Transcriptional Activation of the Adenoviral Genome Is Mediated by Capsid Protein VI

Gene expression of DNA viruses requires nuclear import of the viral genome. Human Adenoviruses (Ads), like most DNA viruses, encode factors within early transcription units promoting their own gene expression and counteracting cellular antiviral defense mechanisms. The cellular transcriptional repressor Daxx prevents viral gene expression through the assembly of repressive chromatin remodeling complexes targeting incoming viral genomes. However, it has remained unclear how initial transcriptional activation of the adenoviral genome is achieved. Here we show that Daxx mediated repression of the immediate early Ad E1A promoter is efficiently counteracted by the capsid protein VI. This requires a conserved PPxY motif in protein VI. Capsid proteins from other DNA viruses were also shown to activate the Ad E1A promoter independent of Ad gene expression and support virus replication. Our results show how Ad entry is connected to transcriptional activation of their genome in the nucleus. Our data further suggest a common principle for genome activation of DNA viruses by counteracting Daxx related repressive mechanisms through virion proteins

Targeting Expression of the Leukemogenic PML-RAR alpha Fusion Protein by Lentiviral Vector-Mediated Small Interfering RNA Results in Leukemic Cell Differentiation and Apoptosis

Acute promyelocytic leukemia (APL) results from a chromosomal translocation that gives rise to the leukemogenic fusion protein PML-RAR alpha (promyelocytic leukemia-retinoic acid alpha receptor). Differentiation of leukemic cells and complete remission of APL are achieved by treatment of patients with pharmacological doses of all-trans retinoic acid (ATRA), making APL a model disease for differentiation therapy. However, because patients are resistant to further treatment with ATRA on relapse, it is necessary to develop alternative treatment strategies to specifically target APL. We therefore sought to develop a treatment strategy based on lentiviral vector-mediated delivery of small interfering RNA (siRNA) that specifically targets the breakpoint region of PML-RAR alpha. Unlike treatment with ATRA, which resulted in differentiation of leukemic NB4 cells, delivery of siRNA targeting PML-RAR alpha into NB4 cells resulted in both differentiation and apoptosis, consistent with the specific knockdown of PML-RAR alpha. Intraperitoneal injection of NB4 cells transduced with lentiviral vectors delivering PML-RAR alpha-specific siRNA but not control siRNA prevented development of disease in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Taken together, these results indicate that development of PML-RAR alpha-specific siRNA may represent a promising treatment strategy for ATRA-resistant APL

Regulation of MEF2 by Histone Deacetylase 4- and SIRT1 Deacetylase-Mediated Lysine Modifications

The class II deacetylase histone deacetylase 4 (HDAC4) negatively regulates the transcription factor MEF2. HDAC4 is believed to repress MEF2 transcriptional activity by binding to MEF2 and catalyzing local histone deacetylation. Here we report that HDAC4 also controls MEF2 by a novel SUMO E3 ligase activity. We show that HDAC4 interacts with the SUMO E2 conjugating enzyme Ubc9 and is itself sumoylated. The overexpression of HDAC4 leads to prominent MEF2 sumoylation in vivo, whereas recombinant HDAC4 stimulates MEF2 sumoylation in a reconstituted system in vitro. Importantly, HDAC4 promotes sumoylation on a lysine residue that is also subject to acetylation by a MEF2 coactivator, the acetyltransferase CBP, suggesting a possible interplay between acetylation and sumoylation in regulating MEF2 activity. Indeed, MEF2 acetylation is correlated with MEF2 activation and dynamically induced upon muscle cell differentiation, while sumoylation inhibits MEF2 transcriptional activity. Unexpectedly, we found that HDAC4 does not function as a MEF2 deacetylase. Instead, the NAD(+)-dependent deacetylase SIRT1 can potently induce MEF2 deacetylation. Our studies reveal a novel regulation of MEF2 transcriptional activity by two distinct classes of deacetylases that affect MEF2 sumoylation and acetylation

Inhibition of Different Lassa Virus Strains by Alpha and Gamma Interferons and Comparison with a Less Pathogenic Arenavirus

The high pathogenicity of Lassa virus is assumed to involve resistance to the effects of interferon (IFN). We have analyzed the effects of alpha IFN (IFN-α), IFN-γ, and tumor necrosis factor alpha (TNF-α) on replication of Lassa virus compared to the related, but less pathogenic, lymphocytic choriomeningitis virus (LCMV). Three low-passage Lassa virus strains (AV, NL, and CSF), isolated from humans with mild to fulminant Lassa fever, were tested. Lassa virus replication was inhibited by IFN-α and IFN-γ, but not TNF-α, in Huh7 and Vero cells. The degree of IFN sensitivity of a Lassa virus isolate did not correlate with disease severity in human patients. Furthermore, cytokine effects observed for Lassa virus and LCMV (strains CH-5692, Armstrong, and WE) were similar. To address the mechanisms involved in the IFN effect, we used cell lines in which overexpression of IFN-stimulated proteins promyelocytic leukemia protein (PML) and Sp100 could be induced. Both proteins reside in PML bodies, a cellular target of the LCMV and Lassa virus Z proteins. Overexpression of PML or Sp100 did not affect replication of either virus. This, together with the previous finding that PML knockout facilitates LCMV replication in vitro and in vivo (M. Djavani, J. Rodas, I. S. Lukashevich, D. Horejsh, P. P. Pandolfi, K. L. Borden, and M. S. Salvato, J. Virol. 75:6204-6208, 2001; W. V. Bonilla, D. D. Pinschewer, P. Klenerman, V. Rousson, M. Gaboli, P. P. Pandolfi, R. M. Zinkernagel, M. S. Salvato, and H. Hengartner, J. Virol. 76:3810-3818, 2002), describes PML as a mediator within the antiviral pathway rather than as a direct effector protein. In conclusion, the high pathogenicity of Lassa virus compared to LCMV is probably not due to increased resistance to the effects of IFN-α or IFN-γ. Both cytokines inhibit replication which is relevant for the design of antiviral strategies against Lassa fever with the aim of enhancing the IFN response

PIC-1/SUMO-1-modified PML-retinoic acid receptor alpha mediates arsenic trioxide-induced apoptosis in acute promyelocytic leukemia.

Fusion proteins involving the retinoic acid receptor alpha (RARalpha) and PML or PLZF nuclear protein are the genetic markers of acute promyelocytic leukemia (APL). APLs with PML-RARalpha or PLZF-RARalpha fusion protein differ only in their response to retinoic acid (RA) treatment: the t(15;17) (PML-RARalpha-positive) APL blasts are sensitive to RA in vitro, and patients enter disease remission after RA treatment, while those with t(11;17) (PLZF-RARalpha-positive) APLs do not. Recently it has been shown that complete remission can be achieved upon treatment with arsenic trioxide (As2O3) in PML-RARalpha-positive APL, even when the patient has relapsed and the disease is RA resistant. This appears to be due to apoptosis induced by As2O3 in the APL blasts by poorly defined mechanisms. Here we report that (i) As2O3 induces apoptosis only in cells expressing the PML-RARalpha, not the PLZF-RARalpha, fusion protein; (ii) PML-RARalpha is partially modified by covalent linkage with a PIC-1/SUMO-1-like protein prior to As2O3 treatment, whereas PLZF-RARalpha is not; (iii) As2O3 treatment induces a change in the modification pattern of PML-RARalpha toward highly modified forms; (iv) redistribution of PML nuclear bodies (PML-NBs) upon As2O3 treatment is accompanied by recruitment of PIC-1/SUMO-1 into PML-NBs, probably due to hypermodification of both PML and PML-RARalpha; (v) As2O3-induced apoptosis is independent of the DNA binding activity located in the RARalpha portion of the PML-RARalpha fusion protein; and (vi) the apoptotic process is bcl-2 and caspase 3 independent and is blocked only partially by a global caspase inhibitor. Taken together, these data provide novel insights into the mechanisms involved in As2O3-induced apoptosis in APL and predict that treatment of t(11;17) (PLZF-RARalpha-positive) APLs with As2O3 will not be successful

Regulation of p53 activity in nuclear bodies by a specific PML isoform

Covalent modification of the promyelocytic leukaemia protein (PML) by SUMO-1 is a prerequisite for the assembly of nuclear bodies (NBs), subnuclear structures disrupted in various human diseases and linked to transcriptional and growth control. Here we demonstrate that p53 is recruited into NBs by a specific PML isoform (PML3) or by coexpression of SUMO-1 and hUbc9. NB targeting depends on the direct association of p53, through its core domain, with a C-terminal region of PML3. The relocalization of p53 into NBs enhances p53 transactivation in a promoter-specific manner and affects cell survival. Our results indicate the existence of a cross-talk between PML- and p53-dependent growth suppression pathways, implying an important role for NBs and their resident proteins as modulators of p53 functions

Forced retinoic acid receptor α homodimers prime mice for APL-like leukemia

SummaryRARA becomes an acute promyelocytic leukemia (APL) oncogene by fusion with any of five translocation partners. Unlike RARα, the fusion proteins homodimerize, which may be central to oncogenic activation. This model was tested by replacing PML with dimerization domains from p50NFκB (p50-RARα) or the rapamycin-sensitive dimerizing peptide of FKBP12 (F3-RARα). The X-RARα fusions recapitulated in vitro activities of PML-RARα. For F3-RARα, these properties were rapamycin sensitive. Although in vivo the artificial fusions alone are poor initiators of leukemia, p50-RARα readily cooperates with an activated mutant CDw131 to induce APL-like disease. These results demonstrate that the dimerization interface of RARα fusion partners is a critical element in APL pathogenesis while pointing to other features of PML for enhancing penetrance and progression