Pierre Boulanger

Crystallization, enzymatic cleavage, and the polarity of the adenovirus type 2 fiber

Crystals of the fiber protein of adenovirus type 2 have been grown. Analysis of these crystals (type I crystals) showed that they were composed of fiber polypeptide with a lower apparent molecular weight (60 kDa) than that of the soluble or virion-incorporated fiber (62 kDa). N-terminal sequencing revealed that the fiber polypeptide chain of 60 kDa was cleaved at tyrosine17 from the N-end. The C-terminus remained intact. Assays with protease inhibitors suggested that the spontaneous cleavage of the fiber occurring upon its crystallization was due to a cellular, calcium-dependent, chymotrypsin-like protease co-purifying with the fiber and activated during hydroxyapatite chromatography. Crystallization of fiber purified in the presence of chymostatin provided crystals of a different structure under the electron microscope (crystals of type II), composed of 62-kDa fiber polypeptide units. The 62-kDa fiber from the type II crystals, as well as the 62-kDa fiber isolated from infected cell extracts, were able to associate with the penton base in vitro to form a penton capsomer. The 60-kDa fiber has lost this capacity. The accessibility of the N- and C-termini of the fiber inside the penton structure was probed by anti-peptide sera after limited proteolysis. The results are consistent with a polarity of the fiber in which its N-terminus is oriented toward the penton base, the C-terminal domain corresponding to the distal knob.

HEED, the product of the human homolog of the murine eed gene, binds to the matrix protein of HIV-1.

heed, the human homolog of mouseeed and Drosophila esc, two members of thetrithorax (trx) and Polycomb group (Pc-G) of genes, was isolated by screening an activated lymphocyte cDNA library versus the immunodeficiency virus type 1 (HIV-1) MA protein used as a bait in a two-hybrid system in yeast. The human EED protein (HEED) had 99.5% identity with the mouse EED protein and contained seven WD repeats. Two heedgene transcripts were identified, with a putative 407-nucleotide-long intron, giving rise to two HEED protein isoforms of 535 and 494 residues in length, respectively. The shorter HEED isoform, originated from the unspliced message, lacked the seventh WD repeat. HEED was found to bind to MA protein in vitro, as efficiently asin vivo in yeast cells. Site-directed mutagenesis and phage biopanning suggested that the interaction between HEED and MA involved the N-terminal region of the MA protein, including the first polybasic signal, in a MA conformation-dependent manner. In the HEED protein, however, two discrete linear MA-binding motifs were identified within residues 388–403, overlapping the origin of the fifth WD repeat. Deletion of the C-terminal 41 residues of HEED, spanning the seventh WD repeat, as in the 494-residue HEED protein, was detrimental to HEED-MA interaction in vivo, suggesting the existence of another C-terminal binding site and/or a conformational role of the HEED C-terminal domain in the MA-HEED interaction. MA and HEED proteins co-localized within the nucleus of co-transfected human cells and of recombinant baculovirus co-infected insect cells. This and the failure of HEED to bind to uncleaved GAG precursor suggested a role of HEED at the early stages of virus infection, rather than late in the virus life cycle.

Assembly of adenovirus penton base and fiber

Abstract Human adenovirus type 2 (H2) fiber isolated from the cellular pool of H2 wild-type (WT) soluble components was found to be capable of assemble in vitro with penton base obtained from the fiber-defective temperature-sensitive (ts) mutant H2 ts-125. This assembly occurred over a relatively broad range of pHs (5.5–9.0) and of ionic strengths (0.05–1.0). The extent of penton formation was estimated by two-dimensional immuno-electrophoresis. The legitimacy of the assembly in vitro was demonstrated by morphological, biochemical, and biological evidence. The assembly of the penton base with the fiber appeared to be a reversible reaction, with a dissociation constant KD = 2 × 10−7 M, in terms of fiber molarity. This value implied a high affinity of the penton base for the fiber. Comparative digestions by carboxypeptidase of uncombined fiber and of penton base-combined fiber suggested that the recognition site for assembly involved the last 20 amino acids of the C-terminal sequence of the fiber polypeptide chain. The same analysis performed on [14C]glucosamine-labeled fiber and penton seemed to indicate that the carbohydrate units carried by the fiber were positioned too far from the C-end to be directly involved in the recognition and assembly process of the penton base with the fiber. Chimeric penton produced in vitro by incubating fiber and penton base from different adenovirus serotypes, as well as in vivo by interserotypic ts+ recombinants suggested a group specificity of the recognition site on the fiber and a likely highly conserved C-terminal amino acid sequence. Assembly of penton base and fiber in vitro resulted in a significant change, in the antigenicity of the penton base-combined fiber, which was thought to reflect some three-dimensional remolding.

Human adenovirus type 2 protein IIIa. II. Maturation and encapsidation.

Abstract Evidence is presented that a HAd2 protein IIIa precursor (PIIIa) of 67,000 daltons exists in assembly intermediates and young virions and is processed into protein IIIa (66,000 daltons). This cleavage is suggested to occur at the N terminus, and at a late stage of virus maturation, between young and mature virions. Sixty copies of IIIa were found to be present per mature virion or assembly intermediate particle, viz. one molecule per penton base subunit. The PIIIa polypeptide was antigenically different from IIIa, with a weaker antigenic reactivity toward anti-IIIa serum. This difference in antigenicity might reflect a variation in three-dimensional structure between the PIIIa polypeptide and IIIa, resulting from proteolytic cleavage. Immunoprecipitation of deoxycholate-disrupted HAd2 virions using anti-IIIa serum suggests some interaction between IIIa and the core protein VII in mature virions. Four ts mutants defective in virion assembly and accumulating empty particles at 39.5° were studied with regard to the occurrence of IIIa antigen in the cell pool of soluble antigens: H5 ts 58, H2 ts 4, H2 ts 112, and H2 ts 101 were shown to be phenotypically IIIa defective, and to belong to three different complementation groups. H2 ts 4 and H2 ts 112 were in the same complementation group, whereas H5 ts 58 and H2 ts 101 fell into two separate groups. Only the H5 ts 58 lesion has been located in the IIIa gene ( E. Frost and J. F. Williams, 1978 , Virology 91 , 39–50). The IIIa antigen appears, therefore, to be a serological marker for adenovirus assembly.

Hexon trimerization occurring in an assembly-defective, 100K temperature-sensitive mutant of adenovirus 2

Analysis of 100K-defective temperature-sensitive adenovirus mutants confirmed the multifunctional character of the nonstructural, virus-coded 100K protein. In addition to its function in hexon trimerization (altered in H5ts1), and its possible direct or indirect role in hexon transport to nucleus (mutated in H2ts118), genetic and biochemical evidence was presented that 100K play some critical role in the scaffolding process of adenovirus capsid. This function appeared to be defective in H2ts107 and to map between coordinates 69.0 and 69.9, leftward from the H5ts1 lesion (70-73 map units; Arrand, 1978). This corresponded to the central domain of the 100K protein, between amino acid 300 and 400 from the N end. DNA sequencing of cloned fragments of H2ts107 DNA overlapping the mutation revealed two point mutations on the same codon at nucleotide 25,082 and 25,083 (GAC----GCA), corresponding to a nonconservative amino acid change (aspartic acid----alanine) at position 324 in the 100K sequence. 100K of adenovirus 2 wild type (WT) was found to bind in significant amounts to novobiocin-affinity column, and to be coeluted with hexon, penton, IIIa, and cellular topoisomerase II activity, by novobiocin- or ATP-Mg2+-containing buffers. H2ts107 100K also bound to novobiocin column, but the elution pattern differed from that of WT, suggesting some alteration in the affinity of the mutated 100K for novobiocin. The same behavior on affinity column as H2ts107 100K was observed for 90K, a cleavage product of the 100K, found in great abundance in H2ts107 at 39.5 degrees and corresponding to the C-terminal moiety of the 100K molecule. This implied that the novobiocin-binding domain of the 100K was not confined at its N terminus, and was altered in the H2ts107 mutant.

Human adenovirus type 2 protein IIIa I. Purification and characterization

Abstract Protein IIIa from human adenovirus 2 was shown to exist as a major soluble antigenic component in the infected cell pool of virus material when the cell extracts were analyzed in two-dimensional immunoelectrophoresis against hyperimmune antivirion sera. Protein IIIa was purified to apparent homogeneity by three methods, two using virus particles, one using infected cell lysates as starting material. The three methods gave similar yields of IIIa. Protein IIIa behaved as a spherical molecule with a sedimentation coefficient of 6.0 S, a frictional ratio of 1.02, and a molecular weight of 65,430 ± 1800 in its native state, suggestive of the existence of one single polypeptide unit. The N terminus of the IIIa was found to be glycine. The amino acid composition revealed an unusually high content of serine, glycine, and proline and a low content of lysine. Isoelectrophoretic analysis revealed a heterogeneous population with a major band at p I 5.95 and a minor band at p l 6.08. Only group-specific antigenic determinants were detected in IIIa, which did not seem to induce neutralizing antibodies.

Molecular weight of adenovirus serotype 2 capsomers. A new characterization.

Abstract We have determined the molecular weight of some of the adenovirus serotype 2 structural proteins: penton, penton base and fibre. Physical techniques, namely neutron scattering and hydrodynamical measurements, indicate that the penton base is a trimer. This is confirmed by analysis of the virion composition based on quantitative gel scanning. This finding implies either that other proteins (e.g. protein IIIa) are essential in the architecture of the fivefold vertex of the virion, or that the usual assumption that icosahedral symmetry involves identical interactions related to the symmetry of the virion does not hold.

Cytoskeletal proteins associated with intracytoplasmic human adenovirus at an early stage of infection

Taking advantage of the sedimentation properties of adenovirus particles, adenovirus-infected baby hamster kidney (BHK21) cells were reversibly fixed with cleavable diimidoester dimethyl 3,3-dithiobispropionimidate (DTBP) at early times of infection (30 min). Cytoskeletal proteins associated with/or in close vicinity to virions were isolated as a complex cross-linked with carrier virus. Four major cellular proteins were thus found to co-purify with adenovirus particles. They were characterized by their coordinates on 2D maps and immunological reactivity. Two of them were identified as alpha-tubulin (58 kD), and vimentin subunits (56 kD). The two other species 68 and 66 kD might correspond to stress proteins. Affinity blotting on gels showed that both alpha-tubulin and vimentin were capable of binding with intact and penton-less adenovirions. Adenovirus components involved in the binding seemed to be mainly core proteins V and VII, and to a lesser extent, hexon. Analysis of neighbor relationships among proteins of the adenovirus-cytoskeletal protein cross-linked complex suggested that some capsid alterations occurred upon/or after entry of the virus into the cell, and that these structural modifications preferentially concerned the vertex components penton and IIIa, and the core protein V.

Antibody-triggered dissociation of adenovirus penton capsomer

Abstract By employing two-dimensional immunoelectrophoreses and immunoselection techniques on Staphylococcus aureus protein A, evidence was provided showing that adenovirus 2 penton capsomer could be dissociated into its two constituting entities, penton base and fiber. The dissociation was obtained with antifiber antibody, but not with antipenton base antibody. The dissociating effect was more pronounced with subgroup-specific antibody which is thought to react with the antigenic determinants located on the rod-like portion of the fiber. It was suggested that penton disruption was due to some antibody-induced conformational change of a critical portion of the fiber.

Morphogenesis of human adenovirus type 2: Sequence of entry of proteins into previral and viral particles

The initial steps of adenovirus capsid morphogenesis and the sequence of entry of structural and nonstructural proteins into assembly-intermediate (IM) particles were investigated by pulse-chase labeling, temperature shifts, and cycloheximide inhibition of particle formation. The experiments were performed on wild-type and two assembly-defective, temperature-sensitive mutants, H2 ts 112 and H2 ts 107. The sequence of events in the adenovirus assembly can be schematized as follows. (i) Hexons, pentons, and protein IX assembled with scaffolding proteins 100K, PVIII, and PVII, precursor to the major core protein, to form a previral particle banding at a density of 1.285 in CsCl; (ii) additional incorporation of maturation and/or stabilization proteins IIIa, 50K, 39K, 28K, and PVI led to 1.295 IM; (iii) exit of 100K, 39K, and 28K, and entry of viral DNA gave rise to 1.370 IM; (iv) dephosphorylation and/or exit of 50K and exchange with core protein V and processing of precursors to VII, VI, VIII, and DNA-terminal protein resulted in formation of infectious 1.345 virion. The polypeptide composition of the new class of assembly-intermediate particles elicited by H2 ts 107 (1.285 IM), suggested that 100K, PVIII, and also PVII might serve as scaffold components for adenovirus capsid building.