Suppression of albumin enhancer activity by H-ras and AP-1 in hepatocyte cell lines.

We demonstrated, using a transient transfection assay, that the albumin enhancer increased the expression of the albumin promoter in a highly differentiated, simian virus 40 (SV40)-immortalized hepatocyte cell line, CWSV1, but was not functional in two ras-transformed cell lines (NR3 and NR4) derived from CWSV1 by stable transfection with the T24ras oncogene. A transient cotransfection assay showed that T24ras and normal c-Ha-ras were each able to inhibit the activity of the albumin enhancer in an immortal hepatocyte cell line. DNase I footprinting and gel mobility shift assays demonstrated that the DNA binding activities specific to the albumin enhancer were not decreased in the ras-transformed cells. ras also did not diminish the expression of HNF1 alpha, C/EBP alpha, HNF3 alpha, HNF3 beta, or HNF3 gamma but did significantly increase AP-1 binding activity. Three AP-1 binding sites were identified within the albumin enhancer, and DNA binding activities specific to these AP-1 sites were induced in the ras-transformed hepatocytes. Subsequent functional assays showed that overexpression of c-jun and c-fos inhibited the activity of the albumin enhancer. Site-directed mutagenesis of the AP-1 binding sites in the albumin enhancer partially abrogated the suppressing effect of ras and c-jun/c-fos on the enhancer. These functional studies therefore supported the results of the structural studies with AP-1. We conclude that the activity of the albumin enhancer is subject to regulation by ras signaling pathways and that the effect of ras on the albumin enhancer activity may be mediated by AP-1.     

TAR-independent replication of human immunodeficiency virus type 1 in glial cells.

The molecular mechanisms involved in the replication of human immunodeficiency virus type 1 (HIV-1) may differ in various cell types and with various exogenous stimuli. Astrocytic glial cells, which can support HIV-1 replication in cell cultures and may be infected in vivo, are demonstrated to provide a cellular milieu in which TAR mutant HIV-1 viruses may replicate. Using transfections of various TAR mutant HIV-1 proviral constructs, we demonstrate TAR-independent replication in unstimulated astrocytic cells. We further demonstrate, using viral constructs with mutations in the tat gene and in the nuclear factor kappa B (NF-kappa B)-binding sites (enhancer) of the HIV-1 long terminal repeat, that TAR-independent HIV-1 replication in astrocytic cells requires both intact NF-kappa B moiety-binding motifs in the HIV-1 long terminal repeat and Tat expression. We measured HIV-1 p24 antigen production, syncytium formation, and levels and patterns of viral RNA expression by Northern (RNA) blotting to characterize TAR-independent HIV-1 expression in astrocytic glial cells. This alternative regulatory pathway of TAR-independent, Tat-responsive viral production may be important in certain cell types for therapies which seek to perturb Tat-TAR binding as a strategy to interrupt the viral lytic cycle.     

Defective HIV-1 Particle Assembly in AP-3-Deficient Cells Derived from Patients with Hermansky-Pudlak Syndrome Type 2

Adaptor protein complex 3 (AP-3) is a heterotetramer that is involved in signal-mediated protein sorting to endosomal-lysosomal organelles. AP-3 deficiency in humans, induced by mutations in the AP3B1 gene, which encodes the β3A subunit of the AP-3 complex, results in Hermansky-Pudlak syndrome 2 (HPS2), which is a rare genetic disorder with defective lysosome-related organelles. In a previous study, we identified the AP-3 complex as an important contributor to HIV-1 assembly and release. We hypothesized that cells from patients affected by HPS2 should demonstrate abnormalities of HIV-1 assembly. Here we report that HIV-1 particle assembly and release are indeed diminished in HPS2 fibroblast cultures. Transient or stable expression of the full-length wild-type β3A subunit in HPS2 fibroblasts restored the impaired virus assembly and release. In contrast, virus-like particle release mediated by MA-deficient Gag mutants lacking the AP-3 binding site was not altered in HPS2 cells, indicating that the MA domain serves as the major viral determinant required for the recruitment of the AP-3 complex. AP-3 deficiency decreased HIV-1 Gag localization at the plasma membrane and late endosomes and increased the accumulation of HIV-1 Gag at an intermediate step between early and late endosomes. Blockage of the clathrin-mediated endocytic pathway in HPS2 cells did not reverse the inhibited virus assembly and release imposed by the AP-3 deficiency. These results demonstrate that the intact and stable AP-3 complex is required for HIV-1 assembly and release, and the involvement of the AP-3 complex in late stages of the HIV-1 replication cycle is independent of clathrin-mediated endocytosis.     

DNA conformation driven by AP-1 triggers cell-specific expression via a strong epithelial enhancer

We report here the characterization of the regulatory region of the human LAMA3 gene, coding for the α3A chain of laminin-5. A 202 bp fragment is sufficient to confer epithelial-specific expression to a thymidine kinase promoter through the cooperative effect of three AP-1 binding sites. Remarkably, removal of the sequences located between the AP-1 sites does not modify the promoter activity in keratinocytes but allows strong expression in fibroblasts. Replacement of the deleted sequences by non-homologous ones fully restores the restricted enhancement in keratinocytes. Functional analysis and mutagenesis experiments demonstrate that a minimal distance between the AP-1 sites is required for the enhancer DNA fragment to adopt a particular conformation driven by the binding of Jun–Fos heterodimers. In non-permissive cells, this conformation leads to the anchorage of non-DNA-binding fibroblastic cofactors to form an inhibitory ternary complex. Therefore, our results describe for the first time an unusual conformation-dependent epithelial-specific enhancer.     

Distinct factors bind the AP-1 consensus sites in gibbon ape leukemia virus and simian virus 40 enhancers.

We have demonstrated that the gibbon ape leukemia virus (GALV) enhancer AP-1 element and the simian virus 40 AP-1 enhancer element bind different factors in HeLa nuclear extracts. A 39-kilodalton HeLa nuclear protein and the c-fos protein bind to the GALV element. Antibodies to c-fos abolish binding to the GALV AP-1 site. In contrast, anti-c-fos immunoglobulin fails to inhibit formation of the simian virus 40-specific complex from extracts of HeLa cells. Thus, AP-1-binding complexes are subject to compositional variation at different binding sites.