HIV-1 integrase inhibitors that compete with the target DNA substrate define a unique strand transfer conformation for integrase.

Diketo acids such as L-731,988 are potent inhibitors of HIV-1 integrase that inhibit integration and viral replication in cells. These compounds exhibit the unique ability to inhibit the strand transfer activity of integrase in the absence of an effect on 3' end processing. To understand the reasons for this distinct inhibitory profile, we developed a scintillation proximity assay that permits analysis of radiolabeled inhibitor binding and integrase function. High-affinity binding of L-731,988 is shown to require the assembly of a specific complex on the HIV-1 long terminal repeat. The interaction of L-731,988 with the complex and the efficacy of L-731, 988 in strand transfer can be abrogated by the interaction with target substrates, suggesting competition between the inhibitor and the target DNA. The L-731,988 binding site and that of the target substrate are thus distinct from that of the donor substrate and are defined by a conformation of integrase that is only adopted after assembly with the viral end. These results elucidate the basis for diketo acid inhibition of strand transfer and have implications for integrase-directed HIV-1 drug discovery efforts.

Inhibitors of strand transfer that prevent integration and inhibit HIV-1 replication in cells.

Integrase is essential for human immunodeficiency virus-type 1 (HIV-1) replication; however, potent inhibition of the isolated enzyme in biochemical assays has not readily translated into antiviral activity in a manner consistent with inhibition of integration. In this report, we describe diketo acid inhibitors of HIV-1 integrase that manifest antiviral activity as a consequence of their effect on integration. The antiviral activity of these compounds is due exclusively to inhibition of one of the two catalytic functions of integrase, strand transfer.

The role of F-cadherin in localizing cells during neural tube formation in Xenopus embryos.

The cell adhesion molecule F-cadherin is expressed in Xenopus embryos at boundaries that subdivide the neural tube into different regions, including one, the sulcus limitans, which partitions the caudal neural tube into a dorsal and ventral half (alar and basal plate, respectively). Here we examine the role of F-cadherin in positioning cells along the caudal neuraxis during neurulation. First, we show that ectopic expression of F-cadherin restricts passive cell mixing within the ectodermal epithelium. Second, we show that F-cadherin is first expressed at the sulcus limitans prior to the extensive cell movements that accompany neural tube formation, suggesting that it might serve to position cells at the sulcus limitans by counteracting their tendency to disperse during neurulation. We test this idea using an assay that measures changes in cell movements during neurulation in response to differential cell adhesion. Using this assay, we show that cells expressing F-cadherin localize preferentially to the sulcus limitans, but still disperse when located away from the sulcus limitans. In addition, inhibiting cadherin function prevents cells from localizing precisely at the sulcus limitans. These results indicate that positioning of cells at the sulcus limitans is mediated in part by the differential expression of F-cadherin.

NF-protocadherin, a novel member of the cadherin superfamily, is required for Xenopus ectodermal differentiation.

BACKGROUND: The assembly of complex tissues during embryonic development is thought to depend on differential cell adhesion, mediated in part by the cadherin family of cell-adhesion molecules. The protocadherins are a new subfamily of cadherins; their extracellular domains comprise cadherin-like repeats but their intracellular domains differ significantly from those of classical cadherins. Little is known about the ability of protocadherins to mediate the adhesion of embryonic cells, or whether they play a role in the formation of embryonic tissues. RESULTS: We report the isolation and characterization of a novel protocadherin, termed NF-protocadherin (NFPC), that is expressed in Xenopus embryos. NFPC showed a striking pattern of expression in early embryos, displaying predominant expression within the deep, sensorial layer of the embryonic ectoderm and in a restricted group of cells in the neural folds, but was largely absent from the neural plate and surrounding placodal regions. Ectopic expression in embryos demonstrated that NFPC could mediate cell adhesion within the embryonic ectoderm. In addition, expression of a dominant-negative form of NFPC disrupted the integrity of embryonic ectoderm, causing cells in the deep layer to dissociate, though leaving the outer layer relatively intact. CONCLUSIONS: Our results indicate that NFPC is required as a cell-adhesion molecule during embryonic development, and its function is distinct from that of classical cadherins in governing the formation of a two-layer ectoderm. These results suggest that NFPC, and protocadherins in general, are involved in novel cell-cell adhesion mechanisms that play important roles in tissue histogenesis.

Xenopus F-cadherin, a novel member of the cadherin family of cell adhesion molecules, is expressed at boundaries in the neural tube.

Development of the vertebrate CNS begins during neurulation when the neural plate gives rise to the neural tube. During neurulation, the different regions of the CNS can be identified in part by the appearance of flexures in the walls of the neural tube. Here we report the isolation and characterization of F-cadherin; a novel member of the cadherin family of cell adhesion molecules, which is expressed at flexures in the neural tube of Xenopus embryos. We show that F-cadherin is first expressed at neural plate stages, that its expression is altered when patterning of the neural tube is perturbed, and that its expression marks boundaries in the neural tube where cell mixing is restricted. These observations suggest that F-cadherin contributes to the regionalization of the neural tube perhaps by mediating differential cell adhesion.

Retinoic acid receptor expression vector inhibits differentiation of F9 embryonal carcinoma cells.

Expression vectors have been constructed for a region of the human retinoic acid receptor-alpha (hRAR-alpha) and transferred into F9 embryonal carcinoma (EC) cells. When the vectors are overexpressed in F9 cells, clones can be selected for resistance to retinoic acid-induced differentiation. This effect is obtained even when the hRAR-alpha region is expressed as a beta-galactosidase fusion protein. Using the beta-galactosidase component of the fusion protein as a marker, overexpression of the fusion protein has been correlated with the retinoic acid-resistance effect. The clones resistant to retinoic acid no longer exhibit the normal retinoic acid induction of endo B cytokeratin, laminin B-1, and tissue plasminogen activator mRNAs observed with normal F9 cells. Retinoic acid induction of type IV alpha-1 collagen and Hox-1.3 RNAs is observed with these clones. When transfected with a thyroid receptor DNA-binding sequence (TRE)/thymidine kinase promoter/luciferase construct, the retinoic acid-resistant clones do not yield the same retinoic acid-induced level of luciferase obtained with F9 cells. It is hypothesized that the RAR vectors are interfering with endogenous RAR(s) in a dominant-negative manner to inhibit retinoic acid-induced differentiation of F9 EC cells.