Controlling gene expression in living cells through small molecule-RNA interactions.

Short RNA aptamers that specifically bind to a wide variety of ligands in vitro can be isolated from randomized pools of RNA. Here it is shown that small molecule aptamers also bound their ligand in vivo, enabling development of a method for controlling gene expression in living cells. Insertion of a small molecule aptamer into the 5' untranslated region of a messenger RNA allowed its translation to be repressible by ligand addition in vitro as well as in mammalian cells. The ability of small molecules to control expression of specific genes could facilitate studies in many areas of biology and medicine.

Homocysteine-induced endoplasmic reticulum stress causes dysregulation of the cholesterol and triglyceride biosynthetic pathways.

Hepatic steatosis is common in patients having severe hyperhomocysteinemia due to deficiency for cystathionine beta-synthase. However, the mechanism by which homocysteine promotes the development and progression of hepatic steatosis is unknown. We report here that homocysteine-induced endoplasmic reticulum (ER) stress activates both the unfolded protein response and the sterol regulatory element-binding proteins (SREBPs) in cultured human hepatocytes as well as vascular endothelial and aortic smooth muscle cells. Activation of the SREBPs is associated with increased expression of genes responsible for cholesterol/triglyceride biosynthesis and uptake and with intracellular accumulation of cholesterol. Homocysteine-induced gene expression was inhibited by overexpression of the ER chaperone, GRP78/BiP, thus demonstrating a direct role of ER stress in the activation of cholesterol/triglyceride biosynthesis. Consistent with these in vitro findings, cholesterol and triglycerides were significantly elevated in the livers, but not plasmas, of mice having diet-induced hyperhomocysteinemia. This effect was not due to impaired hepatic export of lipids because secretion of VLDL-triglyceride was increased in hyperhomocysteinemic mice. These findings suggest a mechanism by which homocysteine-induced ER stress causes dysregulation of the endogenous sterol response pathway, leading to increased hepatic biosynthesis and uptake of cholesterol and triglycerides. Furthermore, this mechanism likely explains the development and progression of hepatic steatosis and possibly atherosclerotic lesions observed in hyperhomocysteinemia.

Role of hyperhomocysteinemia in endothelial dysfunction and atherothrombotic disease.

Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular disease, including ischemic heart disease, stroke, and peripheral vascular disease. Mutations in the enzymes responsible for homocysteine metabolism, particularly cystathionine beta-synthase (CBS) or 5,10-methylenetetrahydrofolate reductase (MTHFR), result in severe forms of HHcy. Additionally, nutritional deficiencies in B vitamin cofactors required for homocysteine metabolism, including folic acid, vitamin B6 (pyridoxal phosphate), and/or B12 (methylcobalamin), can induce HHcy. Studies using animal models of genetic- and diet-induced HHcy have recently demonstrated a causal relationship between HHcy, endothelial dysfunction, and accelerated atherosclerosis. Dietary enrichment in B vitamins attenuates these adverse effects of HHcy. Although oxidative stress and activation of proinflammatory factors have been proposed to explain the atherogenic effects of HHcy, recent in vitro and in vivo studies demonstrate that HHcy induces endoplasmic reticulum (ER) stress, leading to activation of the unfolded protein response (UPR). This review summarizes the current role of HHcy in endothelial dysfunction and explores the cellular mechanisms, including ER stress, that contribute to atherothrombosis.

A non-canonical base pair within the human immunodeficiency virus rev-responsive element is involved in both rev and small molecule recognition.

BACKGROUND: Human immunodeficiency virus (HIV) replication depends on the interaction of an HIV regulatory protein, Rev, with a viral RNA element (the Rev-responsive element, RRE). The high affinity RRE core region contains a non-canonical base pair (G48:G71) that is important for Rev recognition. Aminoglycoside antibiotics, specifically neomycin B, bind to the RRE and selectively block Rev-RRE interactions in vivo and in vitro. We attempted to generate an in vitro model for the establishment of HIV-1 resistance to neomycin B. RESULTS: We have used in vitro genetic selection to evolve RRE variants that bind to Rev in the presence of neomycin B. Most of the RRE variants selected in the presence of 10 microM neomycin B contain a G48:G71 to A48:A71 substitution. Those selected in 100 microM neomycin B contain either C:A or A:A substitutions at this position. Binding constants for the interaction of neomycin B with the wild-type RRE and a subset of the selected RRE variants were determined using a novel ultrafiltration procedure. CONCLUSIONS: A purine-purine base pair within the bulge region of the RRE core elements is critical for neomycin B binding as well as Rev binding. RRE variants that survive in high concentrations of neomycin do so either by binding Rev better than wild-type (this correlates with the sequence A48:A71) or by binding neomycin poorly (correlating with the sequence C48:A71). Other sequences must also influence both Rev and neomycin B binding.

Mutational analysis of the herpes simplex virus trans-inducing factor Vmw65.

Vmw65 is a structural component of herpes simplex virus which, in conjunction with host factors, trans-activates the expression of the viral immediate-early genes. In order to examine the relationship between the primary structure of Vmw65 and its trans-activating function, we generated in-frame insertion, deletion, and nonsense mutations in a cloned copy of the gene. The ability of the mutant polypeptides to function as transcriptional activators was assessed by transient transfection of Vero cells using, as the reporter gene, the Escherichia coli chloramphenicol acetyltransferase (cat) gene linked to the promoter-regulatory region from the HSV-1 immediate-early gene coding for Vmw175. These studies have demonstrated that a highly acidic region near the C-terminus of Vmw65 as well as the structural integrity of several other regions of the polypeptide are essential for its transactivating properties, whereas a small region near the N-terminus of the polypeptide is dispensible for activity. Finally, in vivo competition studies using inactive deletion mutants suggest that a domain of the polypeptide located between amino acids 141 and 185 may be involved in protein-protein interactions.

Hyperglycaemia is associated with impaired vasa vasorum neovascularization and accelerated atherosclerosis in apolipoprotein-E deficient mice.

OBJECTIVE: A direct correlation between blood glucose levels and the microvascular complications of diabetes is well established. However, the effects of hyperglycaemia on the vasa vasorum, a microvascular network which surrounds and supplies the walls of large arteries, is not known. The objective of this study is to investigate the effects of hyperglycaemia on the vasa vasorum and to examine correlations between these effects and the development of atherosclerosis in a mouse model. METHODS: The micro- and macrovascular effects of hyperglycaemia were examined in streptozotocin-injected apolipoprotein-E deficient (ApoE(-/-)) mice. Retina and aortic sinus were isolated from hyperglycaemic mice and normoglycaemic controls at 5-20 weeks of age. Retinal and vasa vasorum microvessel densities were quantified and correlated to atherosclerotic lesion development. The expression levels of pro-angiogenic factors including vascular endothelial growth factor (VEGF) and VEGF receptor 2 were examined. RESULTS: In normoglycaemic ApoE(-/-) mice atherogenesis is associated with vasa vasorum expansion, which likely corresponds to the increasing blood supply demands of the thickening artery wall. In hyperglycaemic ApoE(-/-) mice there is no significant neovascularization of the vasa vasorum, despite the fact that lesions are significantly larger. This defect may result from a localized deficiency in VEGF. CONCLUSIONS: These findings are the first evidence that hyperglycaemia alters the structure of the vasa vasorum. Such microvascular changes directly correlate, and may contribute to, the development and progression of atherosclerosis in hyperglycaemic ApoE-deficient mice.

Synthesis of the herpes simplex virus type 1 trans-activator Vmw65 in insect cells using a baculovirus vector.

Vmw65, the Herpes Simplex Virus trans-activator of immediate-early genes, was expressed in insect cells using a recombinant baculovirus expression vector and partially purified. Insect cell-derived Vmw65 was shown to be indistinguishable from authentic Vmw65 present in purified HSV-1 virions based on electrophoretic mobility, immunoreactivity with a monoclonal antibody, and ability to interact with cellular factors to form a protein/DNA complex with oligonucleotides containing a TAATGARAT element.

An unusual cellular factor potentiates protein-DNA complex assembly between Oct-1 and Vmw65.

The herpes simplex virus trans-activator Vmw65 interacts with the cellular factors Oct-1 and VCAF-1 to generate a multicomponent DNA binding complex that specifically recognizes conserved enhancer elements found upstream of the viral immediate-early genes, resulting in potent stimulation of their transcription. We have identified a HeLa cell factor, distinct from Oct-1 or VCAF-1, which significantly enhances the stability or formation of Vmw65-dependent complexes, as judged by mobility shift analysis using either nuclear extracts or bacterially expressed Oct-1 and Vmw65. This factor, designated SF (stimulatory factor), was partially purified from HeLa cell postnuclear extracts and has an apparent molecular weight of 1500-3000, based on ultrafiltration and size-exclusion chromatography. SF was shown to be resistant to inactivation by heat treatment, protease, nuclease, and phospholipase digestions, and extraction with organic solvents. Pretreatment of SF with beta-glucuronidase did not affect its ability to stimulate Vmw65-dependent complex formation but did reduce the electrophoretic mobility of the resulting complex. These data indicate that SF is probably a component of the Vmw65-induced complex and may be composed, at least partially, of carbohydrate.

Identification of a domain of the herpes simplex virus trans-activator Vmw65 required for protein-DNA complex formation through the use of protein A fusion proteins.

In order to identify structural domains of the herpes simplex virus trans-activator Vmw65 required for protein-DNA complex formation, subfragments of Vmw65 were expressed in Escherichia coli as fusion polypeptides with protein A of Staphylococcus aureus, and the purified hybrids were used in a band shift assay. The results indicate that a region near the amino terminus of Vmw65 between amino acids 141 and 185 is necessary for complex formation.

Enhanced infectivity of herpes simplex virus type 1 viral DNA in a cell line expressing the trans-inducing factor Vmw65.

Vmw65 is a structural component of herpes simplex virus (HSV) which is involved in transactivating the expression of the viral immediate-early (IE) genes. To gain further insight into the function of this protein, a cell line, BSV65, was established which expresses biologically active Vmw65 under control of the Moloney leukemia virus long terminal repeat. This cell line was shown to specifically activate IE genes as demonstrated by transient transfection assays with reporter genes linked to HSV IE or delayed-early promoter-regulatory regions. Furthermore, by using mobility shift assays, cell extracts were shown to be capable of forming a Vmw65-containing complex with oligonucleotides that contained a TAATGARAT motif, a conserved cis-acting IE regulatory element which is required for Vmw65-mediated trans induction. BSV65 cells were able to complement HSV type 1 in 1814, a mutant which is unable to trans-induce IE gene expression and whose growth is impaired at low multiplicities of infection. Transfection of purified HSV type 1 viral DNA into BSV65 cells resulted in an approximately 200-fold increase in virus production compared with the parental cell line. In addition, in comparison to wild-type cells, infectious virus production occurred sooner and efficiency of plaque formation was higher in BSV65 cells following transfection of viral DNA but not following infection with virus. Northern (RNA) dot blot analysis of cells transfected with viral DNA showed that transcription of the IE gene Vmw175 was approximately 10-fold greater in BSV65 cells compared with wild-type cells. These results indicate that, in the presence of functional Vmw65, there is a greater probability that transfected viral DNA will lead to a productive infection.