Promyelocytic leukemia (PML) is the organizer of nuclear matrix domains, PML nuclear bodies (NBs), with a proposed role in apoptosis control. In acute promyelocytic leukemia, PML/retinoic acid receptor (RAR) α expression disrupts NBs, but therapies such as retinoic acid or arsenic trioxide (As2O3) restore them. PML is conjugated by the ubiquitin-related peptide SUMO-1, a process enhanced by As2O3 and proposed to target PML to the nuclear matrix. We demonstrate that As2O3 triggers the proteasome-dependent degradation of PML and PML/RARα and that this process requires a specific sumolation site in PML, K160. PML sumolation is dispensable for its As2O3-induced matrix targeting and formation of primary nuclear aggregates, but is required for the formation of secondary shell-like NBs. Interestingly, only these mature NBs harbor 11S proteasome components, which are further recruited upon As2O3 exposure. Proteasome recruitment by sumolated PML only likely accounts for the failure of PML-K160R to be degraded. Therefore, studying the basis of As2O3-induced PML/RARα degradation we show that PML sumolation directly or indirectly promotes its catabolism, suggesting that mature NBs could be sites of intranuclear proteolysis and opening new insights into NB alterations found in viral infections or transformation.

A three-step mild procedure is described, that permits isolation of an adenovirus type-2 soluble hexon, penton and fiber antigens in an extensively purified form. This procedure comprises ammonium sulfate precipitation, Sephadex ion-exchange chromatography and adsorption chromatography on an hydroxyapatite column. The final antigen preparations were homogeneous with regard to immunological, electrophoretic and electron microscopic criteria.Adenovirus, which can be produced in high yield in tissue culture is an attractive model for studies of animal virus uncoating [l-61 and assembly [7,8], virion-cell interactions [9-111 and time-course biosynthesis of virus structural components within the host cell [13,14]. The infected cell synthesizes a considerable excess of viral material and only loo/, of virus proteins and DNA are incorporated into mature adenovirus particles [15]. Most of the nonincorporated proteinic material is extractible as "soluble" antigens which have been immunologically characterized and identified with capsid structural components hexon, penton and fiber : hexon is the hexagonal capsomer located on the faces and ridges of the viral icosahedron; the vertex projection called penton, is constituted of the penton base prolonged by the fiber [16-201. Several methods have been described for isolation and purification of these capsid subunits [21--251 We propose here a simple mild three-step procedure which permits isolation of large amounts of highly purified adenovirus type-2 hexon, penton and fiber available for biophysical [26-281, biochemical [21-251 and physiological studies [lo]. This method comprises neutral-salt precipitation, ion-exchange chromatography and adsorption chromatography. MATERIALS AND METHODS Production of Viral AntigensAdenovirus type-2, a representative of Rosen's subgroup I11 [29] was propagated in KB cell suqpension culture. KB cells were maintained a t 3 to 5 x lo5 cells in Eagle's basal medium supplemented with 5O/, horse serum and infected with a multiplicity of infection of 50 particles per cell. Cells were harvested 32 h post-infection and washed three times in phosphate-buffered saline. Cell pellet was resuspended in ten volumes hypotonic 0.01 M Tris-HC1 buffer pH 8.1, I mM EDTA and subjected to five cycles of quick freezing and thawing. The cell lysate was then mixed in a Waring-blendor with an equal volume of fluorocarbon Freon 113 [30]. The aqueous phase obtained by low-speed centrifugation containing the viral material was centrifuged for 1 h at 20000rev./min on a caesium chloride cushion (density, 1.43 g/ml) in a swinging bucket rotor (Beckman SW 27). Mature virions appeared as a visible opalescent band a t the top of the CsCl cushion. The band containing the virions was collected and the virus particles were further purified by density-gradient centrifugation in caesium chloride [31]. The supernatant above the virion band in the two succesive cycles of centrifugation, was the source of adenovirus-soluble antigens. Precipitation by Ammonium SulfateA saturate...

Cross-linking of adenovirus hexon capsomers by glutaraldehyde in vitro has been studied as a chemical probe for quaternary structure of hexon protein and virus capsid assembly. Glutaraldehyde cross-linking of hexons gave rise to three main oligomeric species, trimer, hexamer and nonamer, as analyzed on dodecylsulfate-acrylamide gels, which corresponded in terms of complete hexon capsomers, to single hexon capsomer, capsomer dimer and trimer. The major species was found to be the trimer of the hexon polypeptide unit, viz. the intramolecularly cross-linked hexon capsomer. This result indicated that intraoligomeric cross-linking took place more readily than the interoligomeric reactions. As shown by electron microscopy the spatial arrangement of the crosslinked hexon capsomer trimer was reminiscent of the structure of groups of three hexons clustered with %fold symmetry to form the groups of nine hexons isolated from disrupted adenovirus capsid.Virus assembly constitutes a crucial problem in molecular virology and many in vitro systems have been developed in an attempt to reproduce the morphogenesis of the virion within the host cell [l]. Very little is known about the mechanism of adenovirus assembly [2,3]. Hexon capsomer is the major subunit of the adenovirus icosahedral capsid and presents the advantage of being extractable from the infected cell as soluble native antigen, as well as the other capsid components, penton and fibre [4,5]. Although it has been reported that free hexons isolated from the intracellular pool could differ from those incorporated in the virion [ 5 ] , the identity of tryptic finger-prints of adenovirus type 2 hexon antigen crystallized from infected cell extracts and of hexon polypeptide isolated from mature virus particles implies a structural identity between incorporated and non-incorporated hexons [6].Hexon capsomer is an oligomeric protein, 300000 in molecular weight I n this study, the cross-linking reagent glutaraldehyde [16], has been employed to obtain information regarding the fashion in which the hexon polypeptide subunits interact and the resulting structural and biological changes. To prevent confusion in terms between cross-linked oligomers of complete hexon capsomers and cross-linked oligomers of hexon polypeptide units, these latter were referred to as hexon monomer, dimer, trimer, etc. the terms of capsomer dimer and trimer being reserved to assemblies of several hexon capsomers. MATERIALS AND METHODS Isolation and Purification of Adenovirus HexonsThe method of isolation and purification of adenovirus type2 soluble hexons has been described in details elsewhere [17]. It comprised ammonium sulfate precipitation of an infected cell freon extract, DEAE-Sephadex(A 50) chromatography and chromatography on hydroxyapatite column. The final hexon preparation was homogeneous with regard to the following criteria : electron microscopy, immunoEm.