Both Virus and Host Components Are Important for the Manifestation of a Nef- Phenotype in HIV-1 and HIV-2

AbstractWhile it has been demonstrated that the Nef protein of simian immunodeficiency virus is obligatory for the establishment of high viral loads and the development of simian AIDS in rhesus macaques, demonstrating a critical role for the human immunodeficiency virus (HIV) Nef protein in tissue culture has been elusive. Data have been contradictory as to whether Nef has a negative or positive influence on in vitro virus replication. In an attempt to define a role for Nef during virus propagation in tissue culture and to obtain virus-host systems that could distinguish between the Nef mutant and wildtype viruses, we have introduced mutations into the nef genes of infectious molecular clones of three HIV-1 strains and two isolates of the HIV-2ROD strain and have investigated the capacity of viruses derived from them to infect a number of CD4-positive T-cell lines and peripheral blood mononuclear cells (PBMC). Mutating the nef gene of all viruses had a modest negative effect on virus production in activated PBMC. In some T-cell lines with some viruses, the effects were severe, and little or no Nef mutant virus could be detected. In other cell lines, the result of mutating the nef gene either had no effect or had a slight negative effect on the replication kinetics. Therefore, whether the consequences of loss of Nef activity can be demonstrated in vitro depends on both the particular virus and the host cell used, suggesting that Nef is exerting its activity on some cellular pathway. In addition, we describe the construction and properties of hitherto unreported infectious molecular clones of the ROD strain of HIV-2

Biochemical aspects of plant virus infection of cucumbers: a studyof the viral-induced protein and RNA species, and the RNA-dependant RNA polymerase

An investigation has been carried out on the biochemical aspects of the infection of cucumbers by two plant viruses Cucumber Mosaic Virus (CMV) and Tobacco Ringspot Virus (TRSV). 1. Attempts were made to characterise the TRSV-induced RNA species (replicative form, replicative intermediate and viral RNA genome); RNA was extracted from cucumber cotyledons using the phenol method and analysed on 2.4e" polyacrylamide gels. No differences in gel patterns between healthy and infected plants could be found in either single labelling studies (plants labelled through the roots with 32p-orthophosphate) or with double-labelling studies (plants labelled through the roots with either 32p-orthophosphate or 3H-uridine); actinomycin D, although completely abolishing ribosomal RNA synthesis, did not show up any differences between healthy and TRSV-infected cucumber RNA extracts. 2. Attempts were also made to characterise the TRSV-induced protein species. As radioactive amino acids could not be incorporated into cucumber proteins by absorption through the roots, 35s-=sulphate was used as the label. No differences could be found between healthy and infected protein extracts analysed. by polyacrylamide gel electrophoresis. Further attempts to incorporate radioactive amino acids using tissue slices and other methods were also unsuccessful. An alternative approach of differentially labelling healthy and infected plant proteins after extraction by reduction and carboxymethylation with either 3H or I4c-io.loacetic acid produced variable and unreliable radioactive protein profiles on polyacrylamide gels. 3. A comparison of the properties was made between the CMV-induced and. the TRSV-induced RNA polymerases, both soluble and particulate enzymes; these enzymes were undetectable in healthy plants. Both the soluble and particulate RNA polymerases from TRSV-infected cucumbers were detectable two days after infection and declined rapidly after four days, whereas the CMV-induced enzymes appear after about five days, reach a plateau level at 10 days and remained at this 1evel for several more days. The mol. wt. of the soluble CMV-induced RNA polymerase was calculated by sucrose gradient centrifugation to be 123,000 daltons while the soluble TRSV-induced enzyme sedimented over a wide range of 120-180,000 daltons. Solubilising the particulate CMV-induced RNA polymerase could be accomplished by incubating with 50-100mM MgSO4 or by freezing and thawing, but these methods did not release the particulate TRSV-induced enzyme. All other properties tested v/ere similar in both cases. This provided circumstantial evidence for the differences being due to viral-coded functions in the RN polymerase molecules. 4. Several- purification methods have been tried in an attempt to purify the CMV-induced RNA polymerase. Those methods that gave reasonable recoveries were protamine sulphate precipitation, phosphocellulose chromatography (stepwise elution with either KCI or (NH4) 2SO4) and poly c-sepharose chromatography (stepwise elution with M9SO4). A possible method of purification is outlined.Thesis (M.Sc.) -- University of Adelaide, Dept of Biochemistry, 197

Chemical-genetic disruption of clathrin function spares adaptor complex 3-dependent endosome vesicle biogenesis

A role for clathrin in AP-3–dependent vesicle biogenesis has been inferred from biochemical interactions and colocalization between this adaptor and clathrin. The functionality of these molecular associations, however, is controversial. We comprehensively explore the role of clathrin in AP-3–dependent vesicle budding, using rapid chemical-genetic perturbation of clathrin function with a clathrin light chain–FKBP chimera oligomerizable by the drug AP20187. We find that AP-3 interacts and colocalizes with endogenous and recombinant FKBP chimeric clathrin polypeptides in PC12-cell endosomes. AP-3 displays, however, a divergent behavior from AP-1, AP-2, and clathrin chains. AP-3 cofractionates with clathrin-coated vesicle fractions isolated from PC12 cells even after clathrin function is acutely inhibited by AP20187. We predicted that AP20187 would inhibit AP-3 vesicle formation from endosomes after a brefeldin A block. AP-3 vesicle formation continued, however, after brefeldin A wash-out despite impairment of clathrin function by AP20187. These findings indicate that AP-3–clathrin association is dispensable for endosomal AP-3 vesicle budding and suggest that endosomal AP-3–clathrin interactions differ from those by which AP-1 and AP-2 adaptors productively engage clathrin in vesicle biogenesis

Patterns of microRNA Expression in Non-Human Primate Cells Correlate with Neoplastic Development In Vitro

MicroRNAs (miRNAs) are small noncoding RNAs that negatively regulate gene expression post-transcriptionally. They play a critical role in developmental and physiological processes and have been implicated in the pathogenesis of several diseases including cancer. To identify miRNA signatures associated with different stages of neoplastic development, we examined the expression profile of 776 primate miRNAs in VERO cells (a neoplastically transformed cell line being used for the manufacture of viral vaccines), progenitor primary African green monkey kidney (pAGMK) cells, and VERO cell derivatives: spontaneously immortalized, non-tumorigenic, low-passage VERO cells (10-87 LP); tumorigenic, high-passage VERO cells (10-87 HP); and a cell line (10-87 T) derived from a 10-87 HP cell tumor xenograft in athymic nude mice. When compared with pAGMK cells, the majority of miRNAs were expressed at lower levels in 10-87 LP, 10-87 HP, and 10-87 T cells. We identified 10 up-regulated miRNAs whose level of expression correlated with VERO cell evolution from a non-tumorigenic phenotype to a tumorigenic phenotype. The overexpression of miR-376a and the polycistronic cluster of miR-376a, miR-376b and miR-376c conferred phenotypic changes to the non-tumorigenic 10-87 LP cells that mimic the tumorigenic 10-87 HP cells. Thirty percent of miRNAs that were components of the identified miRNAs in our spontaneously transformed AGMK cell model are also dysregulated in a variety of human tumors. These results may prove to be relevant to the biology of neoplastic development. In addition, one or more of these miRNAs could be biomarkers for the expression of a tumorigenic phenotype

GWAS meta-analysis of intrahepatic cholestasis of pregnancy implicates multiple hepatic genes and regulatory elements

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific liver disorder affecting 0.5–2% of pregnancies. The majority of cases present in the third trimester with pruritus, elevated serum bile acids and abnormal serum liver tests. ICP is associated with an increased risk of adverse outcomes, including spontaneous preterm birth and stillbirth. Whilst rare mutations affecting hepatobiliary transporters contribute to the aetiology of ICP, the role of common genetic variation in ICP has not been systematically characterised to date. Here, we perform genome-wide association studies (GWAS) and meta-analyses for ICP across three studies including 1138 cases and 153,642 controls. Eleven loci achieve genome-wide significance and have been further investigated and fine-mapped using functional genomics approaches. Our results pinpoint common sequence variation in liver-enriched genes and liver-specific cis-regulatory elements as contributing mechanisms to ICP susceptibility

Genetic associations at 53 loci highlight cell types and biological pathways relevant for kidney function.

Reduced glomerular filtration rate defines chronic kidney disease and is associated with cardiovascular and all-cause mortality. We conducted a meta-analysis of genome-wide association studies for estimated glomerular filtration rate (eGFR), combining data across 133,413 individuals with replication in up to 42,166 individuals. We identify 24 new and confirm 29 previously identified loci. Of these 53 loci, 19 associate with eGFR among individuals with diabetes. Using bioinformatics, we show that identified genes at eGFR loci are enriched for expression in kidney tissues and in pathways relevant for kidney development and transmembrane transporter activity, kidney structure, and regulation of glucose metabolism. Chromatin state mapping and DNase I hypersensitivity analyses across adult tissues demonstrate preferential mapping of associated variants to regulatory regions in kidney but not extra-renal tissues. These findings suggest that genetic determinants of eGFR are mediated largely through direct effects within the kidney and highlight important cell types and biological pathways