co-expression of the wild-type Dbf4 rescues only the G1/S defect and results in accumulation of those cells with less than 1C DNA as well as 2C DNA. On the other hand, co-expression of Cdc28 alone leads to increase of those cells arrested at the G1/S boundary, as found typically in the wild-type. We also report that overexpression of T281A, a 'weak' allele of Cdc7, causes growth arrest in cdc28-1N (ts) cells, but not in the CDC28 wild-type cells. This suggests that T281A is inactive in cdc28-1N (ts) and is consistent with the idea that Cdc28 activates Cdc7 by phosphorylation.
Conclusion: We conclude that two essential serine-threonine kinases, Cdc28 and Cdc7, genetically inter-act for initiation of the S phase and possibly for G2/M progression and/or S phase checkpoint control.     

Abortive HIV-1 RNA induces pro-IL-1β maturation via protein kinase PKR and inflammasome activation in humans

The proinflammatory cytokine IL-1β mediates high levels of immune activation observed during acute and chronic human immunodeficiency virus 1 (HIV-1) infection. Little is known about the mechanisms that drive IL-1β activation during HIV-1 infection. Here, we have identified a crucial role for abortive HIV-1 RNAs in inducing IL-1β in humans. Abortive HIV-1 RNAs were sensed by protein kinase RNA-activated (PKR), which triggered activation of the canonical NLRP3 inflammasome and caspase-1, leading to pro-IL-1β processing and secretion. PKR activated the inflammasome via ROS generation and MAP kinases ERK1/2, JNK, and p38. Inhibition of PKR during HIV-1 infection blocked IL-1β production. As abortive HIV-1 RNAs are produced during productive infection and latency, our data strongly suggest that targeting PKR signaling might attenuate immune activation during acute and chronic HIV-1 infection.     

A high throughput RNAi screen reveals determinants of HIV-1 activity in host kinases

Drug resistance remains a great challenge in HIV/AIDS treatment despite the recent advances in novel therapeutics. It may be a good strategy to develop drugs targeting the essential host factors to decrease the risk of drug resistance. Previous studies suggested that so many host kinases play roles in HIV life cycles. More importantly, many kinase genes are drugable targets, therefore, it is vital to figure out host kinases responsible for HIV-1 infection and replication to provide novel therapeutic regimens and to deepen our understanding to HIV-host interaction. In present work, a high throughput RNAi screen with a shRNA library against 474 kinases was applied to HEK293T cells stably expressed a HIV-1 LTR (long terminal repeat)-driven reporter plasmid. Four genes, AK1, EphB2, PRKACB and CDK5R2, were found to specifically suppress the HIV-1 LTR activity following effective knockdown. Furthermore, overexpression of AK1 and PRKACB upregulated the HIV-1 LTR activity. Therefore, AK1 and PRKACB are in positive control of HIV-1 activity. DNA microarray analysis demonstrated that overlapped genes between AK1-silenced and PRKACB-silenced cells were mainly enriched in several amino acid biosynthesis pathways, TGF-β signaling and p53 signaling pathways. These alterations may repress the viral infection by the downregulation of ERK1/2, p38MAPK and NFκB signaling pathways. Collectively, our work uncovers several host kinases involving the HIV-1 infection and may provide potential therapeutic targets for AIDS treatment in future.     

The Th1 immune response against HIV-1 Gag p24-derived peptides in mice expressing HLA-A02.01 and HLA-DR1

Using HLA-DR1-transgenic H-2 class II knockout mice, we identified two new HLA-DR1-restricted HIV-1 Gag p24-derived epitopes (Gag(321-340 )and Gag(331-350)) and confirmed the immunogenicity of seven that have been previously described. The human relevance was confirmed for the two new ones (Gag(321-340 )and Gag(331-350)) assaying peripheral blood mononuclear cells from HLA-DR1(+) HIV-1-infected long-term asymptomatic subjects and showing that Gag(331-350) could prime CD4(+) T cells from two HLA-DR1(+) HIV-1 seronegative donors in vitro. Seven of these epitopes, structurally conserved among HIV-1 clade B isolates, were selected for a comparative evaluation of their Th1 helper potential by immunizing HLA-A02.01/HLA-DR1-transgenic, H-2 class I/class II knockout mice with recombinant mouse invariant chain constructs in which each helper epitope was inserted in association with two reporter HIV-1-derived HLA-A02.01-restricted CD8(+) T cell epitopes. A T helper effect was demonstrated in all cases, and was particularly strong with epitopes Gag(301-320),Gag(321-340 )and Gag(271-290), which should, therefore, be considered in the design of new vaccines.     

A recombinant vesicular stomatitis virus encoding HIV-1 receptors and human OX40 ligand efficiently eliminates HIV-1-infected CD4-positive T cells expressing OX40

OX40 protein is highly expressed on activated CD4-positive T cells that are susceptible for human immunodeficiency virus type 1 (HIV-1) infection. To target and kill HIV-1-infected OX40(+) T cells, we used a recombinant vesicular stomatitis virus (rVSV) lacking its envelope glycoprotein (ΔG) and instead expressing HIV-1 receptors CD4/CXCR4 and OX40 ligand (OX40L). Expression of OX40L as well as HIV-1 receptors on the VSV particles led to specific infection of OX40(+) T cells, including primary cells, either acutely or chronically infected with X4 HIV-1. Consequently, the rVSV rapidly eliminated these infected cells and caused a marked reduction of HIV-1 viral load in culture. Inclusion of the OX40L gene in the VSV recombinant led to significantly better infection and HIV-1 elimination compared with an rVSVΔG expressing only HIV-1 receptors. A novel rVSVΔG encoding both HIV-1 receptors and OX40L has a potentially greater therapeutic value than an rVSVΔG expressing only HIV-1 receptors.     

TopBP1 activates ATR through ATRIP and a PIKK regulatory domain

The ATR (ATM and Rad3-related) kinase and its regulatory partner ATRIP (ATR-interacting protein) coordinate checkpoint responses to DNA damage and replication stress. TopBP1 functions as a general activator of ATR. However, the mechanism by which TopBP1 activates ATR is unknown. Here, we show that ATRIP contains a TopBP1-interacting region that is necessary for the association of TopBP1 and ATR, for TopBP1-mediated activation of ATR, and for cells to survive and recover DNA synthesis following replication stress. We demonstrate that this region is functionally conserved in the Saccharomyces cerevisiae ATRIP ortholog Ddc2, suggesting a conserved mechanism of regulation. In addition, we identify a domain of ATR that is critical for its activation by TopBP1. Mutations of the ATR PRD (PIKK [phosphoinositide 3-kinase related kinase] Regulatory Domain) do not affect the basal kinase activity of ATR but prevent its activation. Cellular complementation experiments demonstrate that TopBP1-mediated ATR activation is required for checkpoint signaling and cellular viability. The PRDs of ATM and mTOR (mammalian target of rapamycin) were shown previously to regulate the activities of these kinases, and our data indicate that the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) PRD is important for DNA-PKcs regulation. Therefore, divergent amino acid sequences within the PRD and a unique protein partner allow each of these PIK kinases to respond to distinct cellular events.