Cutting edge: LFA-1 integrin-dependent T cell adhesion is regulated by both ag specificity and sensitivity

Ab stimulation of the TCR rapidly enhances the functional activity of the LFA-1 integrin. Although TCR-mediated changes in LFA-1 activity are thought to promote T cell-APC interactions, the Ag specificity and sensitivity of TCR-mediated triggering of LFA-1 is not clear. We demonstrate that peptide/MHC (pMHC) tetramers rapidly enhance LFA-1-dependent adhesion of OT-I TCR transgenic CD8(+) T cells to purified ICAM-1. Inhibition of src family tyrosine kinase or PI3K activity blocked pMHC tetramer- and anti-CD3-stimulated adhesion. These effects are highly specific because partial agonist and antagonist pMHC tetramers are unable to stimulate OT-I T cell adhesion to ICAM-1. The Ag thresholds required for T cell adhesion to ICAM-1 resemble those of early T cell activation events, because optimal LFA-1 activation occurs at tetramer concentrations that fail to induce maximal T cell proliferation. Thus, TCR signaling to LFA-1 is highly Ag specific and sensitive to low concentrations of Ag.     

Aquaporin-2 trafficking is regulated by PDZ-domain containing protein SPA-1

Targeted positioning of water channel aquaporin-2 (AQP2) strictly regulates body water homeostasis. Trafficking of AQP2 to the apical membrane is critical to the reabsorption of water in renal collecting ducts. Controlled apical positioning of AQP2 suggests the existence of proteins that interact with AQP2. A biochemical search for AQP2-interacting proteins led to the identification of PDZ-domain containing protein, signal-induced proliferation-associated gene-1 (SPA-1) which is a GTPase-activating protein (GAP) for Rap1. The distribution of SPA-1 coincided with that of AQP2 in renal collecting ducts. The site of colocalization was concomitantly relocated by hydration status. AQP2 trafficking to the apical membrane was inhibited by the SPA-1 mutant lacking Rap1GAP activity and by the constitutively active mutant of Rap1. AQP2 trafficking was impaired in SPA-1-deficient mice. Our results show that SPA-1 directly binds to AQP2 and regulates at least in part AQP2 trafficking.     

Specific translocation of protein kinase Calpha to the plasma membrane requires both Ca2+ and PIP2 recognition by its C2 domain

The C2 domain of protein kinase Calpha (PKCalpha) controls the translocation of this kinase from the cytoplasm to the plasma membrane during cytoplasmic Ca2+ signals. The present study uses intracellular coimaging of fluorescent fusion proteins and an in vitro FRET membrane-binding assay to further investigate the nature of this translocation. We find that Ca2+-activated PKCalpha and its isolated C2 domain localize exclusively to the plasma membrane in vivo and that a plasma membrane lipid, phosphatidylinositol-4,5-bisphosphate (PIP2), dramatically enhances the Ca2+-triggered binding of the C2 domain to membranes in vitro. Similarly, a hybrid construct substituting the PKCalpha Ca2+-binding loops (CBLs) and PIP2 binding site (beta-strands 3-4) into a different C2 domain exhibits native Ca2+-triggered targeting to plasma membrane and recognizes PIP2. Conversely, a hybrid containing the CBLs but lacking the PIP2 site translocates primarily to trans-Golgi network (TGN) and fails to recognize PIP2. Similarly, PKCalpha C2 domains possessing mutations in the PIP2 site target primarily to TGN and fail to recognize PIP2. Overall, these findings demonstrate that the CBLs are essential for Ca2+-triggered membrane binding but are not sufficient for specific plasma membrane targeting. Instead, targeting specificity is provided by basic residues on beta-strands 3-4, which bind to plasma membrane PIP2.     

Nucleic acid binding and chaperone properties of HIV-1 Gag and nucleocapsid proteins

The Gag polyprotein of HIV-1 is essential for retroviral replication and packaging. The nucleocapsid (NC) protein is the primary region for the interaction of Gag with nucleic acids. In this study, we examine the interactions of Gag and its NC cleavage products (NCp15, NCp9 and NCp7) with nucleic acids using solution and single molecule experiments. The NC cleavage products bound DNA with comparable affinity and strongly destabilized the DNA duplex. In contrast, the binding constant of Gag to DNA was found to be ~10-fold higher than that of the NC proteins, and its destabilizing effect on dsDNA was negligible. These findings are consistent with the primary function of Gag as a nucleic acid binding and packaging protein and the primary function of the NC proteins as nucleic acid chaperones. Also, our results suggest that NCp7's capability for fast sequence-nonspecific nucleic acid duplex destabilization, as well as its ability to facilitate nucleic acid strand annealing by inducing electrostatic attraction between strands, likely optimize the fully processed NC protein to facilitate complex nucleic acid secondary structure rearrangements. In contrast, Gag's stronger DNA binding and aggregation capabilities likely make it an effective chaperone for processes that do not require significant duplex destabilization.     

HIV-1 gp120-mediated apoptosis of T cells is regulated by the membrane tyrosine phosphatase CD45

The molecular mechanism of the human immunodeficiency virus type 1 (HIV-1) gp120-induced apoptosis of bystander T cells is not well defined. Here, we demonstrate that CD45, a key component of the T cell receptor pathway, plays a crucial role in apoptosis induced by HIV-1 gp120. We observed that HIV-1 gp120-induced apoptosis was significantly reduced in a CD45-deficient cell line and that reconstitution of CD45 in these cells restored gp120-induced apoptosis. However, expression of a chimeric protein containing only the intracellular phosphatase domain was not able to restore the apoptotic function in the CD45-negative clone, indicating an important role for the extracellular domain of CD45 in this function. The role of CD45 in gp120-induced apoptosis was further confirmed in T cell lines and peripheral blood mononuclear cells using a selective CD45 inhibitor as well as CD45-specific small interfering RNA. We also observed that gp120 treatment induced CD45 association with the HIV coreceptor CXCR4. Further elucidation of downstream signaling events revealed that CD45 modulates HIV-1 gp120-induced apoptosis by regulating Fas ligand induction and activation of the phosphoinositide 3-kinase/Akt pathway. These results suggest a novel CD45-mediated mechanism for the HIV envelope-induced apoptosis of T cells.     

The HIV-1 Gag precursor Pr55gag synthesized in yeast is myristoylated and targeted to the plasma membrane

Myristoylation of the Pr65gag protein from Moloney murine leukemia virus has been shown to be essential for virus particle formation [Rein et al., Proc. Natl. Acad. Sci. USA 83 (1986) 7246-7250], and by analogy, myristoylation of the human immunodeficiency virus (HIV) Gag precursor could possibly play a similar role. We have investigated the expression and myristoylation of the complete HIV Gag precursor Pr55gag in yeast, the subcellular localization of that protein, and the contribution of the myristoyl-glycine residue to this localization. Immunogold labelling of myristoylated Pr55gage with antibodies directed against HIV Gag products was apparent in the vicinity of the plasma membrane. On the contrary, non-myristoylated derivatives of Pr55gag were only detected in relatively well-defined regions of the cytoplasm. These results show that targeting and accumulation of the HIV Gag precursor, Pr55gag, at the plasma membrane occurs in yeast in the absence of other viral components and requires the N-myristoyl-glycine residue.     

Critical differences in HIV-1 and HIV-2 protease specificity for clinical inhibitors

Clinical inhibitor amprenavir (APV) is less effective on HIV‐2 protease (PR₂) than on HIV‐1 protease (PR₁). We solved the crystal structure of PR₂ with APV at 1.5 Å resolution to identify structural changes associated with the lowered inhibition. Furthermore, we analyzed the PR₁ mutant (PR_1M) with substitutions V32I, I47V, and V82I that mimic the inhibitor binding site of PR₂. PR_1M more closely resembled PR₂ than PR₁ in catalytic efficiency on four substrate peptides and inhibition by APV, whereas few differences were seen for two other substrates and inhibition by saquinavir (SQV) and darunavir (DRV). High resolution crystal structures of PR_1M with APV, DRV, and SQV were compared with available PR₁ and PR₂ complexes. Val/Ile32 and Ile/Val47 showed compensating interactions with SQV in PR_1M and PR₁, however, Ile82 interacted with a second SQV bound in an extension of the active site cavity of PR_1M. Residues 32 and 82 maintained similar interactions with DRV and APV in all the enzymes, whereas Val47 and Ile47 had opposing effects in the two subunits. Significantly diminished interactions were seen for the aniline of APV bound in PR_1M and PR₂ relative to the strong hydrogen bonds observed in PR₁, consistent with 15‐ and 19‐fold weaker inhibition, respectively. Overall, PR_1M partially replicates the specificity of PR₂ and gives insight into drug resistant mutations at residues 32, 47, and 82. Moreover, this analysis provides a structural explanation for the weaker antiviral effects of APV on HIV‐2.     

Mutations in capsid major homology region affect assembly and membrane affinity of HIV-1 Gag

We introduced mutations into the HIV-1 major homology region (MHR; capsids 153-172) and adjacent C-terminal region to analyze their effects on virus-like particle (VLP) assembly, membrane affinity, and the multimerization of the Gag structural protein. Results indicate that alanine substitutions at K158, F168 or E175 significantly diminished VLP production. All assembly-defective Gag mutants had markedly reduced membrane-binding capacities, but results from a velocity sedimentation analysis suggest that most of the membrane-bound Gag proteins were present, primarily in a higher-order multimerized form. The membrane-binding capacity of the K158A, F168A, and E175A Gag proteins increased sharply upon removal of the MA globular domain. While demonstrating improved multimerization capability, the two MA-deleted versions of F168A and E175A did not show marked improvement in VLP production, presumably due to a defect in association with the raft-like membrane domain. However, K158A bound to detergent-resistant raft-like membrane; this was accompanied by noticeably improved VLP production following MA removal. Our results suggest that the HIV-1 MHR and adjacent downstream region facilitate multimerization and tight Gag packing. Enhanced Gag multimerization may help expose the membrane-binding domain and thus improve Gag membrane binding, thereby promoting Gag multimerization into higher-order assembly products.     

Coassembly and complementation of Gag proteins from HIV-1 and HIV-2, two distinct human pathogens

Approximately one million people in the world are dually infected with both HIV-1 and HIV-2. To identify potential interactions between these two human pathogens, we examined whether HIV-1 and HIV-2 Gag proteins can coassemble and functionally complement each other. We generated HIV-1- and HIV-2-based vectors with mutations in Gag; compared with wild-type vectors, these mutants had drastically decreased viral titers. Coexpression of the mutant HIV-1 and HIV-2 Gag could generate infectious viruses; furthermore, heterologous complementation in certain combinations showed efficiency similar to homologous complementation. Additionally, we used bimolecular fluorescence complementation analysis to directly demonstrate that HIV-1 and HIV-2 Gag can interact and coassemble. Taken together, our results indicate that HIV-1 and HIV-2 Gag polyproteins can coassemble and functionally complement each other during virus replication; to our knowledge, this is the first demonstration of its kind. These studies have important implications for AIDS treatment and the evolution of primate lentiviruses.     

HIV-1 envelope glycoproteins-mediated apoptosis is regulated by CD4 dependent and independent mechanisms

The progressive loss of CD4 T lymphocytes is one of the hallmarks of HIV infection. The reverse correlation observed in vivo, between plasmatic HIV levels and CD4 T lymphocyte counts, supports the concept that direct HIV-mediated cell death contributes to this depletion. Previously, we and others have demonstrated, in vitro, that interactions between membrane-expressed HIV-envelope glycoprotein complexes and CD4 ecto-molecules are critical to cell killing which occurs mainly by apoptosis. Here, by the use of a co-culture model, in which chronically HIV-1 infected cells trigger apoptosis in uninfected CD4+ target cells, we have investigated the role of different CD4 domains in HIV envelope-mediated apoptosis. Target cells were A201 lymphoblastoid cell lines expressing wild-type CD4 or mutant forms of CD4. We show that the cytoplasmic domain of CD4 was not required for apoptosis induction. In contrast, the HIV permissive cell line expressing a CD4/CD8 chimeric molecule which contains only the first 171 amino acids of CD4, appeared to be resistant to HIV-induced apoptosis; thus suggesting that the D3-D4 CD4 module plays somewhat a regulatory role. Pre-treatment of wild-type CD4 expressing target cells by the phorbol ester PMA which leads to down-regulation of CD4, completely abolished apoptosis. Interestingly, in cells expressing CD4 devoid of its cytoplasmic domain, PMA blocked partially cell death without affecting, as expected, the CD4 expression. Taken together, these results demonstrate that although CD4 expression is essential for HIV envelope induced apoptosis, the apoptotic signal could be delivered in the absence of its cytoplasmic domain. Consistent with this, we suggest that other membrane associated molecule(s) are recruited for the signalling to initiate apoptosis.     

KIBRA protein phosphorylation is regulated by mitotic kinase aurora and protein phosphatase 1

Recent genetic studies in Drosophila identified Kibra as a novel regulator of the Hippo pathway, which controls tissue growth and tumorigenesis by inhibiting cell proliferation and promoting apoptosis. The cellular function and regulation of human KIBRA remain largely unclear. Here, we show that KIBRA is a phosphoprotein and that phosphorylation of KIBRA is regulated in a cell cycle-dependent manner with the highest level of phosphorylated KIBRA detected in mitosis. We further demonstrate that the mitotic kinases Aurora-A and -B phosphorylate KIBRA both in vitro and in vivo. We identified the highly conserved Ser(539) as the primary phosphorylation site for Aurora kinases. Moreover, we found that wild-type, but not catalytically inactive, protein phosphatase 1 (PP1) associates with KIBRA. PP1 dephosphorylated Aurora-phosphorylated KIBRA. KIBRA depletion impaired the interaction between Aurora-A and PP1. We also show that KIBRA associates with neurofibromatosis type 2/Merlin in a Ser(539) phosphorylation-dependent manner. Phosphorylation of KIBRA on Ser(539) plays a role in mitotic progression. Our results suggest that KIBRA is a physiological substrate of Aurora kinases and reveal a new avenue between KIBRA/Hippo signaling and the mitotic machinery.     

F-box protein specificity for g1 cyclins is dictated by subcellular localization

Levels of G1 cyclins fluctuate in response to environmental cues and couple mitotic signaling to cell cycle entry. The G1 cyclin Cln3 is a key regulator of cell size and cell cycle entry in budding yeast. Cln3 degradation is essential for proper cell cycle control; however, the mechanisms that control Cln3 degradation are largely unknown. Here we show that two SCF ubiquitin ligases, SCF(Cdc4) and SCF(Grr1), redundantly target Cln3 for degradation. While the F-box proteins (FBPs) Cdc4 and Grr1 were previously thought to target non-overlapping sets of substrates, we find that Cdc4 and Grr1 each bind to all 3 G1 cyclins in cell extracts, yet only Cln3 is redundantly targeted in vivo, due in part to its nuclear localization. The related cyclin Cln2 is cytoplasmic and exclusively targeted by Grr1. However, Cdc4 can interact with Cdk-phosphorylated Cln2 and target it for degradation when cytoplasmic Cdc4 localization is forced in vivo. These findings suggest that Cdc4 and Grr1 may share additional redundant targets and, consistent with this possibility, grr1Δ cdc4-1 cells demonstrate a CLN3-independent synergistic growth defect. Our findings demonstrate that structurally distinct FBPs are capable of interacting with some of the same substrates; however, in vivo specificity is achieved in part by subcellular localization. Additionally, the FBPs Cdc4 and Grr1 are partially redundant for proliferation and viability, likely sharing additional redundant substrates whose degradation is important for cell cycle progression.     

In vitro expression of the HIV-2 genomic RNA is controlled by three distinct internal ribosome entry segments that are regulated by the HIV protease and the Gag polyprotein

The HIV-2 genomic RNA serves both as a messenger for protein synthesis and as a genome for viral assembly and particle production. Our previous work has shown that the HIV-2 genomic RNA encodes two additional Gag proteins that are N-terminal truncated isoforms of the p57 Gag polyprotein. In this study, by the use of mono- and bicistronic RNAs we show that translation at the three AUGs is driven by three distinct and independent internal ribosome entry segments both in vitro and ex vivo. Furthermore we used the recombinant Gag and HIV-2 protease to show that, in vitro, translation is tightly regulated by these two viral proteins. This regulation is exerted both at the level of protein production and also on the selection of the AUG initiation site which changes the ratio at which the three different Gag isoforms are produced.