Mordechai Aboud

Molecular mechanisms of cellular transformation by HTLV-1 Tax

The HTLV Tax protein is crucial for viral replication and for initiating malignant transformation leading to the development of adult T-cell leukemia. Tax has been shown to be oncogenic, since it transforms and immortalizes rodent fibroblasts and human T-lymphocytes. Through CREB, NF-κB and SRF pathways Tax transactivates cellular promoters including those of cytokines (IL-13, IL-15), cytokine receptors (IL-2Rα) and costimulatory surface receptors (OX40/OX40L) leading to upregulated protein expression and activated signaling cascades (e.g. Jak/STAT, PI3Kinase, JNK). Tax also stimulates cell growth by direct binding to cyclin-dependent kinase holenzymes and/or inactivating tumor suppressors (e.g. p53, DLG). Moreover, Tax silences cellular checkpoints, which guard against DNA structural damage and chromosomal missegregation, thereby favoring the manifestation of a mutator phenotype in cells.

Rapid purification of extracellular and intracellular Moloney murine leukemia virus

SummaryThe present study demonstrates the advantages of a combination of concentration by polyethylene glycol-6000 and Sepharose Cl-4B chromatography as a rapid procedure for retroviruses purification. This procedure can be completed within 3 hours, providing a high degree of virus purification with minimal damage to its structural and biological properties. Using transmission electron microscopy we observed many intracellular type-C virions in cytoplasmic vacuoles of 3T3/NIH cells chronically infected with Moloney murine leukemia virus. There intracellular virions could be isolated from postmitochondrial cytoplasmic fractions prepared from the infected cells by a procedure which minimized its contamination by extracellular free or membrane-bound virions. SDS-polyacrylamide gel electrophoresis showed that the intracellular and extracellular virions contained similar protein composition.

Topoisomerase I activity associated with human immunodeficiency virus (HIV) particles and equine infectious anemia virus core.

In the present study, we found a topoisomerase I (topo I) activity in two strains of human immunodeficiency virus type 1 (HIV‐1) and equine infectious anemia virus (EIAV) particles. The topo I activity was located in the EIAV cores and differed from the cellular topo I in its ionic requirements and response to ATP, indicating that these were two distinct forms of this enzyme. Topo I activity was removed from the viral lysates and viral cores by anti‐topo I antiserum. The only protein recognized by this antiserum was an 11.5 kd protein in HIV lysate and 11 kd in EIAV lysate. We showed that the 11 kd protein recognized by the anti‐topo I antiserum is the EIAV p11 nucleocapsid protein. Furthermore, purified topo I protein blocked the binding of the antibodies to the p11 protein and vice versa, purified p11 protein blocked the binding of these antibodies to the cellular topo I. These results suggest that the EIAV p11 nucleocapsid protein and the cellular topo I share similar epitopes.

Induction of Cellular Genes Is Mediated by the Bel1 Transactivator in Foamy Virus-Infected Human Cells

ABSTRACT To gain insight into human foamy virus (HFV; also called spumaretrovirus)-induced alterations of cellular genes, the expression profiles of defined genes in HFV-infected primary human cells were analyzed by cDNA array assays. Several distinct cellular genes activated by HFV infection were identified; the identities of the cellular genes were confirmed by RNA blot analyses. Compared with mock-infected controls, the concentrations of cellular Kip2, Egr-1, COUP-TF1, insulin-like growth factor II (IGF-II), and EphB3 mRNAs were significantly increased in HFV-infected cells and showed a gene-specific and time-dependent induction. Immunoblot analyses with antibodies against some of the cellular gene products revealed increased levels of the corresponding proteins. To investigate mechanisms of HFV-induced alterations in cellular gene expression, the capacity of known HFV genes to increase expression of defined cellular genes was analyzed by transient expression experiments. Plasmids that encode the HFV Bel1 transcriptional transactivator were necessary and sufficient to strongly increase expression of p57Kip2, IGF-II, and EphB3 genes in 293T cells. Potential mechanisms and consequences of activation of cellular genes during HFV infection and Bel1 transactivation of the Kip2 gene are discussed.

High levels of cytoplasmic HTLV-1 Tax mutant proteins retain a Tax-NF-kappaB-CBP ternary complex in the cytoplasm.

The oncogenic potential of HTLV-1 Tax protein is partially ascribed to its capacity to activate NF-κB. The current view is that Tax acts first in the cytoplasm to dissociate NF-κB factors from the IκB proteins and enable their nuclear translocation, then Tax links p65(RelA), within the nucleus, to CBP/p300 and P/CAF, which are essential for its optimal transcriptional activity. Our present study challenges the paradigm that Tax–p65(RelA)-CBP/p300 assembly occurs in the nucleus. Using Tax mutants defective for nuclear localization we show that at low levels these mutants induce the nuclear translocation of NF-κB factors but not their transcriptional activity, whereas at high levels they trap CBP and free p65(RelA) in the cytoplasm and block, thereby, their transcriptional function. In contrast, wild-type (w.t.) Tax strongly stimulated NF-κB-dependent gene expression in all tested experimental settings. These data suggest that the Tax–p65(RelA)-CBP ternary complex is established in the cytoplasm rather than in the nucleus. When this complex is formed with w.t. Tax, the entire moiety translocates into the nucleus and exerts high transcriptional activity. However, if the complex is formed with the cytoplasmic Tax mutants, the resulting moiety is retained in the cytoplasm and is, therefore, devoid of transcriptional activity.

Role of protein kinase C and the Spl-p53 complex in activation of p21WAF-1 expression by 12-O-tetradecanoylphorbol-13-acetate in human T cells

Previous reports have shown that, in certain cell types, p21WAF-1, which plays a central role in cell proliferation, can be activated by HTLV-I Tax protein and by TPA. Tax and TPA are also known to stimulate HTLV-I gene expression. Since cell proliferation has a major impact on HTLV-I replication, it was of interest to investigate their effect on p21WAF-1 in human T cells, which are the main target of HTLV-I in human infection. This study demonstrates that p21WAF-1 is activated in such cells by both factors, each acting through a different mechanism that does not influence the other. The effect of TPA is shown to require PKC activity. Notably, however, examination of different PKC isoforms revealed that PKC-α and PKC-ɛ stimulated p21WAF-1 expression, whereas PKC-η was rather inhibitory and PKC-β1 and β2 were ineffective. All these isoforms were found to be activated by TPA in the employed T cells, but this apparent paradox was resolved by the observation that when coexpressed together in these cells, the stimulatory PKCs override the inhibitory isoform. Further experiments demonstrated that the PKC-induced p21WAF-1 activation was mediated by binding of Sp1-p53 complex to the second most upstream of the six Sp1 recognition sites present in its promoter and that this effect did not require the cooperation of an p53-binding site.

Topoisomerase-II activity in human leukemic and lymphoblastoid cells

Topoisomerase-II activity was analyzed in various human leukemic and lymphoblastoid cell-lines with comparison to normal human peripheral blood lymphocytes. All of the examined tumor cells contained this enzyme in both the nuclear and cytoplasmic fractions, whereas no appreciable activity of the enzyme was detected in either fraction of the resting normal lymphocytes. Using pBR322 plasmid as a substrate, undialyzed extracts of the tumor cells exhibited the typical ATP-dependent relaxation of supercoiled circles and formation of linear and catenated structures, as well as the ATP-independent knotting activity. On the other hand, dialyzed extracts exerted only the ATP-dependent supercoil relaxation. Novobiocin inhibited the linearization and catenation but not the supercoil relaxing or knotting activities. This study provides indications for an excessive level of a structurally abnormal topoisomerase-II in these tumor cell-lines.

Effect of interferon on mouse cells chronically infected with murine leukaemia virus: kinetic studies on virus production and virus RNA synthesis.

NIH/3T3 cells chronically infected with the Moloney strain of murine leukaemia virus were incubated with interferon (IF). There was no effect on virus production during the first 4 h, but thereafter an antiviral state gradually developed, reaching a maximum at about 12 h. When IF was removed, the antiviral state (expressed in terms of inhibition of release of virus) remained constant for 10 h, after which there was an abrupt return to the normal rate of virus release. Analysis of IF-treated cells showed that there was a three to fourfold increase in the amount of virus RNA in the nucleus at 48 h after IF addition, and still a slight increase at 72 h. There were no increases in the amounts of virus RNA in the cytoplasm during 72 h after the addition of IF. These results agree with the postulate that IF inhibits a late stage in the maturation of virus in chronically infected cells.

Evidence that protein kinase A activity is required for the basal and tax-stimulated transcriptional activity of human T-cell leukemia virus type-I long terminal repeat

The present study was undertaken to investigate the role of protein kinase A (PKA) in the control of human T‐cell leukemia virus type‐I (HTLV‐I) long terminal repeat (LTR) expression, since this issue is still controversial. For this purpose we employed two human T‐cell lines; the Jurkat cells in which long exposure to diBu‐cAMP severely down‐regulated the catalytic subunit of PKA (PKA‐C), and H‐9 cells in which such exposure markedly increased PKA‐C level. Transient transfection assays revealed that addition of diBu‐cAMP 1 h before or after transfection profoundly increased HTLV‐I LTR directed CAT expression and synergistically enhanced its stimulation by the viral transactivator tax gene product in both cell lines. However longer exposure to diBu‐cAMP before transfection reduced LTR‐CAT expression to below its basal level and completely abolished its stimulation by tax in Jurkat cells, and this diBu‐cAMP inhibitory effect could be abrogated by co‐transfection of a PKA‐C expressing vector. By contrast, in H‐9 cells, this long exposure to diBu‐cAMP continued enhancing LTR‐CAT expression and its tax‐mediated transactivation, and this stimulatory effect of diBu‐cAMP could be diminished by the PKA‐specific inhibitor N‐[2‐(p‐bromocinnamylamine)ethyl]‐5‐ isoquinolinsulfonamide (H‐89). Notably, in the absence of diBu‐cAMP treatment H‐89 reduced LTR‐CAT expression to below its basal level and prevented its stimulation by tax in both cell lines. Together these findings indicate not only that cAMP‐activated PKA stimulates HTLV‐I LTR expression and its transactivation by tax, but even in the absence of PKA activating signals the basal HTLV‐I LTR expression as well as its stimulation by tax are both dependent on a basal PKA activity.

DNA binding properties of the zinc‐bound and zinc‐free HIV nucleocapsid protein: supercoiled DNA unwinding and DNA‐protein cleavable complex formation

The HIV nucleocapsid (NC) protein contains, as those of other retroviruses, two Cys‐His arrays which function as zinc finger binding domains. The nucleic acid binding properties of retroviral NC have been previously demonstrated. In this study, we characterized the DNA binding ability of the zinc‐bound and zinc‐free forms of HIV NC. We found that in addition to binding single‐stranded DNA, both forms bind and unwind supercoiled plasmid DNA. The binding ability of the zinc‐bound form was higher than the zinc‐free form. In addition we showed the formation of NC protein‐DNA cleavable complex which is the result of a presumably covalent bond formed between the protein and the phosphate moiety of the DNA backbone. The NC unwinding activity and the protein‐DNA cleavable complex formation resembles the first step of the relaxing mechanism of DNA topoisomerase. Our results shed light on the possibility of a novel physiological function for the HIV NC protein in the viral life cycle.